Transgenic Res (2011) 20:1139–1189 DOI 10.1007/s11248-011-9540-8
ABSTRACTS
Program and Abstracts of the 10th Transgenic Technology Meeting (TT2011) TradeWinds Island Grand Resort, St Pete Beach, Florida, USA, October 24–26, 2011
The TT2011 Meeting is organized by the International Society for Transgenic Technologies (ISTT) (www.trans techsociety.org)
TT2011 Organizing Committee LIuı´s Montoliu (National Center of Biotechnology—CNB, CSIC, Madrid, Spain), President Thom Saunders (University of Michigan, Ann Arbor, MI, USA), Vice-President Peter Sobieszczuk (University of Miami, Miami, FL, USA) Boris Jerchow (Max-Delbru¨ck-Center for Molecular Medicine, Berlin-Buch, Germany) Carlisle Landel (Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA) Shirley Pease (Caltech, Pasadena, CA, USA) Johannes Wilbertz (KCTT, Karolinska Institute, Stockholm, Sweden) Elizabeth Williams (TASQ, University of Queensland, Brisbane, QLD, Australia) Tom Fielder (University of California-Irvine, CA, USA) Jan Parker-Thornburg (M. D. Anderson Cancer Center, Houston, TX, USA).
TT2011 Registration and Administration (Technical Secretariat) Jenny McMahon, In-Conference Ltd., Edinburgh, UK Stacy Nairn, In-Conference Ltd, Edinburgh, UK Alison Cameron, ISTT Administrative Assistant
TT2011 Sponsoring Companies and Institutions Springer (www.springer.com) Transgenic Research (www.springer.com/biomed/molecular/ journal/11248) genOway (www.genoway.com) inGenious Targeting Laboratory (www.genetargeting. com) Scionics Computer Innovation GmbH (www.scionics.de) Hamilton-Thorne (www.hamiltonthorne.com) NanoInjection Technologies (www.nanoinjectiontech.com) ParaTechs (www.paratechs.com/nset.htm) Sigma-Aldrich (www.sigmaaldrich.com/sigma-aldrich/home. html) Cook Medical (www.cookmedical.com/home.do) Charles River (www.criver.com) TransViragen (www.transviragen.com) Transposagen Biopharmaceuticals (www.transposagenbio. com) Planer (www.planer.com) Eppendorf (www.eppendorf.com) Applied Stem Cell (www.appliedstemcell.com) Allentown (www.allentowninc.com) Labotect (www.labotect.com) Sutter Instrument (www.sutter.com) Taconic (www.taconic.com) Transnetyx (www.transnetyx.com) The Jackson Laboratory (www.jax.org) AAALAC (www.aaalac.org) Australian Animal Care Systems (www.animalcaresystems. com)
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TT2011 Preliminary Program (July 15, 2011) Sunday, October 23, 2011 10:00–12:00 15:00–18:00 18:00–22:00
Karin Blumer Novartis International AG, Basel, Switzerland 14:00–14:30
ISTT Council meeting Registration and Poster Set Up Social event: Cruise and Pre-meeting dinner
New European Directive on the protection of animals used for scientific purposes: Impact on the generation and management of genetically modified models Belen Pintado National Center of Biotechnology (CNB), CSIC, Madrid, Spain
Monday, October 24, 2011 Opening 8:45–9:00
14:30–15:00
Welcome Address: ISTT President, Organizing Committee
Chris Newcomer AAALAC, Frederick, MD, USA
Session 1. Mouse Genetics Chair: Carlisle Landel, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA 9:00–9:30
15:00–15:30 15:30–17:30
Genetic Background and Superovulation
Coat Colour in generation of chimeras Marina Gertsenstein Toronto Centre for Phenogenomics, Toronto, ON, Canada
10:00–10:30
How to Name a Mouse–Mouse Strain, Gene, and Allele Nomenclature
Session 4: ISTT Prize 17:30–17:40
Coffee Break
17:40–18:45
Session 2. Mouse Models of Disease Chair: Jan Parker-Thornburg, M. D. Anderson Cancer Center, Houston, TX, USA 11:00–11:30
As quiet as a mouse: Use of genetically modified mice to study hearing and deafness
Introduction to the ISTT Prize winner Lluı´s Montoliu National Center of Biotechnology (CNB), CSIC, Madrid, Spain
Howard Dene The Jackson Laboratory, Bar Harbor, Maine, USA 10:30–11:00
Coffee Break Round Table: How to Run a Transgenic Unit Chair: Tom Fielder, University of CaliforniaIrvine, CA, USA Aimee B. Stablewski, Roswell Park Cancer Institute, Buffalo, NY, USA Cheryl Bock, Duke Medical Center, Durham, NC, USA Rebecca Haffner, Weizmann Institute of Science, Rehovot, Israel
Carlisle Landel Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA 9:30–10:00
Shifting Research Animal Regulations and Accreditation Standards: Hobbling or Facilitating Sound Science?
8th ISTT Prize for outstanding contributions to transgenic technologies Mammalian germline modification Ralph L. Brinster University of Pennsylvania, School of Veterinary Medicine, Philadelphia, PA, USA
19:00
Wine, Beer, Cheese and Posters
Andy K. Groves Baylor College of Medicine, Houston, TX, USA 11:30–12:00
Development and preclinical evaluation of therapies for spinal muscular atrophy in mice Arthur Burghes The Ohio State University, Columbus, OH, USA
12:00–13:30
Lunch and Posters
Session 3: Animal Ethics, welfare and updated regulations on animal experimentation
Tuesday, October 25, 2011 Session 5: Fundamental Methods for Transgenic Core Facilities Chair: Elizabeth Williams, TASQ, University of Queensland, Brisbane, QLD, Australia 9:00–9:30
Charles Hawkins Johns Hopkins University, Baltimore, MD, USA
Chair: Johannes Wilbertz, Karolinska Institute, Stockholm, Sweden 13:30–14:00
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Animal Ethics
Essentials of Microinjection
9:30–10:00
Factors Affecting In Vitro Fertilization
Transgenic Res (2011) 30:1139–1189 Jan Parker-Thornburg M. D. Anderson Cancer Center, Houston, TX, USA 10:00–10:30
International Germplasm Transfer Kent Lloyd University of California-Davis, CA, USA
10:30–11:00
Coffee Break
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Recombinase mediated promoter switching enables two different transgene expression levels to be achieved in a single line of targeted transgenic mice Ben Davies Wellcome Trust Centre for Human Genetics, University of Oxford, UK
Session 6: International initiatives, web resources for transgenic/KO mice Chair: Peter Sobieszczuk, University of Miami, Miami, FL, USA 11:00–11:30
16:15–16:30
Germline Performance of Gene-Targeted ES Cell Clones Imported from the International Knockout Mouse Consortium by a Transgenic Core Facility
Selected short oral presentation A ‘‘Lymphoreporter’’ mouse for in vivo imaging of lymphangiogenesis in development, inflammation and tumour metastasis
Finding your transgenic mouse/ES cell line/ gene with on-line resources
Sagrario Ortega Spanish National Cancer Research Center (CNIO), Madrid, Spain
Peter Sobieszczuk University of Miami, Miami, FL, USA 11:30–12:00
Selected short oral presentation
16:30–17:30 17:30–19:30 20:00
Posters, ODD numbers ISTT General Assembly Conference Banquet
Thom Saunders University of Michigan, Ann Arbor, MI, USA 12:00–12:30
The IKMC Web Portal: a single access point to mouse knockouts from the International Knockout Mouse Consortium William Skarnes Wellcome Trust Sanger Institute, Hinxton, UK
12:30–14:00
Wednesday, October 26, 2011 Session 8: Beyond Mouse Transgenesis Chair: Lluı´s Montoliu, National Center of Biotechnology (CNB), CSIC, Madrid, Spain 9:00–9:30
Posters & light Lunch, Posters, EVEN numbers
Bruce Whitelaw Roslin Institute, University of Edinburgh, Midlothian, Scotland, UK
Session 7: New technologies Chair: Boris Jerchow, Max-Delbru¨ck-Center for Molecular Medicine, Berlin-Buch, Germany 14:00–14:30
14:30–15:00
Development of vibratory microinjection systems Fujio Miyawaki Tokyo Denki University, Saitama, Japan
15:30–16:00
10:00–10:30
The use of transgenic zebrafish to investigate biological processes in vivo Robert Kelsh Centre for Regenerative Medicine, University of Bath, Bath, UK
10:30–11:00
Coffee Break
Session 9: Animal Biotechnology Chair: Wojtek Auerbach, Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA 11:00–11:30
The JAX Colony Management System (JCMS): An extensible colony and phenotype data management system for transgenic mouse colonies Chuck Donnelly The Jackson Laboratory, Bar Harbor, Maine, USA
Transgenic pigs as models for translational biomedical research Eckhard Wolf University of Munich, Munich, Germany
Pronuclear injection-based mouse targeted transgenesis by site-specific recombination Masato Ohtsuka Tokai University, School of Medicine, Isehara, Kanagawa, Japan
15:00–15:30
9:30–10:00
Authentic Rat Embryonic Stem Cells and Germline Transmission of Targeted Mutations Qi-Long Ying Center for Stem Cell and Regenerative Medicine, University of Southern California, LA, CA, USA
An introduction to non-mouse models
The Regulation and Politics of Food from Genetically Engineered Animals Alison L. Van Eenennaam University of California-Davis, CA, USA
11:30–12:00
Suppression of bird flu transmission in transgenic chickens
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Transgenic Res (2011) 20:1139–1189 Helen Sang The Roslin Institute, University of Edinburgh, Midlothian, Scotland, UK
12:00–12:30
Beyond the Mouse—Novel Engineered Animals
Genetically
Edward Weinstein Sigma Advanced Genetic Engineering Labs, St. Louis, MO, USA 12:30–12:45
Selected short oral presentation Production of transgenic sheep by inhibin a shRNA
productivity of transgenic facilities. It depends on the application of exogenous gonatotropins to entrain and stimulate the ovaries to mature and then release an unusually large number of oocytes. From the time of the early work in the 1970s by Beaumont and Smith, it was apparent that genetic factors play a role in superovulatory responses. This talk will summarize the work to date on the role of genetic background upon this response, with an eye towards improving the production of oocytes and embryos. 2. Coat colour in generation of chimeras Marina Gertsenstein Toronto Centre for Phenogenomics, Toronto, Canada
Xiangyang Miao Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China 12:45–13:00
Selected short oral presentation Targeted transgene integration in mouse embryos using ‘‘open source’’ zinc finger nucleases Mario Hermann Institute of Laboratory Animal Sciences, University of Zu¨rich, Zu¨rich, Switzerland
13:00–14:30
Lunch and Posters
Session 10: Manipulating the Rat Genome Chair: Thom Saunders, University of Michigan, Ann Arbor, MI, USA 14:30–15:00
Mutating the rat genome with transposons Joseph Ruiz Transposagen Inc., Lexington, KY, USA
15:00–15:30
From GWAS to function using sensitized strains, transgenic rescue and gene KO Howard Jacob Medical College of Wisconsin, Milwaukee, WI, USA
15:30
ISTT Young Investigator Award ISTT Registration Awards TT2011 Best Poster awards Presentation of TT2013 meeting Concluding Remarks
16:00
End of TT2011 meeting
Coat colour genetics has a very long history in biomedical research. 1303 currently known annotated genotypes are listed under ‘‘coat color anomalies’’ by Mouse Genome Informatics resources (http://www.informatics.jax.org). Mouse coat colour mutations were instrumental for the initial experimental use of mice by proving Mendelian laws in mammals and establishing first inbred strains. They remain very important in the studies of the melanocyte biology and provide a visible tool for genetic analysis. One of their very useful practical aspects in transgenic technology is to serve as markers during the generation of chimeras with genetically modified embryonic stem (ES) cells. The coat-colour markers of the host embryos differ from those of the ES cells to make coat colour chimerism visually distinguishable and to determine ES cell origin of germ cells during test-breeding of chimeras. Historically most common ES cells originated from 129 strain due to the ease of derivation and efficient colonization of germ cells in chimeras. However, 129 strain is not genetically pure, it has multiple substrains with different combinations of coat colour alleles and breeding chimeras’ offspring may result in complex coat colours. The large scale high-throughput mouse mutagenesis projects co-ordinated by the International Knockout Mouse Consortium (IKMC) are using C57BL/6 (B6) ES cells to mutate all protein coding genes of the mouse genome. The generation of chimeras with targeted B6 ES cells requires different host embryos than B6 typically used for 129 ES cells as they would not result in coat colour chimerism unless agouti B6 ES cells (JM8A3, JM8A1.N3, JM8A3.N1) where the dominant agouti coat colour gene was restored by targeted repair of non-agouti mutation (Pettitt et al., 2009). The interplay of coat colour alleles carried by 129 and B6 ES cells, host embryos and the strains used for germline test-breeding will be reviewed. 3. How to name a mouse-mouse strain, gene, and allele nomenclature
TT2011 Invited Speaker Abstracts
Howard Dene, Lois Maltais, Beverly Richards-Smith, Janan Eppig
1. Genetic background and superovulation
Mouse Genome Informatics, The Jackson Laboratory, Bar Harbor, Maine, USA
Carlisle Landel Thomas Jefferson University, Philadelphia, USA Superovulation of female rodents for the enhanced production of oocytes and embryos is a major tool for increasing the
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There are approximately 25,000 genes in the mammalian genome. Many of these have been named and studied independently in several different organisms over the course of time. Orthologous genes have frequently been given different names in different species (sometimes even different names in one species
Transgenic Res (2011) 30:1139–1189 when studied by different groups of specialists.) When discussing genes, we need to be clear about the gene to which we are referring. This is best accomplished when there is one central repository for standard nomenclature. While one umbrella organization covering all mammalian gene nomenclature does not yet exist, several organizations act as nomenclature repositories for specific model organisms and cooperate with one another to develop a standardized mammalian genetic nomenclature. The Mouse Genome Informatics database (MGI) is one such organization and follows rules of nomenclature established by the International Committee on Standardized Genetic Nomenclature for Mice. These rules are found at http:// www.informatics.jax.org/mgihome/nomen/gene.shtml. Other organizations include the Rat Genome Database (RGD) and the Human Genome Organization (HUGO) Gene Nomenclature Committee. Informal consultations among these organizations, and others, serve to establish nomenclature consistency in mammalian genetics. Mutations too benefit from a standardized representation. Spontaneous and induced mutations in mouse and rat have been traditionally represented with the gene symbol, a superscripted mutation designator and a descriptive name i.e. Lepob for the ‘‘obese’’ mouse mutation in the leptin gene. A more recent symbol for a spontaneous mutation is Ucp1m1H for ‘‘mutation 1, Harwell’’. Technological advances over the last 30 years have led to a spectrum of new, engineered, mutations: random insertions of ever more complex genetic constructs (transgenes), gene traps, insertions or deletions targeted to specific genes, transposon induced mutations, and endonuclease induced mutations. Nomenclature rules exist for all of these engineered mutation types. Standardized nomenclature offers the research community unique allele symbols providing information on where and how alleles were created. We will discuss the process of determining formal nomenclature for various types of mutations starting first with identifying current gene nomenclature. The various components of allele symbols and what they mean will be discussed as well as how alleles are expressed in formal strain nomenclature. Finally, we will look at the ‘‘Mutant Alleles, Strains, and Phenotypes Submission Form’’ found at http://www. informatics.jax.org/mgihome/submissions/amsp_submission.cgi. This submission form is one way in which MGI can help you name your mouse. Supported by NIH grant HG000330. 4. As quiet as a mouse: use of genetically modified mice to study hearing and deafness Andrew Groves Baylor College of Medicine, Houston, TX, USA We rely on our inner ears to hear, but also to provide us with our sense of gravity and ability to maintain balance. Hearing and balance disorders are a major public health issue, with approximately 1 in 1000 children being born with hereditary hearing loss, and 50% of retirees reporting some form of hearing or balance loss. The inner ear senses sound and gravity with highly sensitive mechanoreceptor cells, called sensory hair cells. These cells can be easily killed by environmental noise and certain types of antibiotics and chemotherapy drugs.
1143 Mammals cannot regenerate these cells, and so hearing loss in humans is progressive and permanent. Historically, many of the original ‘‘fancy mouse’’ breeds had inner ear defects manifested by circling or ‘‘waltzing’’ behavior. I will describe some of these in my presentation, and will then mention a number of genetically modified mouse mutants which have been used to understand hereditary defects in the inner ear. I will discuss the use of Cre-Lox and Tet-transactivator technology in studying inner ear development and function, and will also describe the use of transgenic reporter mice to identify and purify specific populations of cells from the inner ear. Finally, I will briefly discuss how new transgenic mouse models have allowed us to study the effects of hair cell loss in mice and to devise strategies for hair cell regeneration in mammals, with the ultimate goal of hearing restoration in humans.
5. Development and preclinical evaluation of therapies for spinal muscular atrophy in mice Arthur Burghes1, Kevin Foust2,1, Vicki McGovern1, Paul Poresnsky1, Adam Bevan2,1, Sandra Duque1, Thanh Le1, Chitra Iyer1, Aurelie Laporte1, Isaac Alwine1, Chalermchai Mitrpant3, Steve Wilton3, Brian Kaspar2,1 1 Ohio State University, Columbus OH, USA, 2Nationwide Children’s Hospital, Columbus OH, USA, 3University of Western Australia, Perth WA, Australia
Spinal muscular atrophy (SMA) is caused by loss of the SMN1 gene and retention of SMN2 which results in low SMN protein levels. We have mimicked this situation in mice creating mice with SMA. SMA mice can be corrected by expression of SMN in the nervous system but expression in muscle has no effect. Removal of functional Smn from muscle in the presence of SMN2 using Cre drivers results in no phenotype and replacement of Smn in muscle of SMA mice does afford any rescue. The data indicates that two copies of SMN2 provides sufficient SMN for most tissues and that nerve has a particular requirement for high SMN levels. We have also produced transgenes with inducible SMN these demonstrate that postnatal induction of SMN does rescue SMA mice but early induction is necessary for maximum affect. Furthermore removal of SMN induction later in life indicates two groups of animals those that die within a month and those that survive on their SMN2 neither group showing a marked motor neuron phenotype. This indicates that 2 copies of SMN2 produces SMN levels on the edge of what is required by all cells in adult live. We have investigated therapeutic approaches to SMA using drug compounds that induce SMN from SMN2, gene therapy using scAAV9 and antisense morpholino oligonucleotides (ASOs) directed against ISS-N1(intron splice silencer). To date drug compounds have shown minimal effect in SMA mice due to their limited ability to induce SMN, thus further drug compound development is required. Antisense morpholinos when given early into the neural system corrected splicing of SMN2, increased SMN levels and resulted in mice that survived for over 100 days after a single dose of ASOs. We are investigating whether repeat dosing or delivery outside the nervous system is of further benefit. We have shown that scAAV9-SMN is highly effective at correcting SMA mice when given early with mice surviving over 300 days and
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1144 possess a normal neuromuscular junction phenotype. In primates even of older age vascular delivery of scAAV9-GFP results in a remarkably efficient delivery of GFP to motor neurons and no marked toxicity. Indeed scAA9-SMN also showed no toxicity. Furthermore scAAV9-SMN can also be efficiently delivered to motor neurons by intrathecal injections in pigs. This sets the stage for clinical trials of scAAV9-SMN and ASOs in SMA.
6. Animal ethics Karin Blumer Novartis International, Basel, Switzerland Since the beginning of philosophical thinking the relationship between humans and animals has been at the center of intellectual debate. Early philosophy considered that only mankind was deserving of basic moral rights. With the views of famous philosophers such as Rene´ Descartes and Francis Bacon, this anthropocentric view even intensified during the Enlightment. However, with increasing knowledge about animal biology and behavior, views started to change. A key moment was the publication of Darwin’s theory of evolution, which probably laid the foundations for the dramatic shift we have seen in the last few decades. Today we hear a growing call for animal rights, and it is often argued that it is only the ability to experience pain and suffering that confers moral status. In their most radical form, such views assume that man and most animal species are fully equal in a moral sense. These positions obviously condemn the genetic modification of most species used in biomedical research. However, neither scientific evidence nor philosophical argumentation is adequate to justify the fundamental assumptions of the animal rights movement. This talk will discuss inconsistencies of contemporary animal rights concepts and outline that the fundamental dilemma of biomedical research is not adequately represented in the debate about ‘‘what constitutes a right’’. As it is undisputed that most animals can experience pain and suffering in a way which is comparable to humans and that some may even have higher cognitive abilities the talk will conclude with a call for a moderate biocentric approach as it is used in most modern legal systems. This approach stresses the strong responsibility man has towards animals and the ban to impose any unjustified or unnecessary pain, suffering or distress on them. Thus any use of all animals—not only of certain species—in research has to follow the 3R principles of Russell and Burch.
7. New European directive on the protection of animals used for scientific purposes: impact on the generation and management of genetically modified models Belen Pintado CNB-CBMSO transgenic Service CSIC-UAM, Madrid, Spain The long time expected revision of the European Directive 86/609/ECC, adopted on September 22nd, 2010 will be effectively applied by Member States on January 1st, 2013. The new directive regulations will affect Member States in a different degree, since national legislation, more stringent than
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Transgenic Res (2011) 20:1139–1189 the Directive may remain active, but it intends to avoid disparities as much as possible. The effect of the Directive on the generation and management of genetically modified models will be found at different levels and it marks some common measures that will need to be adopted by animal facilities in all Member States. These measures will affect different aspects: (a) Care and accommodation of animals; (b) Evaluation and authorisation of projects and procedures including specific features as classification of severity, compliance with the 3 Rs principle or ethical evaluation of cost-benefits among others; (c) Specific requirements for personnel. Some of these requirements focus on formation and training of personnel carrying out procedures or designing projects, but also the mandatory in situ presence of an animal welfare officer and an animal welfare body with very specific tasks; (d) Defined records to be kept by establishments and new statistical reports to be sent to the Commission; (e) Regular inspections to be carried out by competent authorities. The adoption of laws and regulations to comply with this Directive by Member States poses the risk of different interpretations. For that reason the European Commission has set periodical meetings in order to provide guidelines to accomplish a coherent regulation. The presentation will compile the evolution of these guiding principles that will need to be incorporated as national laws by November 10th 2012.
8. Shifting research animal regulations and accreditation standards: hobbling or facilitating sound science? Christian Newcomer Association for Assessment and Accreditation of Laboratory Animal Care International, Maryland, USA In the past 2 years, three important animal research guidance documents with international reach have been revised substantially. These include the FASS Guide for the Care and Use of Agricultural Animals in Research and Teaching (Ag Guide), the European Directive 2010/63/EU and the Guide for the Care and Use of Laboratory Animals, Eight Edition (Guide, 8th Edition). Each of these documents has been attentive to the advances in research animal care and use since their last revision and offers special emphasis on the topic of genetically-modified animal models. At this time, only the European Directive has been adopted as a regulation, and the Guide, 8th Edition, is pending final consideration by the Office of Laboratory Animal Welfare at the U.S. National Institutes of Health. The Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International), an organization providing a confidential, independent, voluntary peer review accreditation program has indicated its intent to adopt the Ag Guide, the Guide, 8th Edition and Appendix A of the European Directive as primary standards for its assessment and accreditation activities. AAALAC International’s site visit review experts worked concertedly for more than a year to identify important caveats and points of clarification on the content and use of these three standards in an effort to aid institutions in using these standards effectively and collaboratively to advance research animal welfare and to promote the programmatic conditions for success in the experimental use of today’s increasingly specialized animal models. This presentation will
Transgenic Res (2011) 30:1139–1189 review the new themes, requirements and recommendations of the Guide, 8th Edition, the European Directive and the Ag Guide and emphasize particularly the new developments of special interest and importance to the maintenance and use of genetically-modified animals in the areas of programmatic oversight, laboratory animal management practices, veterinary care and facilities.
9. Running a transgenic unit Aimee Stablewski Roswell Park Cancer Institute, Buffalo, NY, USA To successfully generate transgenics, knockouts and perform other mouse reproductive services, there are a myriad of things that require preciseness and the ability to adapt to the everchanging literature and scientific demands. In the classic section of the Transgenic Technology meetings: Running a Transgenic Unit, myself and several others will talk about these intricacies and how to overcome and troubleshoot problems that each of us face on a daily basis. Several things that we will focus on will include the increasing use of the embryonic stem cell clones and vectors from the various consortium/large-scale efforts around the world, sperm and embryo cryopreservation and how it has changed over the last several years, IVF and much more. The majority of this section of the TT2011 meeting will be a question and answer section, on everything to make a Transgenic Unit successful, from the most basic thing, to the most advanced.
1145 microinjection and also for gene targeting applications using embryonic stem cells. The transgenic facility now focuses on and specializes in embryonic stem cell experiments as a means to create transgenic mice. Also, we have been able to coordinate with personnel in the Division of Laboratory Animal Resources to now handle routine mouse work such as: rederivation, cryopreservation of sperm and embryos, accelerated reproduction by in vitro fertilization, embryo transfer from frozen or live mouse embryos, mouse breeding and genotyping, if these services requested by investigators. Currently, in a year, the facility runs about 60 DNA microinjection days, BAC recombineers about 20 projects, transfects 36 stem cell experiments and microinjects ES cells from our own targettings or cells obtained other sources. We assist about 40 investigators per year. The largest challenges we are currently facing as a facility are how to successfully help investigators who would like to obtain DNA constructs and targeted ES cells from repositories. The DNA constructs usually do not fit our PCR system for rapid screening of putative positive clones, and the quality of the ES clones available for microinjection is extremely variable, and often poor. We work very hard for success and customer satisfaction, but have found it difficult with these imported materials.
11. The facility for genetically modified animals at the Weizmann Institute, Israel Rebecca Haffner Weizmann Institute of Science, Rehovot, Israel
10. Evolution of the transgenic mouse facility at Duke University Medical Center over 23 years Cheryl Bauman Bock1,2 Duke Cancer Institute, Durham, NC, USA, 2Duke University Medical Center, Durham, NC, USA
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The Duke Cancer Institute Transgenic Mouse Shared Resource began formal operation in January 1989. The facility is funded by the Duke Comprehensive Cancer Center grant and by user fee payments for services. Initially, the facility provided one service: microinjection of DNA constructs into one-cell mouse embryos to create transgenic mice. Investigators quickly requested other services, including: cryopreservation of mouse gametes, isolation of preimplantation mouse embryos for various applications, rederivation of mouse lines, microinjection of targeted mouse embryonic stem cells and targeting of stem cells as a service. The facility responded to most of these requests, however, it was not until 1995 that we began offering gene targeting as a service. Since then, the facility has carried out several hundred gene targeting experiments and has an excellent record of successful targeting and germline transmission in all experiments using our 129 ES cells. Over the past several years, investigators at Duke voiced their need for DNA construction services. We have now been able to add Bacterial Artificial Chromosome Recombineering as a service for production of BAC constructs for DNA
The Transgenic Unit at the Weizmann Institute is a core facility for generating genetically modified animals. Core services include the production of transgenic mice, via DNA microinjection, gene targeting in ES cells, and the generation of mice carrying targeted mutations, via ES injection or aggregation. The majority of mice generated carry mutations in genes targeted in our in house ES targeting facility. However recently, international high throughput targeting consortia have made available many ES clones carrying specific targeted mutations. Our facility now routinely imports and expands such targeted ES clones, and produces mutant mice from these resources. Support services include: embryo cryopreservation, assisted fertility (IVF, artificial insemination and ovary transfer), import and export of new strains from fresh or cryopreserved embryos and sperm, and tetraploid aggregation to investigate maternal effect mutations. The ES facility also offers karyotyping, and de novo establishment of new ES lines. We provide services to the Weizmann Institute and to academic institutions throughout Israel.
12. Mammalian germline modification Ralph Brinster University of Pennsylvania, Philadelphia, USA My research in the area of mammalian germline modification began more than 40 years ago and can be divided into four
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1146 overlapping phases, all focused on the germline. Initially, I began studies with fertilized mouse eggs and early embryos and developed a culture system based on metabolic characteristics of the cells. These studies were essential for my later research, including the generation of transgenic animals. The second series of experiments involved transplantation of foreign stem cells into the mouse blastocyst, which resulted in chimeric mice. These experiments demonstrated a method by which one could generate transgenic animals, but unfortunately the transgenes were not transmitted to progeny. However, the studies demonstrated the feasibility of transgenesis and led to the development of embryonic stem cells. The third series of experiments involved the introduction of RNA and DNA into fertilized mouse eggs, which depended on the culture system and the availability of purified genes. These are the studies for which I am perhaps best known, but they would have been impossible without my earlier research on egg culture and the demonstration using teratocarcinoma cells that it was possible to introduce new genes into a mouse. The fourth series of experiments involved male germline stem cells, spermatogonial stem cells. I initiated these studies because germline cells are of enormous importance, and spermatogonial stem cells are the only cells in the adult body that self-renew and can transmit genes to the next generation. The first requirement was to develop an assay system that would allow experiments and quantitative analysis of the cells, much like the mouse egg culture system. I reasoned that if donor testis cells were placed in recipient seminiferous tubules depleted of germ cells only the stem cells would colonize. Subsequent studies demonstrated this to be true and established the spermatogonial transplantation system in mice. The obvious next step was to develop a culture system, which proved to be difficult and took almost 10 years. Many exciting challenges and opportunities remain, including understanding the regulation of spermatogonial stem cell self-renewal and differentiation, the development of in vitro spermatogenesis, and the extension of the culture and differentiation systems to higher species, including humans. Our research in these areas will be discussed during my lecture.
Transgenic Res (2011) 20:1139–1189 transgenic mice via pronuclear injection and targeted mutants through ES cell injection.
14. Factors affecting in vitro fertilization Jan Parker-Thornburg University of Texas M. D. Anderson Cancer Center, Houston, TX, USA In vitro fertilization has become an essential tool for working with genetically modified mice. A short list of procedures that can be enhanced using IVF would include: rederivation, line rescue, mouse transfer between institutions, tests of spermcryopreservation procedures, cryopreservation and line expansion. At the Berlin TT2010 meeting, Rob Taft and Jane Farley presented a number of IVF procedures that are used at The Jackson Laboratory. This discussion will follow up on that presentation by examining issues of sperm quality, obtaining oocytes capable of being fertilized and making pups, the timing between superovulation and getting the oocytes into fertilization dishes and other issues that we have encountered, including ‘‘slow sperm’’ in some samples. One basic requirement for successful IVF is having high quality sperm. While males of prime breeding age are considered best for the procedure, we have also found that very old males can be used successfully. Our facility attempts to insure having good, motile sperm by setting up pre-matings with the males identified for the procedure. Once good sperm is obtained, a second requirement is to obtain oocytes with optimal fertilization capacity. Here we consider the age of the female, the hormones used for superovulation, the timing of the hormones, and the timing of oocyte collection after HCG injection. Our recent results have delineated major differences between mouse strains, with oocytes from129SvIm/J strains having much stricter time requirements than those from FVB strains. Methods of handling oocyte and sperm collection and culture conditions will be discussed. In addition, we will examine external factors that can influence the success of the procedure, including transit times, time of handling outside of the incubator, culture media and volatiles in the environment.
13. Essentials of microinjection Charles Hawkins Johns Hopkins University, Baltimore MD, USA It is an interesting aspect of the field of producing genetically modified mice—a field that is 30+ years old—that there is very little standardization among the labs that provide this service. Variables include: how to prepare DNA for injection; how to prepare cells for injection; the strain and/or age of the embryos injected; how the embryos are injected or manipulated; the media used for microinjection, embryo culture, and ES cell culture prior to injection; the temperature at which injection occurs; and the mouse strains and techniques associated with transferring the embryos into psuedopregnant females. The Johns Hopkins Transgenic Mouse Core Laboratory has been successfully generating genetically modified mice for over 15 years. In an attempt to generate further discussion I will describe the techniques used by this lab to produce
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15. International germplasm transfer Kent Lloyd University of California, Davis, CA, USA One of the primary roles of a fully functioning transgenic laboratory is the preservation, maintenance, and recovery of murine sperm and embryos. Techniques ranging from cryopreservation (embyos and sperm), vitrification (embryos), and evaporative and freeze drying (sperm) are used to facilitate the archiving, protection, and perpetuity of mutant mouse lines. For example, frozen germplasm is used as an alternative to storing and maintaining mice as live breeding colonies ‘‘on the shelf’’. Cryopreservation of embryos is considered the gold standard for maintaining an inanimate archive of mutant mouse lines, but the process is lengthy, costly, and laborious. Cryopreserving sperm is simpler, faster, and cheaper, but some strains, notably C57BL/6, without special treatment, may
Transgenic Res (2011) 30:1139–1189 not be viable for in vitro fertilization (IVF) due to poor postthaw recovery of sperm motility. In either case, transgenic laboratories today must be facile with the derivation of mice by IVF using revitalized and/or rehydrated sperm and with the recovery of preserved embryos. In addition, the use of germplasm rather than live animals facilitates access to mouse models. This is particularly important as literally thousands of new knockout, transgenic, and other genetically-altered mutant mouse models are being produced around the world. These valuable mouse resources will be available from dedicated repositories in North America, Europe, and Australasia, primarily as preserved germplasm which can be converted into live mice and distributed or, preferably, distributed directly as either sperm or embryos. Compared to live mice, germplasm greatly facilitates high-throughput archiving as well as ready and fast access by scientists, as most laboratories and/ or institutions implement quarantine of incoming live mice. In contrast, importation of germplasm avoids delays associated with quarantine, although rederivation procedures are required on-site to generate cohorts of live mice for research studies. Further, refinements in germplasm preservation and recovery procedures are improving distribution and rederivation efficiencies. For example, instead of using bulk dry shippers, freshly-harvested embryos can be packed and shipped overnight on wet ice and rederived successfully. Similarly for shipment of vitrified embryos on dry ice. In addition, evaporative and freeze-dried sperm can be shipped at room temperature and used for ICSI. Many of these procedures are applicable not only for 24–36 h transnational transport, but also for international shipping occurring over several days.
16. Finding your transgenic mouse/ES cell line/gene with on-line resources Peter Sobieszczuk University of Miami, Miami, FL, USA Complete genomic sequencing information of human, mouse and several other species has been deposited as raw electronically-processed data at http://www.ensembl.org/index.html, with a manually annotated subset available at the Havana/Vega site http://vega.sanger.ac.uk/index.html. These data are also available from NCBI, the successor to the EMBL and Genebank databases. Physical DNA equivalents of mouse sequences can be obtained from a BAC collection generated from CHORI, and commercially from Invitrogen, and many of the BAC libraries are annotated in the Ensembl database. Creation of the International Knockout Mouse Consortium1 with its web site http://www.knockoutmouse.org/about brought together four individual projects with the idea of making all gene cell lines and KO mouse publicly available from the single portal. The undertaking, originally supported by the US NIH and UK Wellcome Trust/European Commission, to systematically knock out the majority of mouse genes within the next 5 years is underway. This project now includes Genome Canada and the Texas Institute for Genomic Medicine. When searching for the current status of your favourite gene(s) a good starting point would be International Mouse Strain Resource accessible and maintained at http://www.findmice.org//IMSR SearchForm.jsp and Mouse Genome Informatics’ site at Jax
1147 laboratories http://www.informatics.jax.org/. Federation of International Mouse Resources coordinate repositories and resource centers around the world, members include in North America: TJL, MMRRC, MMHCC, CMC and CMMR, Europe: EMMA, Japan: RIKEN and CARD and in Australia, multiple members of the APN network http://www.fimre.org/. All the above listed sites provide on-line help; however additional assistance can be sought via the World Wide Web electronic group, transgenic list2 at http://www3.imperial.ac. uk/lifesciences/old/services/research/transgeniclist. Like many other web based discussion groups, the transgenic list aims to bring together people with the same interests through an easy and unrestricted exchange of information on the e-mail network. Established in its present form in July 1996, it includes amongst its members active researchers in transgenesis, from the novice to experts in the field. Major keywords cover the wide spectrum of disciplines discussed: homologous recombination, targeted mutagenesis, inducible expression, ES cells, microinjection, mouse genetics, and animal husbandry. 1. Collins F., et al. A mouse for All Reasons. [2007] Cell. 128, 9–13. 2. Sobieszczuk P. [1997] Transgenic e-mail list. Transgenic Research 6, 308
17. Germline performance of gene-targeted ES cell clones imported from the International Knockout Mouse Consortium by a transgenic core facility Thomas Saunders, Keith Childs, Debora Vanheyningen, Elizabeth Hughes University of Michigan, Ann Arbor, MI, USA Genetically modified mouse models are valuable tools for the understanding of gene function. The International Knockout Mouse Consortium (IKMC) is establishing an ES cell resource to knockout every gene in the mouse. Access to the genetargeted ES cell clones eliminates the need to design genetargeting vectors, to manipulate pluripotent ES cells in culture and to screen ES cell clones for desired genetic changes. Targeted ES cells are sent to transgenic facilities around the world for the production of ES cell-mouse chimeras and germline transmission of novel mouse models of gene function. One question about IKMC ES cell lines is their germline potency. The University of Michigan Transgenic Core imported over 100 gene-targeted and gene-trapped ES cell lines from IKMC members (EUCOMM, NorCOMM, SIGTR, BayGenomics, FHCRC, GGTC, CMHD, Regeneron, and TIGM). ES cell clones were analyzed with respect to euploid chromosome numbers, Mycoplasma contamination, verification of gene targeting/trapping event, generation of ES cell-mouse chimeras, and germline transmission of modified alleles. The majority of ES cell clones (79%) had euploid chromosome counts. The majority of euploid ES cell clones (73%) formed germline chimeras. Rarely, clones were incorrectly targeted or required subcloning to isolate gene-targeted subclones. The most common reason that ES cells did not go germline was because of aneuploid chromosome makeup. Additional clones
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1148 failed to form germline chimeras for unspecified reasons. The data presented suggest that in order for an investigator to be assured of obtaining a knockout mouse model at least three ES cell clones with mutations in the gene of interest will need to be imported. When it is necessary to achieve germline transmission from two independent ES cell clones so that confounding phenotypes arising from spurious spontaneous mutations during ES cell culture can be eliminated then four or five ES cell clones should be imported for chimera production and germline breeding. The IKMC ES cell repository is a valuable resource. The ability to quickly retrieve targeted ES cell lines for genes of interest and to generate mouse-models accelerates the discovery of gene function.
18. The IKMC Web Portal: a single access point to mouse knockouts from the International Knockout Mouse Consortium William Skarnes The Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK The International Knockout Mouse Consortium aims to provide a public resource of thousands of lacZ-tagged, conditional mutations in C57BL6 ES cells. This effort requires the design and construction of vectors and the production of targeted ES cells on an unprecedented scale, beyond the scope of conventional methodologies. Several high-throughput pipelines have been established for the generation of reporter-tagged conditional alleles1, gene deletions2, and trapped alleles3. To date, knockout alleles are available for more than 16,000 proteincoding genes. For this program, highly germline-competent ES cells from the C57BL/6 N substrain were established3-6 and validated by the generation of more than 1000 lines of mutant mice. In my talk, I will describe the resources available from the IKMC and explain how researchers can search for and access material generated by all pipelines through a common web portal (www.knockoutmouse.org).7 1. Skarnes, W. C. et al. A conditional knockout resource for genome-wide analysis of mouse gene function. Nature 474, 337–342 (2011). 2. Valenzuela, D.M. et al. High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nature Biotechnol. 21, 652–659 (2003). 3. Hansen, G. M. et al. Large-scale gene trapping in C57BL/ 6 N mouse embryonic stem cells. Genome Res 18, 1670–1679 (2008). 4. Pettitt, S. J. et al. Agouti C57BL/6 N embryonic stem cells for mouse genetic resources. Nat Methods 6, 493–495 (2009). 5. Poueymirou, W. T. et al. F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses. Nat Biotechnol 25, 91–99 (2007). 6. Gertsenstein, M. et al. Efficient generation of germ line transmitting chimeras from C57BL/6 N ES cells by aggregation with outbred host embryos. PLoS One 5, e11260 (2010). 7. Ringwald, M. et al. The IKMC web portal: a central point of entry to data and resources from the International
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Transgenic Res (2011) 20:1139–1189 Knockout Mouse Consortium. Nucleic Acids Res 39, D849–855 (2011).
19. Authentic rat embryonic stem cells and germline transmission of targeted mutations Qi-Long Ying University of Southern California, Los Angeles, California, USA Rats are a proven model organism for the study of human disease. Researchers have taken advantage of their physiological and pharmacological similarities to humans for over 150 years. The demonstration of robust true pluripotent germline competent rat embryonic stem (ES) cells is one of the most highly anticipated developments in the field. Access to such cells will allow investigators to interrogate gene function in highly characterized rat models of disease. Using a combination of specific protein kinase inhibitors, we have recently established germline competent rat ES cells. We have also demonstrated that gene-targeted rat ES cells can be efficiently generated via homologous recombination. Furthermore, the targeted mutation in the rat ES cell genome can transmit through the germline to create gene-targeted rats. The establishment of ES cell-based gene targeting technology in the rat allows us to address fundamental biological questions related to human diseases through the creation of superior animal models.
20. Pronuclear injection-based mouse targeted transgenesis by site-specific recombination Masato Ohtsuka Tokai University, School of Medicine, Isehara, Kanagawa, Japan During the past three decades, transgenic (Tg) mouse technology has widely been recognized as a useful tool for analysis of in vivo gene function and generation of mouse models for human disease. They are basically generated by pronuclear injection (PI) of a transgene, but have some drawbacks such as the inability to control the integration site and copy number of the transgene, which often leads to the variable and unstable transgene expression. Homologous recombination-based targeted transgenesis through embryonic stem (ES) cells appears to be better than PI-mediated transgenesis, since the former can confer predictable and reproducible transgene expression. However, it requires isolation of ES clones exhibiting targeted insertion of a transgene and blastocyst injection of those clones for chimeric mice production, those of which are very laborious, costly and time-consuming than PI-based transgenesis. To improve these problems, we recently established a PI-based targeted transgenesis (PITT) system, in which gene of interest (GOI) is inserted into the predefined locus through CreloxP site-specific recombination in fertilized eggs obtained from ‘‘seed mice’’. These seed mice are generated through chimeric mouse production via engineered ES cells that harbor mutant loxP sites in the predetermined locus (Rosa26 or
Transgenic Res (2011) 30:1139–1189 H2-Tw3 locus in this study). To generate targeted Tg mice, PI of the donor vector containing GOI and Cre expression vector is performed using hemizygous eggs obtained from the cross between homozygote male seed mice and wild-type female mice. As a result, we obtained more than 25 Tg lines so far, including those with various types of fluorescent genes. Our PITT method showed 4–5% efficiency of correct transgene integration per newborn, when donor vectors with 10–15 kb in size were used. The germ-line transmission rate was very high ([90%). More importantly, all the PITT mice generated exhibited stable and reproducible transgene expression even after ten generations. We also demonstrated that production of artificial miRNA-based knockdown mice showing reproducible knockdown of expression of GOI (Tyrosinase and eGFP genes in this study) was possible by this technique. The PITT method could therefore provide a strong basis for systematically generating a series of targeted Tg lines via direct modification of zygotic genome, not only for reliable transgene expression but also for reproducible gene knockdown.
1149 swelling the pronucleus by 26–30%. The incidences of ‘‘pulling-out event’’ were significantly lower in the cases of 35 and 44 kHz but slightly higher in the case of 40 kHz. VMS showed slightly better embryonic development in culture and considerably lower death rate. The third version of VMS: This is the brand-new version and its amplitude was augmented for large DNA microinjection. We are now evaluating its effectiveness using BAC vector (8.1 kb). We have confirmed that VMS is capable of injecting even 8 ng/ll BAC solution into a pronucleus at a pressure of 30 hPa.
22. The JAX Colony Management System (JCMS): an extensible colony and phenotype data management system for transgenic mouse colonies Charles Donnelly, Michael McFarland, Beth Sundberg, Dave Springer, Kavitha Rama, Carol Bult, Peter Blauth, Abigail Ames The Jackson Laboratory, Bar Harbor, Maine, USA
21. Development of vibratory microinjection systems Fujio Miyawaki1, Dilidaer Kudereti1, Jun Hasegawa2 1 Tokyo Denki University, Saitama, Japan, 2Takushoku University, Tokyo, Japan
To make pronuclear microinjection more efficient, we have been developing vibratory microinjection systems (VMSs). VMSs can provide a micropipette with longitudinal vibration, the amplitude of which is controlled by voltage applied to the vibrator. Basically, the higher the voltage, the larger the amplitude. The first version of VMS: The frequency of vibration was within an audible range (*18 kHz). Compared with the ordinary (non-vibratory) microinjection, we evaluated two frequencies: 5 and 10 kHz. The applied voltages were fixed to 15 Vp-p for 5 kHz and 8 Vp-p for 10 kHz. A total of 1,448 BDF-1 mouse fertilized eggs were injected with 2.5 ng/ll Venus gene solution. Commercially-available micropipettes (FemtotipÒ, Eppendorf) were used to minimize individual difference in opening of the tip (0.3–0.7 lm). VMS achieved zygote injection with significantly less cellular deformation and significantly lower incidence of the lethal event that a micropipette pulled nuclear RNA and/or DNA out of a fertilized egg. The VMS also resulted in significantly better embryonic development although its death rate was higher. The reason for this seemed to result from a bend of each micropipette at an angle of 208. The second version of VMS: This VMS provided longitudinal vibration up to 100 kHz. Compared with the audiblerange VMS, we reduced the amplitudes of vibration at the same applied voltages in order to make possible fine tuning of amplitudes. However, unintentional lateral vibration was visible at lower voltages than expected, probably due to instability of this vibrator, but we evaluated three frequencies: 35, 40 and 44 kHz. The applied voltage was set at a value which slightly vibrated the tip of each micropipette that was used unbent. GFP solution (2.0 ng/ll) was injected into 1,341 BDF-1 fertilized eggs at a pressure of 30 hPa. VMS shortened the injection time by 23–38% and reduced the time spent
In this talk we will present the rationale for using a relational database management system for managing animal colonies and we will present an introduction to The Jackson Laboratory Colony Management System (JCMS). JCMS is a software application for managing data and information related to research mouse colonies, associated bio-specimens, and experimental protocols. JCMS runs directly on computers that run one of the PC WindowsÒ operating systems, but can be accessed via web browser interfaces from any computer running a Windows, MacintoshÒ, or LinuxÒ operating system. JCMS can be configured for a single-user or multiple-users in small to medium size work groups. The target audience for JCMS includes laboratory technicians, animal colony managers, and principal investigators. The application provides operational support for colony management and experimental workflows, sample, and data tracking through transactionbased data entry forms and date-driven work reports. Flexible query forms allow researchers to retrieve database records based on user-defined criteria. Recent advances in handheld computers with integrated barcode readers, middleware technologies, web-browsers, and wireless networks add to the utility of JCMS by allowing real-time access to the database from any networked computer. JCMS is a freely available and supported research tool. Since its initial public release in 2005 the user community for the software has grown steadily; there are currently over 1900 active subscribers to the JCMS user support list. The JCMS software application and associated tools and documentation are made available from the project web site (http://colonymanagement.jax.org/). The JCMS web site includes an on-line discussion forum for users that is monitored by the JCMS development team.
23. An introduction to non-mouse models Bruce Whitelaw The Roslin Institute, Scotland, UK Transgenesis is a highly practiced art in the field of mouse biology. A number of transgene delivery strategies are
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1150 available which are now reliable and efficient. This is not always the case for larger animals. For example, no fully validated livestock ES cells exist. Not to say that the mouse leads in all areas of transgenesis. Cloning technology provides a robust transgene delivery strategy which has yet to be widely used by the mouse research community. Indeed through the application of nuclear transfer, site-specific genetic changes can be produced, just as such allelic variation can be engineered in the mouse when using ES cell based strategies. Another example of where the research community has found solutions to overcome the limitations in methodology associated with different species, is the use of primordial germ cells in the production of transgenic birds, a species where exciting engineered to combat disease progression could have important implications for human health. Indeed, there seems to be a never ending stream of new ways to engineer the genome of animals, with one of the most exciting recent developments being zinc finger nucleases. This technology has been widely taken up by the rodent community—but for the rat rather than then mouse—and is likely to see much application in farm animal species. When thinking about transgenesis in animals bigger than the mouse, the opportunity for biotechnological applications often comes to the fore. It would be certainly be challenging, if transgenic methodology was simply restricted to the mouse, to conceive of development of engineered animal bioreactors. Regardless of where differences in application or methodology exist much can be learned from dialogue between research groups working on transgenesis in the mouse and other animal species. Each species provides exiting opportunities for research, technical development and commercialisation. This presentation will introduce non-mouse transgenesis focussing on the challenges associated with different species with respect to gene transfer and identify the areas where non-mouse models are leading the way in the field of transgenesis.
24. Transgenic pigs as models for translational biomedical research Eckhard Wolf, Simone Renner, Barbara Kessler, Mayuko Kurome, Tuna Gu¨ngo¨r, Valeri Zakhartchenko, Annegret Wu¨nsch, Anne Richter, Nikolai Klymiuk, Bernhard Aigner Institute of Molecular Animal Breeding, and Laboratory for Functional Genome Analysis (LAFUGA), LMU Munich, Germany The translation of novel discoveries from basic research to clinical application is a long, often inefficient and thus costly process. Accordingly, the process of drug development requires optimization both for economic and for ethical reasons, in order to provide patients with appropriate treatments in a reasonable time frame. Consequently, ‘‘Translational Medicine’’ became a top priority in national and international roadmaps of human health research. Appropriate animal models for the evaluation of efficacy and safety of new drugs or therapeutic concepts are critical for the success of translational research. Transgenic pigs are increasingly being established as large animal models for selected human diseases. The first pig whole genome sequence and many
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Transgenic Res (2011) 20:1139–1189 other genomic resources will be available in the near future. Importantly, efficient and precise techniques for the genetic modification of pigs have been established, facilitating the generation of tailored disease models (Aigner et al., J Mol Med 88:653–664, 2010). Using lentiviral transgenesis (Hofmann et al., EMBO J 4:1054–1060, 2003), we established transgenic pigs expressing a dominant negative receptor (GIPRdn) for the incretin hormone glucose-dependent insulinotropic polypeptide (GIP) in the pancreatic beta-cells (Renner et al., Diabetes 59:1228–1238, 2010). These pigs mimic important features of human type 2 diabetes mellitus: impaired insulinotropic action of GIP, progressive deterioration of glucose control and reduction of pancreatic beta-cell mass. The progressive nature of the phenotypic changes in GIPRdn transgenic pigs together with the opportunity to take serial blood samples of sufficient volume from unrestrained animals creates a unique model to screen for metabolomic footprints associated with early prediabetic stages. A targeted metabolimics study revealed characteristic changes in plasma concentrations of specific amino acids, sphingomyelins, phospholipids, and acylcarnitines, which may be interesting markers to discover early stages of prediabetes in humans. Clinically manifest diabetes in pig models was achieved by expression of mutant insulin (INS) genes in the pancreatic beta-cells. These models exhibit markedly reduced pancreatic beta-cell mass leading to reduced insulin secretion and highly elevated fasting blood glucose levels. Transgenic pigs expressing mutant INS genes are interesting models for studying secondary lesions of diabetes mellitus, for regenerative medicine (e.g. allogeneic transplantation of porcine pancreatic islets to test their functionality before they are used for pig-to-primate xenotransplantation), and for studying consequences of maternal diabetes mellitus on the development of embryos and fetuses, and their interactions with the maternal environment.
25. The use of transgenic zebrafish to investigate biological processes in vivo Robert Kelsh University of Bath, Bath, Banes, UK Over the last 10 or more years fish, especially zebrafish and medaka, have become recognised as major biomedical model systems. Their advantages, especially of high fecundity, external development, and optical transparency combine with the strong conservation of gene function to make these animals excellent for in vivo investigation of biological processes. These fish have long been favoured for toxicology and developmental biology, but are now increasingly being used to study behaviour, for drug screening, to dissect gene regulation, to examine disease mechanisms, to investigate stem cells, and for dissecting cell biology, all in vivo and often with exquisite resolution. To take full advantage of fish model systems, transgenesis techniques have become vital and they are now mainstream and increasingly sophisticated. I will illustrate some of the power of zebrafish transgenesis techniques using examples from my own and others’ labs. Transgenesis in zebrafish is performed by simple injection of DNA constructs into the very early cleavage stages, and is aided by the relatively large size of the 1-cell stage. Varied
Transgenic Res (2011) 30:1139–1189 plasmid, retroviral, PAC and BAC vectors are now in routine use, the latter being particularly useful for ensuring that distant regulatory elements can be included. The utilisation of the meganuclease I-SceI or transposons (e.g. Tol2) has revolutionised the efficiency of the transgenesis process, so that up to 50% of founders can give germ-line transmission. Transgenic animals can be identified using expression of the transgene itself (e.g. where the construct mediates expression of GFP), using a second tissue specific promoter (heart and lens are both favoured versions), or by standard PCR techniques to analyse fin-clips or offspring. For some purposes uniform expression of the transgene is highly desirable and hence considerable effort has gone into identifying promoters that show ubiquitous expression. In other cases, tissue/stage-specific expression is vital and many well-characterised promoter/enhancer constructs are now available. The development of increasingly sophisticated enhancer trap lines and of high-throughput dissection of regulatory elements is rapidly expanding this repertoire. Temporal control of gene expression can readily be achieved using drug-based inducible systems, ideally suited to the aquatic and external development of these organisms. Following on from the successful application of retrovirus transgenes for mutagenesis, other transgenic approaches have now been developed to allow gene-trapping, expression analysis and mutagenesis. This transgenic tool-kit helps make the zebrafish a powerful and flexible model for dissecting biological processes in the living animal.
26. The regulation and politics of food from genetically engineered animals Alison Van Eenennaam Department of Animal Science, University of California, Davis, CA 95616, USA Although the first transgenic, or genetically engineered (GE) animals were developed almost 30 years ago, no GE animals have yet been commercialized for food production. The United States Food and Drug Administration (FDA) clarified its legal authority to regulate GE animals under the new animal drug provisions of the Federal Food Drug and Cosmetics Act in a 2009 guidance document. Included in this guidance was FDA’s stated intent to increase the transparency of its deliberations and actions by holding public advisory committee meetings prior to approving any GE animal. An application to allow a fast-growing GE salmon (AquAdvantage salmon) to enter the food supply, under regulatory review since 1995, was the subject of such a public advisory committee meeting in September 2010. The unanimous conclusion of the FDA scientists presented at this meeting was that AquAdvantage salmon were as safe to eat as food from conventional Atlantic salmon, and that they were not expected to have a significant environmental impact when raised and reared with the multiple physical, biological, and geographic/geophysical containment measures detailed in the application. Unfortunately for the sponsor this public meeting was used an opportunity for opponents of GE animals to cherry pick data from the application and formulate menacing sound bites about cancer risks, allergens, and Trojan horses. There is little benefit to society if attempts to increase public participation in the
1151 regulatory process are used as an opportunity to demonize technology. Absent from the debate over the AquAdvantage salmon was any balancing discussion of the risks associated with obtaining Atlantic salmon from alternative sources. In principle, there is no difference between the types of concerns and environmental risks associated with the escape of GE fish and those related to the annual escape of the millions of salmon that are genetically divergent from native populations in other ways, e.g. strains selected for enhanced growth. Subjecting conventionally bred and GE animals to different regulatory standards in the absence of unique risks is inconsistent from a scientific perspective. The protracted evaluation of the AquAdvantage salmon and continuing uncertainties in the regulatory process and time line have essentially halted commercial and public investment in the development of GE animals for agricultural applications in the United States, although transgenic food animal research is ongoing in other countries. This outcome has broad implications for the competitiveness of U.S. agriculture and the future geographic location of GE animal research, development, and production.
27. Suppression of bird flu transmission in transgenic chickens Helen Sang1, Jon Lyall2, Adrian Sherman1, Ian Brown3, Laurence Tiley2 1 The Roslin Institute, University of Edinburgh, Midlothian, UK, 2Department of Veterinary Medicine, University of Cambridge, Cambridge, UK, 3Vereinary Laboratories Agency, Weybridge, Surrey, UK
We have developed a method for genetic modification of the chicken using lentiviral vectors and culture to hatch of microinjected embryos in surrogate shells. We are using this technology in a range of applications, in developmental biology, biotechnology and modification of production traits of importance to the poultry breeding and production industries. Avian influenza is a major economic and animal and human health challenge, due to the potential development of new pandemic strains of virus. We have designed and tested in vitro two transgenes expressing short RNAs to inhibit avian influenza virus replication. We then established transgenic lines carrying these transgenes and tested the response of the genetically modified birds to direct infection by H5N1 virus and the transmission of infection from the directly infected birds to cohoused birds that were nontrangenic or transgenic. Results of this challenge experiment with transgenic birds expressing miRNAs targeting conserved regions of the flu genome were disappointing as no protection was seen. The results with the second transgenic line expressing a decoy RNA were more encouraging. Transgenic and non-transgenic birds succumbed to H5N1 infection with no obvious difference in susceptibility between the two groups of birds. However, the in-contact groups of birds, transgenic and non-transgenic, cohoused with the infected nontransgenic birds developed lethal bird flu infection while the in-contact groups co-housed with infected transgenic birds did not. This study indicates that transgenic expression of the decoy RNA reduced significantly transmission of avian flu from infected birds to cohoused birds. Although these results
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1152 are only a step on the way to producing fully resistant chickens they form a proof of principle that a GM approach can be taken to developing disease resistance to major infectious diseases in a farm animal species. Future research will build on this to develop additional transgenes to block infection of avian influenza and to extend this approach to other significant diseases of poultry.
28. Beyond the mouse: novel genetically engineered animals Xiaoxia Cui, Lara Carbery, Daniel Fisher, Aaron McCoy, Diana Ji, Rachel Henry, Edward Weinstein Sigma-Aldrich, St. Louis, MO, USA The rat, rabbit, zebrafish, and pig have long been important experimental models in multiple fields of study. Unlike the mouse, efficient gene targeting in these species has remained a near impossibility with researchers forced to rely on random methods of mutagenesis, such as ENU and transposon-based manipulation. The zinc finger nuclease technology is a wellestablished tool for targeted manipulation of genomes and has been utilized extensively for a broad array of in vitro applications. We have now taken this technology and applied it to an in vivo setting. We will present data on the creation and characterization of targeted ‘‘knockout’’ mice, rats, and rabbits where key genes have been removed from the genome. Furthermore, we will present data on the addition (‘‘knock-in’’) of genes, in a targeted manner, into the rodent genomes through use of this technology and discuss the potential far-reaching implications.
29. Mutating the rat genome with transposons Joseph Ruiz Transposagen Biopharmaceuticals, Inc, Lexington, KY, USA Over the last 20 years, the mouse has developed into the leading rodent research model. Mice have become the model of choice mainly due to cost and the ease of creating transgenics and performing other genetic modifications. As the pharmaceutical industry is focused on more targeted and complicated therapeutics, they are looking towards biotech companies to help deliver innovative animal models that more closely predict the performance of therapeutics in humans. The laboratory rat is used extensively by the pharmaceutical industry for preclinical efficacy and toxicology studies. From a physiological, behavioral, toxicological, and biological standpoint, rats are oftentimes more similar to humans than the mouse. Their larger size facilitates procedures otherwise difficult in mice, including instrumentation, blood sampling, and surgeries. Transposagen Biopharmaceuticals, Inc., is a leader in the creation of unique genetically modified rat models. Transposagen’s signature product, the TKOTM Knockout Rat Model, is a laboratory rat with a transposon-mediated single gene trap disruption that mimics a human disease and can be used for drug discovery and development research. We are continually developing new technologies to enable more sophisticated genetic studies in the rat.
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Transgenic Res (2011) 20:1139–1189 We will outline numerous applications that utilize our piggyBacTM Genetic Modification System for efficient modification of the rat germ line. We will present a novel and costeffective strategy that combines piggyBacTM technology with spermatogonial stem cells (SSCs) to generate transgenic rats. piggyBacTM -transduced SSCs are transplanted into the testes of DAZL-deficient sterile males, which will differentiate in situ to generate spermatids derived from the modified stem cells. Offspring are rats with stable and heritable transgenes that are typically present as single copy insertions. This system has been used to generate random conditional gene trap knockout mutations, tissue-specific Cre-recombinase expressing rat lines, and single copy transgenic rats that express miRNAs, shRNAs, or inducible transgenes. Representative projects will be presented. The cutting-edge piggyBacTM-SSC technology has allowed us to deliver the next generation of animal research models with a high rate of reproducibility, in less time and most importantly at a considerable costs savings to the client.
30. From GWAS to function using sensitized strains, transgenic rescue and gene KO Howard J. Jacob1,3,4, Aron M. Geurts1,2,3, Rebecca Schilling1, Angela Lemke1, Shawn Kalloway1,4, Jamie Foeckler1,5, Jason Klotz1, Hartmut Weiler1,5, Jozef Lazar1,6, Melinda R. Dwinell1,3, Carol Moreno1,3, for the GO Grant Team
1
Human & Molecular Genetics Center, 2Cardiovascular Research Center, 3Department of Physiology, and 4 Department of Pediatrics, 5Department of Dermatology, 6 Blood Center of Wisconsin, Milwaukee, WI, USA GWAS and other types of genetic studies have nominated genes contributing to hypertension and renal failure. Functional studies must now be undertaken for these genes. The rat is the dominant model for the physiological assessment of the cardiovascular system, but has lacked the ability to target genes. Using zinc finger nucleases, we successfully applied 100 ZFN reagents across seven genetic backgrounds in 24 months. Across all strains and targets, injection and transfer of 140 (range 10–867) embryos resulted in 3.8 (range 1–14) genemodified founder (F0) pups per gene target. The overall gene modification rate averaged 2.7% per injected and transferred embryo (range 0.3–40%) and 17.3% of screened founders (range 1–100%), demonstrating a 100-fold range of ZFN activity in rat embryos. For five ZFN reagents where at least 200 embryos were injected, we were unable to identify genemodified offspring. For four of those five targets, alternative ZFNs were successful in modifying all three genes. In total, 99% (83/84) of gene targets were successfully modified in at least one strain. As hypertension is caused by genes, multiple risk factors and environmental factors, we are using a sensitized screening strategy whereby we deploy candidate gene KO on top of a genetically susceptible, but not necessarily hypertensive strain. For select genes we also use transgenic rats over expressing a gene of interest. Phenotyping of the first few strains using our high throughput physiology program for BP, renal function, and
Transgenic Res (2011) 30:1139–1189 vascular function. We have studied Rab38 a gene responsible for a QTL for renal failure and a host of other traits associated with protein trafficking using both transgenic rescue and site directed gene KO, and replicated the entire range of phenotypes originally found in the congenic animal. Finally, as proof-of-concept we targeted SH2B adaptor protein 3 (Sh2b3) and show that it is a gene that impacts blood pressure and cardiac and renal function in the sensitized SS rat model of hypertension, validating the GWAS associations in humans.
1153 100 lg/ml. Moreover, homozygous transgenic mice possessed higher amounts of the recombinant protein in the milk than their hemyzygous counterparts. The secreted proteins were correctly glycosylated and exhibited full biological activity. In addition, for each line we determined the transgene copy number and its integration site in mouse genome using q-PCR and TAIL-PCR techniques respectively. To investigate whether the transgenes has any adverse effects on mice health we performed peripheral blood cell analysis and found no differences between the transgenic and control mice.
TT2011 Poster Abstracts 0
31. 5 -regulatory region of the goat a-s1-casein gene drives tissue-specific expression of highly active human G-CSF and GM-CSF proteins in the mammary gland of transgenic mice Ivan Burkov1, Irina Serova1, Gennady Dvoryanchikov2, Lyudmila Andreeva3, Nariman Battulin1, Alexandr Smirnov1, Oleg Serov1 1
Institute of Cytology and Genetics, Novosibirsk, Russia, 2 University of Miami, Miller School of Medicine, Miami, FL, USA, 3Institute of Molecular Genetics, Moscow, Russia One of the most established systems for the production of pharmaceutical human proteins is the milk of transgenic animals. A number of researchers believe that the secretion of recombinant protein of 1–2 mg/ml in milk is economically acceptable. This estimation is relevant for proteins which are normally present in high concentration such as albumin or fibrinogen. For proteins with a high biological activity, i.e. hematopoietic factors, it is not suitable since the recombinant proteins may have a deleterious effect on transgenic animals. In light of this, it is unsurprising that a list of human proteins produced by transgenic farm animals does not include any growth factors or cytokines. We are of the belief that the strategy for the production of human active biological proteins should be modified, so that a level of their secretion in milk would be moderate in order to safeguard transgenic animals from the potentially harmful effect of the transgene expression. In accordance with these considerations we have created two vectors providing stable moderate synthesis of highly active human granulocyte colony-stimulating factor (hG-CSF) and human granulocyte–macrophage colony-stimulating factor (hGM-CSF) proteins in the milk of transgenic mice with the absence of ectopic expression. Both pGoatCas-GCSF and pGoatCas-GM-CSF constructs comprised of the following parts: (1) 3,387 bp 50 -regulatory sequence of the goat a-S1casein gene; (2) full-sized gene encoding hG-CSF or hGMCSF respectively; (3) 30 -UTR of the bovine a-S1-casein gene. The DNA was then purified and microinjected into the pronuclei of fertilized mouse eggs. A total of eight independent transgenic mouse lines were generated: four carrying the pGoatCas-GCSF construct and another four carrying the pGoatCas-GM-CSF. The transgenic lines were then subjected to detailed analysis utilising various approaches. RT-PCR and immunohistochemical assay demonstrated tissue-specific expression of hG-CSF and hGM-CSF in the mammary glands of the transgenic mice. The concentration of the recombinant proteins in the milk of the transgenic lines varied from 1 to
32. Focal adhesion kinase governs cardiac concentric hypertrophic growth by activating the AKT/mTOR pathway Carolina Clemente1, Jose´ Xavier Neto1, Ana Paula Dalla Costa2, Silvio Consonni2, Joa˜o Antunes2, Silvana Rocco1, Michele Pereira2, Carla Judice2, Jose´ Matos-Souza2, Kleber Franchini1 1
Brazilian Association for Synchrotron Light Technology, Campinas, SP, Brazil, 2State University of Campinas, Campinas, SP, Brazil The heart responds to sustained overload by hypertrophic growth in which the myocytes distinctly thicken or elongate on increases in systolic or diastolic stress. Though potentially adaptive, hypertrophy itself may predispose to cardiac dysfunction in pathological settings. The mechanisms underlying the diverse morphology and outcomes of hypertrophy are uncertain. Here we used a focal adhesion kinase (FAK) cardiac-specific transgenic mice model (FAK-Tg) to explore the function of this nonreceptor tyrosine kinase on the regulation of myocyte growth. FAK-Tg mice displayed a phenocopy of concentric cardiac hypertrophy, reflecting the relative thickening of the individual myocytes. Moreover, FAK-Tg mice showed structural, functional and molecular features of a compensated hypertrophic growth, and preserved responses to acute and chronic pressure overload. Mechanistically, FAK overexpression resulted in enhanced myocardial FAK activity, which was proven by treatment with a selective FAK inhibitor to be required for the cardiac hypertrophy in this model. Our results indicate that upregulation of FAK does not affect the activity of Src/ERK1/2 pathway, but stimulated signaling by a cascade that encompasses PI3 K, AKT, mTOR, S6 K and rpS6. Moreover, inhibition of the mTOR complex by rapamycin extinguished the cardiac hypertrophy of the transgenic FAK mice. These findings uncover a unique role for FAK in regulating the signaling mechanisms that governs the selective myocyte growth in width, likely my controlling the activity of PI3 K/AKT/mTOR pathway, and suggest FAK activation could be important for the adaptive responses to increases in cardiac afterload.
33. A high-throughput and robust genotyping pipeline at Genentech’s Research Support Facility (gRsf) J. Colin Cox, Robert Schwingendorf, Carol Cain-Hom, Anna Pham, Gregg Sy, Ryan Pabalate, Emily Hunley, Jessenia Perez, Rhonda Wiler Genentech, South San Francisco, CA, USA
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1154 The Genotyping Laboratory in the Mouse Genetics Department at the Genentech Research Support Facility (gRsf) has developed a highly efficient and robust data production pipeline for providing rapid and accurate genotypic analysis of genetic mouse models. Our laboratory extracts genetic material from roughly 350,000 tissue samples per year, and performs nearly 600,000 individual genotyping reactions on those samples covering nearly 600 unique genotyping assays for hundreds of investigators. Here, we present an overview of this pipeline, focusing on the procedures, software, and automation that facilitate such an endeavor. We also present our data production line and typical throughput and turn-around time for genotyping services; generally, tissues samples are consolidated, undergo DNA extraction, are arrayed and PCR amplified, analyzed, and reported to customers in five days or less. Finally, we provide detail into the type of genotypic analysis we offer, such as PCR fragment analysis, 50 -nuclease protection assay (TaqManTM), copy number variation (CNV) assay, single nucleotide polymorphism (SNP) assay, etc.
34. Derivation of chromosomally stable C57BL/6 ES cells Elizabeth Hughes, Virginia Zawistowski, Keith Childs, Thom Saunders University of Michigan, Ann Arbor, MI, USA Mouse embryonic stem cells have proven an invaluable resource to generate gene targeted mice for use in many areas of research. Most of this work has been done with 129-derived ES cells because ES lines are most readily established from 129 strains of mice. More recently, mouse ES lines have been established from other strains of mice, in particular from C57BL/6. Gene targeting experiments in C57BL/6 derived ES cells allow generation of mutations on the C57BL/6 background without the extensive backcrossing required when using 129 derived ES cells. We have found Bruce4-derived clones are more likely to be aneuploid than are 129-derived clones. Our definition of a ‘‘euploid’’ ES clone is that when 20 chromosome spreads are counted at least 60% of the spreads contain 40 chromosomes with normal morphology. Subcloning has been used to recover sub-populations of cells having the desirable properties of low passage ES cells from high passage populations of cells, and we have subcloned Bruce4 to identify a chromosomally stable subline. We seeded Bruce4 ES cells at 3,000 cells per 10 cm dish on feeder layers, and picked clones having morphology consistent with normal ES cells. One subclone, Bruce4.G9, was found to be more genetically stable compared to the parental Bruce4 cell line. Bruce4.G9-derived clones are more likely to be euploid than are Bruce4-derived clones, and retain the germline competence of euploid Bruce4derived clones. In parallel experiments, 79% of expanded Bruce4.G9 derived clones met our criteria for a euploid ES cell line suitable for blastocyst injection compared to only 28% of Bruce4 derived clones. Bruce4.G9 derived clones produced germline competent ES-cell mouse chimeras in 54% of the cell lines used, which is comparable to the 50% germline produced from the Bruce4 lines. We conclude Bruce4.G9 is a chromosomally stable subclone of Bruce4 and is suitable for use in gene targeting experiments.
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Transgenic Res (2011) 20:1139–1189 35. A new human monoclonal antibody platform based on transgenic and knock-out rats Biao MA2, Anne-Laure Iscache3, Michael Osborn2, Severine Menoret3, Suzanne Avis2, Marianne Bruggemann2, Ignacio Anegon3, Roland Buelow1 1
Open Monoclonal Technology, Inc., Palo Alto, CA, USA, Recombinant Antibody Technology, Ltd., Cambridge, UK, 3 Platform Rat Transgenesis IBiSA-CNRS, Nantes, France 2
Proven therapeutic application and safe use of human antibodies attracts an ever-increasing desire of many companies to develop new monoclonal antibodies. However, despite key technologies established over 20 years ago, generating diverse specificities of human monoclonal antibodies is expensive and potential targets are often limited by exclusivity agreements. We developed a new, fully human monoclonal antibody platform based on transgenic rats. The rat is a widely used laboratory animal with a well-characterized immune system, a nearly complete genome sequence, and routinely used transgenesis and hybridoma technologies. Our genetic engineering approach is the result of an improved understanding of B-cell development and a novel approach for the inactivation of endogenous antibody expression without the use of stem cells. We developed a patented zinc-finger-nuclease (ZFN)-mediated technique to generate immunoglobulin knockout rats. Engineered ZFN proteins induce double strand DNA breaks in the chosen target sequence, which stimulate the cell’s natural DNA-repair pathways and can result in site-specific changes in the DNA sequence. Microinjection into embryos of ZFNs specific for rat immunoglobulin Cc, Cj or Cl led to a high frequency of animals born with disruption at the target locus. No off-target effects in other genes were observed and offspring of the ZFN-edited rats carried the mutated genes. Endogenous heavy and light chain immunoglobulin was not produced in homozygous knockout rats. To eliminate suboptimal B-cell receptor signaling, we designed immunoglobulin loci with human antibody genes that allowed the natural assembly of a B-cell receptor complex, normal signaling and high expression of human antibodies. Immunoglobulin transgene loci of 300–600 kb, encoding human kappa or lambda light chains, and heavy chain were introduced sequentially into the rat genome by embryo microinjection of several overlapping BACs or YACs. Breeding of transgenic with knock-out animals resulted in rats expressing antibodies with fully human idiotypes. Each introduced translocus provided a large number of different and functional V genes, and all diversity and joining segments, which actively participate in DNA rearrangement, transcription and human Ig expression. Analysis of monoclonal antibodies including V-gene usage, CDR-length, and hypermutation will be presented.
36. Cryopreservation of laser-treated mouse oocytes and in vitro fertilization using low viability frozen sperm Steve Sansing, Ling Liu Charles River, Wilmington, MA, USA Cryopreservation of mouse sperm has become an essential method for the archiving, distribution and recovery of
Transgenic Res (2011) 30:1139–1189 genetically modified mouse lines. Recent refinements of the traditional sperm cryopreservation method have markedly improved the fertilization efficiency using frozen sperm in mice. However, there still exists variability in laboratories that have frozen sperm using the traditional and refined methods. Sperm frozen by either of these methods may have decreased motility and/or concentration which would make it difficult to recover a line by regular in vitro fertilization (IVF). To overcome these hurdles, laboratories may use laser assisted IVF. In the present study we cryopreserved laser-treated oocytes using slow rate freezing or vitrification and assessed fertilization efficiencies using cryopreserved sperm with low motility. C57BL/6NCrl oocytes were laser-treated to make a single hole of approximately 10–12 lm in the zona pellucida followed by cryopreservation using slow-rate freezing or vitrification. Recovery rate for the treated oocytes was 93.4% and post-thaw survival was 78.1%. The oocytes were then fertilized using frozen-thawed low viability sperm (rapid progressive motility of less than 10%). Following overnight culture, 42.2% of the oocytes were viable and fertilized as evidenced by development to the 2-cell stage. Further culturing resulted in 91.7% of the 2 cell embryos developing to the blastocyst stage. There was no significant difference found between oocytes cryopreserved using slow-rate freezing or vitrification in recovery rate, postthaw survival rate and IVF rate. We also tested the common modification of adding .25 mol/L sucrose to the oocyte media (M2) during laser treatment to see if this had an effect on postthaw oocyte survival or fertilization rates. While the oocyte recovery and survival rates increased (98.2% and 90.5%, respectively) there was no affect on fertilization rate (42.0% without sucrose, vs. 42.3% with sucrose,). While the 42.2% fertilization rate we achieved is considerably lower than the 69.2% fertilization rate we typically observed when using fresh laser-treated oocytes, the results indicate that more than enough embryos are produced for most IVF applications. These data indicate that for those laboratories that do not possess the capability of creating laser drilled oocytes, they would have the option of receiving laser-treated, cryopreserved oocytes from another facility should they be faced with having to use low viability sperm to reconstitute a line.
37. Analysis of structural and functional concepts employed in mouse phenotyping and archiving facilities Heike Kollmus1, Rainer Post2, Markus Brielmeier3, Julia Ferna´ndez4,5, Helmut Fuchs3, Colin McKerlie6, Lluis Montoliu4,5, Pedro Jose Otaegui7, Manuel Rebelo8, Hermann Riedesel1, Jesu´s Ruberte7, Radislav Sedla´cek9, Martin Habre´ d’Angelis3, Klaus Schughart1,10 1 Helmholtz Centre for Infection Research, Braunschweig, Germany, 2doranth post architekten GmbH, Munich, Germany, 3Helmholtz Zentrum Mu¨nchen, Munich, Germany, 4Centro Nacional de Biotecnologı´a, CSIC, Madrid, Spain, 5Centro de Investigacio´n Biome´dica en Red de Enfermedades Raras (CIBERER), ISCIII, Madrid, Spain, 6 The Hospital for Sick Children & Toronto Centre for Phenogenomics, Toronto, Canada, 7Center for Animal Biotechnology and Gene Therapy, Universitat Auto`noma de Barcelona, Barcelona, Spain, 8Instituto Gulbenkian de
1155 Cieˆncia, Oeiras, Portugal, 9Institute of Molecular Genetics, Prague, Czech Republic, 10University of Veterinary Medicine, Hannover, Germany The mouse is a leading experimental model system to understand human biology and to develop new treatments for human disease. More than 35,000 mutant mouse lines and about 4,000 lines from genetic reference populations will become available over the next 10 years. As they become available, they need to be characterized, archived, and disseminated to the scientific community. Infrafrontier, the European infrastructure for phenotyping and archiving of the mouse genome, is a scientific programme funded by the European Commission. The Infrafrontier programme is developing a common European infrastructure for high throughput systemic phenotyping, archiving, and dissemination of these mouse resources across Europe. The availability of highly sophisticated and large capacity infrastructures is essential for the programme; either existing facilities or new facilities to be developed in the future. Therefore, a working group (WP5) of the Infrafrontier programme assembled a comprehensive description of existing large phenotyping and archiving mouse facilities. An expert team visited nine research facilities and three commercial breeders in Europe, Canada, the United States, and Singapore. Detailed data of each facility were collected and subsequently represented in standardized floor plans and descriptive tables in an internal Infrafrontier report. Our report provides detailed standardised information on several of the visited animal facilities dedicated to large-scale and complex basic research projects. Furthermore, we describe several principle functional units that together represent a stateof-the-art phenotyping and archiving mouse facility. These include core breeding, breeding and holding, phenotyping, archiving, transgenics/re-derivation, quarantine, and supporting infrastructure units. Our study and findings will assist in comparing various principles and operational models but also provide an important basis for the planning and designing of new mouse production, phenotyping, and archiving facilities. On-going work is focused on describing the characteristics and the interconnection of the functional units in more detail.
38. Performance standards for DNA microinjection: ISTT survey results Thomas Fielder1, Laura Barrios2, Lluis Montoliu3,4 1
University of California-Irvine, Irvine, CA, USA, Secretaria General Adjunta de Informatica (SGAI), Consejo Superior de Investigaciones (CSIC), Madrid, Spain, 3Centro Nacional de Biotecnologia (CNB), Consejo Superior de Investigaciones (CSIC), Madrid, Spain, 4Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER), Madrid, Spain 2
The efficiency with which transgenic mice can be produced via pronuclear injection of DNA constructs is subject to a large number of variables ranging from human to mechanical to biological. Transgenic core facilities would benefit from knowing how their efficiency compares to that of other facilities, and whether significant improvements in any phase of the process can be realistically achieved, but direct
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1156 comparisons between facilities are complicated by the large number of differences in protocols and strains being used. We have conducted a world-wide survey of core facilities to collect data, for individual injection days, of embryo and pup yields and numbers and strains of mice used as embryo donors and pseudopregnant recipients. Data from individual facilities have been analyzed and compared with the global data set, and recommendations for improving yields are offered. All relevant ratios have been analysed, including the number of harvested, fertilized, and injected embryos per donor, the number of pups per recipient, and the number of total pups and transgenic pups per embryo injected, among others. We have compared such ratios for each embryo donor strain and recipient strain, as well as for the two classes of DNA constructs injected (small plasmid-based transgenes and BAC/PAC/YAC-based transgenes). In addition, we present an analysis of the major sources of variability in these ratios, and have attempted to identify differences among facilities that are most likely to influence average yields and variability of yields.
39. Optimized sperm cryopreservation media for mice Steve Sansing, Ling Liu Charles River, Wilmington, MA, USA Sperm cryopreservation has become an acceptable and costeffective procedure for archiving the genomes of genetically modified animals. During the sperm freezing process, ice formation and reactive oxygen species formation have been proven to be closely associated with post-thawed sperm capability to fertilize oocytes. We examined supplementation of sperm cyroprotective agents (CPA) with free radical scavengers to potentially improve the thawed sperm motility, viability and subsequent fertilizing capacity. Various concentrations of antioxidant supplement (AOS) and polyvinyl alcohol (PVA) and the effects they had on post thaw motility and in vitro fertilization (IVF) efficiency were tested. Sperm were harvested from male mice and 10 ll sperm/CPA suspension was loaded into each 0.25 ml cryopreservation straw. Straws were sealed on both ends and placed into a freezing canister which was floated on liquid nitrogen for 10 min then submerged. Straws were thawed in a 37°C water bath for 2–3 min. After thawing, sperm was diluted and assessed using a computer assisted sperm analysis system for motility and used in an IVF procedure. Embryos generated from IVF were cultured overnight and two-cell stage embryo development was assessed. The results indicated that supplementation of CPA with 10 ll/ml AOS provided a significantly greater protection of sperm motility, and significantly higher IVF rates were achieved with C57BL/6NCrl mouse sperm frozen in CPA + 10 ll/ml AOS + 100 lg/ml PVA. Based on these results, the modified sperm cryopreservation media were used to freeze sperm from 5 inbred strains of mice and perform IVF. The IVF rates were 55.7, 26.4, 87.3, 87.8 and 26.2% in C57BL/ 6NCrl, 129S2/SvPasCrl, FVB/NCrl, DBA/2NCrl, and BALB/ cAnNCrl respectively. These results are consistent with recent reports. Over a 1.5 year period, 49 distinct mouse lines were archived by sperm cryopreservation and later recovered by IVF. The recipient pregnancy and live-birth rates were 93.2 and 39.9% respectively. This new sperm cryopreservation
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Transgenic Res (2011) 20:1139–1189 media will increase success in archiving and recovery of genetically modified mouse models.
40. Msi1-CRE transgenic mice: driving transgene expression to the putative gut stem cell Maria Perez-Caro1, Lucia Mendez-Sanchez1, Jose-Fernando Perez-Fontan3, Juan-Luis Garcia-Hernandez2, Teresa Hernandez2, Enrique de Alava2, Susana Fraile-Martin2, Manuel Sanchez-Martin1 1
University of Salamanca, Salamanca, Spain, 2Centro de Investigacio´n del Ca´ncer, Salamanca, Spain, 3Hospital Universitario de Salamanca, Salamanca, Spain Musashi-1 (Msi1), a highly conserved RNA-binding protein, has been postulated as a neural stem cell marker playing important roles in maintenance of the stem cell state, differentiation and tumorogenesis. In addition, Msi-1 expression has also been detected in cells located just above Paneth cells in the mouse small intestine where the stem cell populations have their niche. At this site, the role of Msi1 is poorly studied and the mechanisms regulating Msi1 expression are not clear yet. To study these topics, we have generated a transgenic mouse line using Msi-1 derived construct which strongly expresses the Cre-recombinase at the bottom of the crypts of the intestinal tract. Thus, to generate the transgene we used a 7 kb fragment of genomic DNA from the Mushashi-1 gene promoter compressing 4 kb upstream and 3 kb downstream from the ATG coding sequences, respectively. The CRE expression of the transgene was evaluated using both RT-PCR and Q-PCR assays. We have crossed the Msi1-Cre transgenic mice with mice that carry a B-Gal cre-reporter to detect the expression of the recombinase in vivo and to localize of Msi1-Cre positive cells. We have observed expression of the Cre-recombinase in the Msi-1 compartment of several tissues such as brain, skin, lung, and specifically, we have also observed Msi-1 Cre expression at the bottom of the crypts of the intestinal tract, where intestinal stem cells localize. This mouse model could be useful to study the biology of the Msi1-compartment where Msi1 + intestinal stem cells localize and it could be instrumental to design new stem cell assays aimed to improve the characterization of the stem cell population of the small intestine.
41. Cryopreservation and distribution of mutant mouse models by the Sanger Mouse Genetics Project Richard Houghton, Michael Woods, Helen Kundi, Laila Pearson, Stuart Newman, Hannah Wardle, Carole Frost, Catherine Ingle, Jennifer Salisbury, James Bussell, Joanna Bottomley, Ramiro Ramirez-Solis Sanger Institute, Cambridge, UK The Wellcome Trust Sanger Institute Mouse Genetics Project (Sanger MGP) is a European Mutant Mouse Archive (EMMA) partner and deposits archived mouse lines generated by the project at EMMA distribution centres for a sustainable resource to the scientific community. Mouse lines are also sent to the KOMP repository for archiving and distribution.
Transgenic Res (2011) 30:1139–1189 The mutant mouse lines are generated from the EUCOMM and KOMP ES cell resource produced at the Sanger Institute. These mouse lines are characterised through primary phenotypic studies and both the data (Sanger Mouse Resource Portal; Europhenome) and mouse resource (IKMC; EMMA; KOMP Repository) are made available to the scientific community. Demand on the archiving of the mouse models has significantly increased over the past 2 years as the number of mutant mouse lines has increased. Development of sperm freezing and IVF techniques for the C57BL/6 N strain is envisaged to compliment the embryo cryopreservation and allow more mouse lines to be cryopreserved more efficiently and economically. Prior to archiving at a distribution centre and whilst being actively maintained on the shelf for phenotypic characterisation we also offer the potential early opportunity to source the mice should they be available beyond our phenotyping requirements. As well as supporting the international academic scientific community our resource has supplied more detailed secondary phenotyping studies to consortia such as Genome Canada and EUMODIC partners. The scientific community has shown significant interest in our mouse lines and Sanger MGP now contributes *25% of the EMMA exports. By the end of 2010 we had completed more than 500 international exports of live mice and embryos to the scientific community and repositories.
42. The development of sperm freezing and IVF techniques by the Sanger Institute Mouse Genetics Project Evelyn Grau, Helen Kundi, Laila Pearson, Stuart Newman, Ellen Shapland, Michael Woods, Richard Houghton, Hannah Wardle, Ramiro Ramirez-Solis Sanger Institute, Cambridge, UK The Sanger Institute Mouse Genetics Project (Sanger MGP) generates mutant mouse lines from the EUCOMM and KOMP ES cell resource produced at the Sanger Institute. These mouse lines are characterised through primary phenotypic studies, archived and made available to the scientific community. Cryopreservation of 400 E2.5 embryos has been the standard method of archiving mutant mouse lines for the MGP, with 200 embryos being shipped to the European Mouse Mutant Archive (EMMA) for distribution to the scientific community. However, demand on the archiving of mouse models has significantly increased over the past 2 years as the number of mutant mouse lines has increased. To limit the impact of this, sperm freezing and IVF techniques for the C57BL/6 N strain have been developed by the Sanger MGP. These techniques compliment embryo cryopreservation and allow mouse lines to be cryopreserved more efficiently and economically for the MGP, whilst also reducing the number of animals required. Historically, sperm frozen from the C57BL/6 N strain has been of very low viability in IVF experiments. However, recent modifications to the cryoprotective medium have made sperm freezing a viable method for archiving mouse lines on a C57BL/6 N
1157 background. The Sanger MGP has proved that sperm freezing is a viable high-throughput option for cryopreserving mouse lines on a C57BL/6 N background. The Sanger MGP has cryopreserved around 100 lines as sperm, with all lines entering a stringent quality control pipeline which currently exceeds a 96% pass rate.
43. EVC2/LIMBIN KO mice as a model for Ellis-van Creveld Syndrome and Bovine Chondrodysplastic Dwarfism Gregory Scott1, Brittany Monceaux3, Yuji Mishina2, Tracy Brown1, Manas Ray1 1 The National Institute of Environmental Health Sciences, RTP, NC, USA, 2University of Michigan, Ann Arbor, MI, USA, 3LSU Health Science Center, Shreveport, LA, USA
Ellis-van Creveld (EvC) syndrome is an autosomal recessive disorder causing skeletal dysplasia. This genetic disorder has been linked to two nonhomologous head-to-head genes, EVC and EVC2/LIMBIN. EvC syndrome is characterized by dwarfism, polydactyly, ectodermal dysplasia (dysplastic nails, teeth, and thin hair) and often atrio-ventricular septal defects. EVC2/LIMBIN is also associated with bovine chondrodyplastic dwarfism (BCD) found in Japanese brown cattle. BCD is an autosomal recessive disorder leading to shortened limbs, joint abnormalities, and ateliosis. Because of both the human significance of EvC, and the comericial costs of BCD, it is important that we further our understanding of the function of these genes. Our objective here is to provide the initial characterization of a new mouse model for EvC. We created this model by inserting a stop codon, followed by an IRES-lacZ-polyA sequence, into exon12 of the Evc2/Limbin gene, which mimics the mutation found in BCD. Initial results showed high postnatal mortality in the mutant animals. Approximately 50% of the mutant pups born died shortly after birth, with subsequent examination showing lungs in this group of mutant mice that never inflated properly. MRI and histological examination revealed a disorganization of the cartilage in the trachea, similar to what is found in the over expression of FGF18, leading to a collapse of the unsupported airway. Mutant mice that survived were small and showed various dental and skin abnormalities. To determine the effect of EVC2/Limbin mutation on expression of other genes, RNA was analyzed from E18.5 embryos in three groups: wild type, mutant mice able to breathe (IB), and mutant mice unable to breathe (NIB). Additionally, RNA was analyzed from various tissues in surviving adult mice. Real Time (RT) PCR was used to analyze the expression of several members of the FGF family, as well as BMP4, PTCH1, Gli1, Gli3, and Glis3. The results from the RT PCR revealed that several of the FGF genes showed altered expression in the above two mutant groups, with the NIB group showing higher FGF18 levels compared to the IB group (p = 0.1). With our new mouse model, we will further our knowledge into the mechanisms behind EvC and BCD.
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1158 44. Efficient generation of ZFN KO and BAC transgenic rats Wanda Filipiak, Margaret Van Keuren, Galina Gavrilina, Michael Zeidler, Thomas Saunders University of Michigan, Ann Arbor, Michigan, USA The rat is an important animal model for cardiovascular, cancer, and pharmacological research. Bacterial artificial chromosome (BAC) transgenes can be used to generate transgenic animal models that express genes at physiological levels with the same developmental timing and expression pattern as endogenous genes. Zinc Finger Nucleases are used to prepare gene knockout/knockin rat models. Transgenic, knockout and knockin rats are used to model human genetic diseases and explore gene function. Transgenic rats were produced in Crl: CD (SD) Sprague– Dawley rats. Two hundred-seven transgenic founders were generated from fifteen BAC transgenes. Results were compared to the transgenic efficiency of nine plasmid transgenes in SD rats. An average of 2.9 transgenic founders were produced for every 100 fertilized eggs that were microinjected with BAC DNA transgenes 101–231 kb in size. BAC efficiency was slightly better but not significantly different from small plasmid DNA (2.5 transgenic founders were produced for every 100 eggs microinjected with transgenes 4–21 kb in size). Also one hundred-eleven transgenic founders were generated from microinjection of six ZFN mRNA transgenes, and the efficiency was significantly better (5.9 founders per 100 microinjected eggs). Anecdotal evidence suggests that the production of transgenic rats is less efficient than transgenic mouse production. BAC transgenic efficiency in B6SJLF2 mice was 2.2 transgenic founders for every 100 microinjected mouse eggs. Since the efficiency of BAC transgenic rats was 2.9, it can be argued that BAC rats can be produced with greater facility than BAC mice. Anecdotes about the difficulty of rat transgenesis should not deter transgenic laboratories from attempts to produce and study BAC transgenic rat models.
Transgenic Res (2011) 20:1139–1189 embryos (Bos indicus) and the production of embryonic chimeras by aggregation of Bos taurus (2n) with Bos indicus (4n) embryos. Oocytes from Nelore cows from abattoir were matured, fertilized with semen from Nelore and Holstein bulls, and cultured in SOF (synthetic oviduct fluid). Two-cell stage Nelore embryos (30 hpi), with a well defined inter-blastomeric axis, were selected for electrofusion procedure (ECM 830-BTX, Harvard Apparatus) to produce tetraploid embryos. For this procedure, some parameters were tested according to the number of pulses (1 or 2), voltage (40, 50, 75, 100, 140, 500 V) and duration of electroshock (20, 25, 50, 60 ls). Nelore tetraploid embryos produced after electrofusion and diploid taurine embryos, both on 8 to 16-cells stage (72 hpi) were subjected to protease treatment to remove the zona pellucida, and subsequently treated with the agglutinant agent phytohemagglutinin. Bos indicus (4n) and Bos taurus (2n) embryos were added in pairs (4n + 2n) into individual wells (WOW) for culture until the blastocyst stage to validate the chimeric embryos formation. Among the tested parameters, the best fusion results (92%) and rates of cleavage after-fusion (66%) were obtained with a single pulse of 75 V for 60 ls. The production rate of expanded 4n blastocysts was 31.5% using these parameters. After four replicates (still in the final stage of standardization) 4 blastocyst chimeras (4n + 2n) were obtained from 31 attempts (13%). The production of bovine embryonic chimeras [Bos indicus (4n) + Bos taurus (2n)], with a non-random pattern of distribution of their cell aggregates, will enable the validation of this technique in applied research, by producing exclusively taurine calves, but with placental elements from Bos indicus breed, following transfer of these chimeras into recipient cows. Financial support: FAPESP, Brazil.
46. Highly-efficient, fluorescent, locus directed cre and flpo deleter mice on a pure c57bl/6n genetic background Marie-Christine Birling1, Andre´e Dierich1,2, Sylvie Jacquot1, Yann He´rault1,2, Guillaume Pavlovic1 1 Institut Clinique de la Souris, Illkirch 67400, France,2 IGBMC, Illkirch 67400, France
45. Aggregation of diploid (Bos taurus) and tetraploid embryos (Bos indicus) to produce bovine embryonic chimeras Eduardo M. Razza1, Rafael A. Satrapa1, Isabele P. Emanuelli2,1, Ciro Moraes Barros1, Marcelo F. Gouveia Nogueira2 1
Dept. of Pharmacology, IB, Sao Paulo State University (UNESP), Botucatu, Sao Paulo, Brazil, 2Dept. of Biological Sciences, FCL, UNESP, Assis, Sao Paulo, Brazil The formation of tetraploid embryos (4n) by electrofusion and their subsequent chimerism with a diploid embryo (2n) must result in a chimeric conceptus, whose inner cell mass (ICM) is entirely 2n. Hence, the aggregation of a zebu embryo (4n, thermotolerant) with a taurine embryo (2n, thermosensitive) would result in an exclusively taurine ICM, but the trophectoderm (future extraembryonic components) would be mostly from zebu embryo, which could support taurine embryo/fetus to adapt—during pregnancy—in tropical climate. The purpose of this study was to standardize the production of 4n Nelore
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In order to facilitate the use of the new mutant resource developed in the mouse, we have generated Cre and FlpO deleter mice on a pure inbred C57BL/6 N background. The new transgenic constructs were designed to drive either the Cre or FlpO recombinase, fused to a specific fluorescent marker (respectively the eGFP or the eYFP) and were inserted by homologous recombination in the neutral Rosa26 locus. These lines allow a rapid, cost-effective and efficient identification of the carrier individuals through the co-expression of the fluorescent marker. The recombination efficiency of the two deleter lines, Rosa26tm1 (AC.cre2AeGFP) Ics and Rosa26tm1 (CAG.FLPo2AeYFP) Ics, was carefully evaluated using five loxP-flanked or four FRT-flanked alleles located at different positions in the mouse genome. For each locus tested so far, we observed a 100% excision rate. The transgenic mice are easily distinguishable from wild type animals by their bright fluorescence that remains detectable until 10 days after birth. Fluorescence is still detectable by observation of the adult unpigmented legs. Furthermore they both display accumulation
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of the specific recombinase during oogenesis. Indeed both Cre or FlpO mRNA and/or protein are retained in mature oocytes irrespectively of the transmission of the pCA-Cre or pCAGGsFlpO transgene, resulting in efficient Cre or Flp-mediated recombination of paternally derived target genes upon fertilization These fluorescent ‘Cre- and Flp- deleter’ transgenic lines are valuable tools for the scientific community by their high and stable recombination efficiency, the simplicity of genotype identification and the preservation of a pure genetic background when used to remove specific selection cassette or to induce complete loss-of-function allele.
47. Comprehensive phenotyping of mouse models Tania Sorg1, Abdel Ayadi1, Sylvie Jacquot1, Elodie Bedu1, Roy Combe1, Bastien Fricker1, Hamid Meziane1, Mohammed Selloum1, Yann Herault1,2 1
2
Institut Clinique de la Souris, Illkirch, France, IGBMC, Illkirch, France The Institut Clinique de la Souris (ICS)/Mouse Clinical Institute (MCI) is a technology platform that provides a comprehensive set of highly specialized mouse services to scientists from academia and industry. The ICS combines the capacity of generating mutant mice on a large scale with a high-throughput and comprehensive phenotypic analysis of mice. The ICS phenotyping platforms are adapted for the study of genetically engineered mouse models (GEMMs), but can also be used for pharmacological and toxicological studies in the mouse. The ICS services will ultimately help the scientific community to use the mouse to develop a complete functional annotation of the human genome and to employ this to better understand human diseases and their underlying physiological and pathological basis. Achieving this goal will underpin and speed the biopharmaceutical and biotech industries that increasingly depend upon advanced, mouse-based analysis in drug development. The ICS has successfully assembled a comprehensive phenotyping platform, which at present is composed of the 5 following core units: – Clinical Chemistry laboratory The activities of this laboratory cover clinical chemistry, hematology, coagulation, immunology, endocrinology and other metabolites. – Metabolic exploration This service is set up to phenotype the metabolic function, such as in diabetes and obesity models. Currently tests are in place to analyze the body composition, glucose homeostasis, and energy expenditure, as well as the skeleto-muscular and the uro-genital systems, and the gastro-intestinal tract. – Cardiovascular and respiratory exploration This service is set up to phenotype the function of the cardio-respiratory system. Currently tests are in place to analyze the cardiac function and anatomy, as well as the respiratory system such as in asthma models. – Behavior and nervous system This service explores the central and peripheral nervous system, sensory systems, as well as behavior in mice. This core has developed a comprehensive tests battery to evaluate: general CNS
function, affective behaviors (anxiety, depression), cognitive function, sensory thresholds and analgesia, as well as the sensory systems (visual and auditory functions) – Histology and pathology This service provides a comprehensive histological and histopathological analysis of mutant and control mice, as well as embryology studies. The assays performed by each core unit will be presented, as well as validated flow schemes for applications in therapeutic areas.
48. Next generation pronuclear injection: MEMS replaces the pump Aubrey Wilson1, Quentin Atten2, Brian Jensen2, Larry Howell2, Susan Tamowski3, Sandra Burnett1 1
Department of Micro and Molecular Biology, Brigham Young University, Provo, Utah, USA, 2Department of Mechanical Engineering, Brigham Young University, Provo, Utah, USA, 3Transgenic and Gene Targeting Mouse Core, University of Utah, Salt Lake City Utah, USA Injecting DNA into the pronucleus of an embryo is an inherently traumatic process to the embryo. We have developed a microelectromechanical system (MEMS) which significantly reduces the trauma to the injected embryos by decreasing the injection needle cross sectional area and eliminating fluid flow into the pronucleus while still delivering sufficient DNA for transgene integration. The MEMS device couples control of electrical charge on an extremely small lance with precision mechanical movement. In this new method called ‘‘nanoinjection’’ DNA is electrically accumulated on the surface of the lance by applying a positive charge. The lance then pierces the zygote’s pronucleus with its DNA-coated tip. The DNA is released into the pronucleus by reversing the charge on the lance and the lance is then withdrawn. Harnessing the DNA molecules’ charge for movement instead of relying on fluid flow allows a smaller solid lance (0.06 lm2) to replace a hollow microinjection needle (0.78 lm2). Elimination of fluid flow removes the strain of pronuclear swelling and the risk of bursting. We compared nanoinjection and microinjection sideby-side over the course of four days injecting 1013 eggs between the two groups. While nanoinjection and microinjection yielded similar rates of integration per pup (17.3 ± 6.2% and 14.1 ± 7.4% respectively) nanoinjected zygotes had higher viability from injection to birth. Nanoinjected embryos were significantly more likely to survive to the two-cell stage (77.3 ± 4.2% vs. 54.7 ± 3.8%) with the odds of an injected embryo developing to the two cell stage being 2.8 times higher for nanoinjection than microinjection. Even when comparing apparently healthy two-cell stage zygotes from both groups, those that had been nanoinjected were significantly more likely to result in live births (52.4 ± 5.7% vs. 24.2 ± 4.5%). The superior embryo survival of nanoinjected zygotes lead to significantly higher rates of integration per injected embryo (6.7 ± 2.5% vs. 1.9 ± 0.8%). The odds ratio for producing a transgenic animal through nanoinjection was 4.2 times higher than through microinjection. Microinjection appears to have lingering effects which reduce embryo viability and these effects are at least partially avoided though nanoinjection
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1160 methods. Nanoinjection proved a much more efficient process requiring fewer injected zygotes and fewer surgeries to produce an equivalent number of transgenic animals.
49. Gene targeting of the rat Csf1r locus using homologous recombination in ES cells Kyle R Upton, Clare Pridans, Stephen Meek, Linda Sutherland, Bruce Whitelaw, Tom Burdon, David Hume The Roslin Institute and R(D)SVS, Easter Bush, Midlothian, UK Colony stimulating factor 1 receptor (Csf1r) is a master regulator of macrophages and highly conserved in mammals. Expression of Csf1r is essential for macrophage growth and differentiation, and its expression is restricted to macrophages and trophoblasts. As such, it is of great interest for the study of the plethora of roles which macrophages play including development, immune response, inflammation and cancer. A knock out of the Csf1r allele, will allow the study of the effect of macrophage ablation on numerous diverse systems. To this effect we have knocked out the Csf1r allele in rat ES cells, by replacing the first exon with an irrelevant sequence, inserting a floxed PGK-NeoR cassette in the first intron. By including a diptheia toxin A negative selection cassette distal to the short homology arm, we achieved a targeting efficiency of 76% (homologous recombinants versus total G418 resistant clones), confirmed by Southern blotting. These ES cell clones are currently being screened to identify pluripotent (differentiation competent) lines with a suitable karyotype for injection. From studies in mice, expression of Csf1r is minimally affected in heterozygous knockouts, with transcriptional compensation from the intact allele. To take advantage of this we are investigating a knock-out-knock-in strategy to replace the Csf1r transcript with eGFP. The Csf1r locus provides a number of challenges for such an approach. The Csf1r gene encodes a 978 amino acid protein containing a signal peptide, 5 Immunoglobulin like domains, a transmembrane region and a cytosolic tyrosine kinase domain. The signal peptide encoded in the first exon would lead to secretion of a fusion protein truncated before the transmembrane region. Alternate splicing before and after the transmembrane encoding region (exon 10) means targeting 30 to this region may not destroy all transcript isoforms. The first intron also contains an integral regulatory element (FIRE) which may require transcriptional processing for correct function. Three alternate vectors have been constructed which deal with these issues in different manners. Design, construction and testing of these vectors will be discussed. Functional constructs will be converted to targeting vectors and used for gene targeting in germline competent DA rat ES cells, with a view to create transgenic rats. 50. Germ line competency of chicken embryonic stem cells to produce genetically modified chickens Mikiharu Nakano1, Masaki Nishimoto2, Ryo Ezaki2, Shuichi Furusawa1, Hiroyuki Horiuchi1,2 1 Hiroshima University, Hiroshima, Japan, 2Hiroshima Industrial Promotion Organization, Hiroshima, Japan
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Transgenic Res (2011) 20:1139–1189 In mice, embryonic stem cells (ESCs) have been used as a powerful tool for elucidate gene function and create genetically modified model animals. Previous studies have demonstrated that chicken ES cell (cESC) lines can differentiate into many cell types in vitro and in vivo. However, because of almost cESCs cannot keep their germ line competency after long term culture, cESCs have not suitable for create transgenic chickens. Why cannot cESCs maintain with germ line competency? Here, we propose several countermeasures for this problem. The first is to maintain expression of chicken vasa homolog (Cvh) in cESCs. Chicken blastodermal cells derived from stage X embryo, which are the origin of cESCs, contain Cvh-positive primordial germ cells (PGCs) or their precursors. However, Cvh-positive PGCs was not detected in almost cESC lines after long term culture. Recently, we established novel cESC lines that maintained the expression of Cvh after long term culture and gene modification. In addition, we confirmed that Cvhpositive cESC lines differentiated into PGCs in chimeric embryos, and into oocyte in female chimeras. These results indicate that Cvh-positive cESCs can contribute to germ line at least. Thus it is important to maintain optimal condition for expression of Cvh in cESCs culture. Failure of mouse ESCs to contribute to the germ line is often caused by chromosomal abnormalities in ESCs. Therefore, we examined the karyotype of macro chromosomes of novel cESCs and found that 2–10% of the cells were polyploidy. However, by gene-transfection with random integration, the chromosomal abnormalities of the cells were raised to 90%. On the other hand, the chromosomal abnormalities of the cells after gene-targeting based on homologous recombination and/or subcloning were the same levels (2–10%) as cESCs before gene-transfection. This result indicates that genetargeting and subcloning may be effective to lower risk of chromosomal abnormalities in cESCs. Although further research will be required to create transgenic chickens by novel cESCs, our data may help to put cESCs into practical use for germ line transmission.
51. Targeted gene insertion into pretested genomic loci of the porcine genome and generation of live offspring Wiebke Garrels1, Lajos Mates2, Stephanie Holler1, Zsuzsanna Izsvak2, Bjo¨rn Petersen1, Zoltan Ivics2, Heiner Niemann1, Wilfried A. Kues1 1 Friedrich-Loeffler-Institut, Institut of Farm Animal Genetics, Neustadt am Ru¨benberge, Mariensee, Germany, 2 Max-Delbru¨ck-Center for Molecular Medicine, Berlin, Germany
The pig is important for modelling human diseases, because physiology, metabolism and genome organisation reflect the human situation closer than small animal models. Genetic engineering can expand the utility of pigs for modelling human diseases, for developing novel therapies, or for providing tissues for xenotransplantation. We generated Sleeping Beauty transgenic pigs1 expressing the Venus fluorophore. The transposase catalysed gene delivery increased the efficiency of chromosomal integration and favoured single copy insertion events into euchromatic regions.
Transgenic Res (2011) 30:1139–1189 The transgenic animals showed normal development and persistent reporter gene expression for [18 month. In the offspring a copy number dependent transgene expression was found. With the aid of splinklerette PCR we confirmed specific SB-catalysed transposition and identified 25 integration sites at the expected TA dinucleotide consensus sites. Five out of the 25 integration sites could be assigned to chromosomal positions and were mapped to porcine chromosomes X, 3, 7, 8 and 13. Four of these integration sites were found in intergenic regions and one was located within intron 2 of the SMARCA 5 gene. The Venus-transposon contains heterospecific loxP sites, which allows a targeted exchange of the Venus transgene cassette in a pretested locus against a transgene of choice by transient expression of Cre recombinase. Primary cells from one F1 fetus with a single integration of the Venus transposon cassette were co-electroporated with a floxed CAGGS-mCherry plasmid and a Cre expression plasmid. The cells were sorted by FACS and the mCherry positive cells were used for somatic cell nuclear transfer (SCNT). A total of 100 reconstructed embryos were transferred to one foster sow. At day 30 of gestation the sow was sacrificed and a total of 12 normally developed fetuses were recovered. All of them showed specific mCherry fluorescence, but no Venus fluorescence. Correct cassette exchange was confirmed by sequencing of the recombined loxP sites. In summary, Sleeping Beauty transposition and recombinase mediated cassette exchange (RCME) in pretested genomic loci (safe habor) are effective in producing transgenic pigs with germline transmission and stable transgene expression and thus improve genetic modification of pigs. Importantly, no antibiotic selection is required. The exploitation of in situ pretested loci and the targeted integration of single transgene copies may allow generating better models for human diseases. Ref: W Garrels, L Mates, S Holler, H Niemann, Z Izsvak, Z Ivics,WA Kues, 2010: Generation of transgenic pigs by Sleeping Beauty transposition in pig zygotes. Reproduction in Domestic Animals 45, 65 (abstr.)
1161 While the immune cell function of CD2BP2 is still elusive, a spliceosomal function of the proteins has come into focus. Consistently, CD2BP2 has been found as a component of the U5 snRNP and was demonstrated to inhibit splicing in vitro. The aim of this study was to analyze the role of the CD2BP2 protein in mice by targeted deletion of its gene. The distribution of CD2BP2 in different mouse organs was analyzed by realtime RT-PCR and Western Blot. The mRNA was ubiquitous expressed whereas the protein was mainly present in spleen, thymus, lymph node, bone marrow and reproductive organs. Intracellular flow cytometry of several immune cells showed an ubiquitous expression in all cells of the respective tissues. For the targeting vector, a neomycin cassette was cloned 50 to the first of the 7 exons that was flanked by frt-sites. One loxP site was cloned in front of the first frt-site, while the second loxP site was introduced behind the third exon. The targeting vector was electroporated in 129 R1/E and JM8A3.N1 cells. Positive clones could be received from both electroporations but only the 129 derived cells lead to germ line transmission. After breeding with PGK-cre mice, heterozygous knockout mice were bred. The first potential null mice are currently born. We will start the analysis of CD2BP2 knockout mice with a focus on general immune cell function and the analysis of alternatively spliced marker proteins. Viral and bacterial infection models in combination with mRNA and proteomic analysis will be performed with the null mice in comparison to the WT control animals. Thereby, this first knockout of a GYF domain containing protein will help to understand the function of CD2BP2 in the living system.
53. Assessment of cell chimerism between pig offspring and their mothers in two reporter transgenic lines Wiebke Garrels1, Stephanie Holler1, Ulrike Taylor1, Doris Herrmann1, Zoltan Ivics2, Heiner Niemann1, Wilfried A. Kues1 1
52. Characterization of the GYF-domain protein CD2BP2 Gesa Ines Albert1, Ronald Naumann2, Florian Herse3, Klaus-Peter Knobeloch4, Christian Freund1 1
Institut fuer Molekulare Pharmakologie and Freie Universitaet Berlin, Berlin, Germany, 2MPI of Molecular Cell Biology and Genetics, Dresden, Germany, 3 Experimental and Clinical Research Center, Charite´ Medical Faculty, Max-Delbrueck Center for Molecular Medicine, Berlin, Germany, 4Neuropathology, University Clinic, Freiburg, Germany GYF-domains (named after a conserved glycine, tyrosine, phenylalanine motif) are small adaptor domains that interact with proline-rich sequences (PRS) in target proteins. The CD2 binding protein 2 (CD2BP2) was initially described as an interaction partner of the cytoplasmic domain of the T cell adhesion molecule CD2. A direct interaction of the CD2BP2 GYF domain with the PRS of CD2 could be demonstrated by structural and biophysical methods.
Friedrich-Loeffler-Institut, Institut of Farm Animal Genetics, Neustadt am Ru¨benberge, Mariensee, Germany, 2 Max-Delbru¨ck-Center for Molecular Medicine, Berlin, Germany In humans, trafficking of human cells between mother and fetus during pregnancy and long-term maintenance of these cells after birth is well documented. In addition, fetal microchimerism has been shown for bovine and human twins sharing a common placenta. The long-term presence of microchimerism can have consequences for the function of the immune system and the regenerative capacity of the host. Whether microchimerism occurs in multiparous mammals such as the pig, where the fetuses have separate placentas, has not been studied in detail. The hemochorial human placenta, where the maternal blood is in direct contact with the chorionic epithelium, facilitates trafficking of cells and antibodies because the placenta barrier is so thin. However, also for species such as the pig with an epitheliochorial placenta, in which the maternal and fetal epithelium forms a tight barrier, cell trafficking has been observed under certain conditions.
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1162 Here, we characterized cell chimerism in five litters of wildtype sows inseminated with semen from two fluorescence reporter transgenic boars (Ni 503 and Ni 505). Due to the segregation of three independent single copy monomeric integrations in both boars an excess of transgenic offspring was found in every litter (n = 5). In total 40 living piglets were born. Transgenic piglets (n = 33) showed robust ubiquitous expression of the reporter in somatic and germ-line cells, and thereby providing a unique resource to assess a potential cell trafficking to their non-transgenic littermates (n = 7) or mothers (n = 4). By performing highly sensitive flow cytometry using a FACScan (BD Bioscience, Heidelberg, Germany) equipped with an argon laser (n = 488 nm, 15 mW), fluorescence microscopy, histological examinations and real-time PCR no evidence for microchimerism between transgenic and nontransgenic porcine littermates, and their mother could be found. This finding is based on detection limits of 1 in 150,000 to 300,000 cells for FACS and for 1 in 20,000 cells in real-time PCR. These data suggest that the epitheliochorial structure of the porcine placenta effectively prevents cellular exchange during prenatal development. Without iatrogenic interventions during gestation, porcine littermates should have a full immune response against tissues of their siblings.
54. Rapid generation of genetically engineered mouse from C57BL/6 ES cell lines using tetraploid complementation Sang Yong Kim, Priscilla Wu, So Young Eom, Raehum Paik, Z.Josh Huang Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA The C57BL/6 (B6) mouse strain is the major reference strain for genome sequence and physiological and behavior studies. However, most mouse embryonic stem (ES) cell lines that are used for gene targeting have been established from 129 and C57B6X129 hybrid strains. A 2008 survey (Thomas Saunders, University of Michigan) indicated that approximately 90% of all published gene targeting were performed in 129 ES cell lines. In studies that require C57BL/6 (B6) strain, the 129 mice have to be extensively backcrossed with B6 often for 10 generations. To data, gene targeting in B6 embryonic stem (ES) cell is often inefficient. To improve the efficiency and throughput of genetic engineering in B6 strain, we have developed new B6 ES cell lines (SY Kim, 2010). These B6 ES cell lines have a number of advantages over those already available. First, they have high percentage of germline transmission (*50–80%). Second, they can used for tetraploid complementation, producing a high percentage (15–20%) of mice that are entirely B6 background. Recently, we have successfully generated knockin lines (R26-LsL/H2BGFP, Pv-2ACreEr and Al-Dual) completely ES cell-derived mice were efficiently produced through tetraploid blastocyst complementation. Our new B6 ES cells can be used to rapidly generate mouse model that are homozygous for the gene of interest in a cost-effective and efficient manner.
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Transgenic Res (2011) 20:1139–1189 55. Lentiviral vector mediated transgenesis: a method to generate an animal model to study TDP-43-related neurodegeneration Paulina Koza, Agata Klejman, Leszek Kaczmarek Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland TDP-43 is a key pathological protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U). Our aim was to generate three kinds of transgenic rats, expressing two mutant variants and wild type of human TDP-43 gene, fused with EGFP and under the control of neuron specific synapsin promoter. Lentiviral vectors carrying all three TDP-43 variants were created and for each construct the number of subzonal injections of one zygote was experimentally tested. Three founders carrying wild type Syn-TDP-43 were generated. The incorporation of transgene into genome was confirmed by specific PCR reaction. Microscopic visualization and western blotting technique showed an abundant expression of GFP—fused TDP-43 protein in the brains of 3 month old animals. Notably, at this age no gross degeneration of neurons was detected. RotaRod test conducted on 5 month old transgenic rats showed no distinct motor deficits. At present we carry out behavioral analyses to verify whether animals exhibit cognitive impairments, which could possibly appear before any motor disorders. As neurodegeration occurs in ALS and FTLDU patients in age-dependent manner, we plan to run further morphological and behavioral analyses on aging transgenic rats. Despite additional attempts, we were unable to generate transgenic lines expressing the mutant variants of human TDP43 protein, neither using lentiviral transgenesis nor standard pronuclei microinjection method. Possibly, the stable expression of mutant forms from early developmental stages is lethal and creating those lines could be possible using inducible promoter system.
56. Fecal DNA for genotyping transgenic animals Zhidong Chen1, Rebecca Mantha2, Janice Chen3, Orazio Slivano4, Hideko Takahashi5 1 MultiTarget Pharmaceuticals LLC, Salt Lake City, Utah, USA, 2Atomic Energy of Canada Ltd, Chalk River, Ontario, Canada, 3Johns Hopkins University, Baltimore, Maryland, USA, 4University of Rochester School of Medicine and Dentistry, Rochester, New York, USA, 5National Institutes of Health, Rockville, Maryland, USA
Stool contains numerous sloughed intestinal epithelial cells and its collection does not cause distress or pain to the animals, making it an attractive DNA source for non-invasive genotyping of transgenic animals. In 1999, Broome and colleagues first reported the use of mouse fecal DNA for animal genotyping. Most IACUC guidelines cite the Broome’s method and encourage investigators to consider its use for genotyping transgenic animals whenever possible. However, fecal DNA genotyping has essentially not been accepted by the transgenic community; in fact, its use for genotyping of transgenic
Transgenic Res (2011) 30:1139–1189 animals has been reported in only a few articles since 1999. There are several obstacles to the adoption of fecal DNA genotyping. First, feces contain an abundance of PCR inhibitors that may be co-purified with fecal DNA and result in failed PCR amplification. Secondly, existing methods for fecal DNA extraction are either costly or involve the use of toxic organic solvents, which are impractical for routine uses. Finally, fecal DNA contains a large excess of bacterial DNA and there is a concern of its interference with the analysis of transgenes. To overcome these obstacles and facilitate the use of fecal DNA genotyping, we developed a multi-functional reagent, AquaStool, for fecal DNA extraction and PCR inhibitor removal. AquaStool can lyse the cells, extract the DNA, and prevent fecal PCR inhibitors from binding to fecal DNA in a single step. In the present study, we obtained fecal samples of transgenic mice from collaborating investigators in other institutions, extracted the fecal DNA with AquaStool, and performed PCR genotyping. Subsequently, we reported our fecal DNA genotyping results to our collaborators to compare with their tail DNA genotyping results. We found that both the transgenic and wild-type animals were identified identically by fecal DNA and tail DNA genotyping. The fecal DNA genotyping specificity and sensitivity were 100 and 98% respectively (n = 75). We showed that AquaStool fecal DNA extraction and genotyping was simple, reliable, and costeffective. Fecal sampling is not limited by the animal’s age and sampling frequency, and after air-drying extra fecal samples can be stored at room temperature for later genotype verification. In addition to animal genotyping, fecal DNA may be used for diagnosis and management of gastrointestinal infections in the animals. This simple non-invasive fecal DNA extraction and genotyping method not only improves the welfare of transgenic animals, but also gives researchers a new opportunity and option to identify and manage transgenic animals.
57. Derivation of rat embryonic stem cells and generation of protease-activated receptor-2 knockout rats Satoshi Yamamoto, Mitsugu Nakata, Reiko Sasada, Yuki Ooshima, Takashi Yano, Tadahiro Shinozawa, Yasuhiro Tsukimi, Michiyasu Takeyama, Yoshio Matsumoto, Tadatoshi Hashimoto Takeda Pharmaceutical Company Limited, Fujisawa City, Kanagawa, Japan During 2008–2010, remarkable achievements have been reported for the production of knockout (KO) rats. One of them is the derivation of authentic embryonic stem (ES) cells from rat blastocysts using a novel culture medium containing GSK3 and MEK inhibitors (2i medium). In this study, we report the establishment of the gene-targeting technology via homologous recombination in rat ES cells. First, we generated the germ-line competent ES cells from Dark Agouti rats using 2i medium. These ES cells have alkaline phosphatase activity and express pluripotent markers. They differentiate into cardiomyocytes in vitro. Furthermore, they can produce chimeras with high ES cells contribution when injected into blastocysts. Next, we introduced the targeting vector with a neomycin resistant gene driven by the CAG promoter to disrupt
1163 protease-activated receptor-2 gene (Par-2). After 7-day drug selection, we obtained 489 neomycin resistant colonies. Following screening by PCR genotyping and qPCR analysis, we confirmed 3 homologous recombinant clones. These recombinant clones resulted in chimeras that transmitted the Par-2 targeted allele to offspring. Par-2 KO rats showed the loss of Par-2 mRNA expression in their stomach and the lack of the smooth muscle relaxation in aorta via PAR-2 function by pharmacological test. Rats are widely used in biomedical research because of a lot of advantages (body size, physiology, pathology, toxicology and so on) which the mouse does not have. The establishment of gene-targeting technology in rat ES cells will be a valuable tool for the production of human disease models and drug discovery.
58. Detection of Neo in copulation plugs from Sox3 polyanine targeted chimeras Sandra Piltz, James Hughes, Paul Thomas University of Adelaide, Adelaide, South Australia, Australia We are studying families with an inherited form of pituitary growth hormone deficiency termed X-linked Hypopituitarism (XH) in which only boys are affected. In addition to poor pituitary function, some boys with XH also have mild intellectual disability. We have previously shown that XH is due to an unusual change in the SOX3 gene in which the number of consecutive alanine residues is increased above a critical threshold (polyalanine expansion mutations). Similar mutations have recently been identified in several other genes that also cause severe birth defects. However, little is currently known about how polyalanine expansion mutations cause these disorders. Here the development of a mouse model is described with particular focus on an unexpected fertility defect in male chimeras. The Sox3 locus was targeted with a 10 kb construct that carried the polyalanine mutation in Sox3 and a downstream Neo cassette downstream. ES cell clones were selected based on Neo integration of the Sox3 locus. These clones were further divided into those with and without the polyalanine mutation. Clones without the mutation served as controls. The only chimeras that transmitted the agouti coat colour through the germline were from the R1 parental cell line (4 males) and targeted clones without the mutation (2 males). This suggested the polyalanine mutation was affecting fertility. To test whether this defect was at the level of sperm production or sperm function, the following technique was employed: Identification of germline competent chimaeras by copulatory plug genotyping, Stephen Wilson & Steven A. Sheardown. Transgenic Research, 2011, Volume 20, Number 2, Pages 429–433 Copulation plugs generated by high percentage chimeric males were collected. Genomic DNA was extracted from the plugs and genotyping PCRs were performed to detect the presence of Neo (indicating the presence of targeted sperm). One of the chimeras (60% agouti) has so far produced 50 black pups. A low level of Neo from 3 copulation plugs from this male has been detected, which implies that there may be a small percentage of Neo positive sperm being produced.
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Breeding from this chimera continues and mid-gestation embryos are also being assayed for Neo by PCR as they may be embryonic lethal.
59. Human heavy-chain antibodies from triple knockout transgenic mice 1
1
2,3
Joyce L. Young , Yanjing Zhang , Almudena Fernandez , H. Lorraine Thompson1, Bryan Edwards1, Philip D. Hayes1, Yumin Teng1, Brian McGuinness1, Sue Edwards4, Rob Norrington4, Lluis Montoliu2,3, Mike Romanos1 1
Crescendo Biologics Ltd., Babraham Research Campus, Cambridge, UK, 2Centro Nacional de Biotecnologia (CNBCSIC), Madrid, Spain, 3CIBER de Enfermedades Raras (CIBERER), ISCIII,, Madrid, Spain, 4Research Models & Services UK, Charles River, Manston, UK We describe here the generation of transgenic mice that can produce antigen-specific human heavy chain-only antibodies. Heavy chain-only antibodies (HcAb) occur naturally in camelidae and unlike conventional immunoglobulins, encode their antigen specificity entirely within a single binding domain (VH region). The VH domain is the smallest portion of an immunoglobulin that retains target specificity and has many properties that make it highly attractive as a therapeutic agent. Pronuclear injection of fertilised oocytes was used to produce 3 transgenic mouse lines using a purified yeast artificial chromosome (YAC) of *200 kb in size. The YAC contains a mini human Ig-locus in germ line configuration comprising a limited number of VH gene segments, the entire set of D and J genes and a single human gamma constant region, adapted to facilitate HcAb production. From 79 pups that were born after transfer of manipulated oocytes, 6 transgenic animals were obtained 3 of which contained the full length, intact YAC as verified by breeding and ALU repeat Southern blot analysis. Genomic Q-PCR and Southern blot probing were consistent with one line having a tandem integration of the transgene and the other two lines bearing single copy integrants. The transgenic mice were cross-bred with our first-in-class knock-out animals from which endogenous immunoglobulin heavy and light chain expression had been silenced through large-scale genomic deletions or neomycin cassette-mediated disruption of coding sequences. These KO animals provide a clean background devoid of mouse antibodies. Thus, mice with the mini-human heavy chain transgene and a further 3 homozygous targeted KO loci were obtained. In all lines, the transgene underwent somatic rearrangement to produce a human heavy chain-expressing B-cell repertoire, with human HcAb detectable in non-immune animals. Following immunisation, an antigen-specific HcAb response was apparent by serum ELISA. Because the immune response was mediated by single binding domains rather than relying on paired heavy and light chains for specificity, the antigenreactive VH sequences could be cloned, expressed and enriched using in vitro display techniques without having to rely on hybridoma production. Despite the limited number of VH gene segments available from this prototype mini-heavy chain locus, diverse sequences were recovered.
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The present transgenic mice provide a blue print for generating candidate-quality human VH fragments with high affinity, stability and solubility that have been matured in vivo.
60. Targeted transgene integration in mouse embryos using ‘‘open source’’ zinc finger nucleases Mario Hermann1,4, J. Keith Joung2, Morgan L. Maeder2, Burkhard Becher3, Kurt Bu¨rki1, Adriano Aguzzi4, Thorsten Buch5,3, Pawel Pelczar1 1 Institute of Laboratory Animal Sciences, University of Zurich, Zurich, Switzerland, 2Molecular Pathology Unit and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA, 3Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland, 4Institute of Neuropathology, University of Zurich, Zurich, Switzerland, 5Institute for Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, Munich, Germany
Gene targeting by homologous recombination (HR) in embryonic stem cells is a technique widely utilized for creating mouse models in biomedical research. Recently described Zinc Finger Nucleases (ZFNs) enable the precise, sequence-specific introduction of double strand breaks (DSBs) in the target genome. Such DSBs can subsequently be repaired by either non-homologous end joining (NHEJ) or HR, turning ZFNs into powerful tools for genome engineering. We have used publicly available ZFNs generated by the Zinc Finger Consortium to perform gene targeting directly in mouse oocytes. A single ZFN pair specific for the mouse Rosa26 locus was used to disrupt the target sequence by inducing NHEJ events at the cleavage site. Furthermore, the same ZFN pair facilitated HR-mediated transgene integration upon co-injection with a Rosa26 targeting vector. This efficient one-step protocol can be applied to generate knock-in as well as knock-out animals independent of stem cell technology and intellectual property restrictions.
61. Deciphering the genetic background of hormonedependent mammary gland cancer in the rat model by Sleeping Beauty Transposon Mutagenesis Sanum Bashir1, Elena Popova1, Lajos Mates2, Boris Jerchow1, Ulrike Ziebold1, Michael Bader1, David Largaespada3, James Shull4, Zoltan Ivics1, Zsuzsanna Izsvak1 1
Max Delbru¨ck Centre for Molecular Medicine, Berlin, Germany, 2Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary, 3University of Minnesota, Minnesota, USA, 4McArdle Laboratory of Cancer Research, Wisconsin, USA The Sleeping Beauty (SB) transposon system is a two component system and is composed of a transposase source and a transposon vector flanked by inverted terminal repeats. SB has been developed as a technology platform for vertebrate
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genetics with application areas including gene therapy, transgenesis, somatic mutagenesis (cancer research), and germline mutagenesis for gene discovery. Here we are using a mutagenic SB transposon T2/Onc and hyperactive transposase SB100X to investigate the complex genetic background of hormone induced mammary cancer in the rat model, where the development of mammary cancer is similar to human as it is also estrogen-dependent most of the time. We have generated transgenic rat lines for the mutagenic Sleeping Beauty transposon T2/Onc (mutator) as well as transgenic rats which express the hyperactive transposase SB100X in the mammary gland (Jump Starter). Insertional Mutagenesis with SB in both germline and somatic tissues has been approached with donor loci containing many copies of the transposon vector in the form of concatomeric arrays. However transposition out of multicopy donor loci also triggers additional genome rearrangements and therefore complicates seriously the analysis of such screens. The availability of hyperactive SB100X system enables us to redesign our genetic screens as the SB100X could be able to remobilize single copy transposon donors more effectively. Instead of rat we are using mouse as the animal model for the single copy remobilization of Sleeping Beauty in the mouse germline.
63. Multi-fragment and multi-site recombinasemediated cassette exchange in mouse ES-cells for F0generation mice
62. Generation of C57BL/6 NTac-Atm1.1Arte Tyrtm1Arte albino mice as embryo host for injection of C57BL/6 N ES cells 1
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Branko Zevnik , Maria da Silva Ramalho , Heidrun Kern , Nathalie Uyttersprot1, Gerald Bothe2, Ana V. Perez2, Gunther Kauselmann1 1
will be inherited by the progeny if not actively screened against and removed from the colony. In this study, we have combined a loss-of-function point mutation at the tyrosinase locus with a reversion of the nonagouti locus to a dominant agouti (A) allele in the C57BL/6 N substrain genetic background. Homozygous C57BL/6 NTac-A tm1.1Arte Tyrtm1Arte double mutated albino mice are used as blastocyst donors for C57BL/6 NTac ES cell injections. Resulting black/albino chimeras can be mated to strain C57BL/6 NTac and still allow recognition of germline transmission in G1 offspring by coat color. In non-germline transmitted offspring, the inherited heterozygous Tyrtm1Arte allele will have no phenotypic effect on coat color, whereas the restored dominant agouti allele leads to agouti coat color. Germline transmission, on the contrary, will produce black C57BL/6 N offspring, without compromising the genetics of the employed ES cell substrain background. Furthermore C57BL/6 NTac-Atm1.1Arte Tyrtm1Arte mice should respond equally well to superovulation as wildtype C57BL/6NTac. The availability of a combined albino/agouti donor mouse strain should aid in the streamlined production of genetically modified mouse mutants from C57BL/6N ES cells keeping the C57BL/6N nature of the substrain intact.
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TaconicArtemis, Cologne, Germany, Taconic, Hudson, NY, USA An increasing number of genetically engineered models are generated using C57BL/6 N embryonic stem (ES) cells, as this is the standard inbred mouse strain for many types of research including large scale gene knockout consortia. The ideal embryonic host for C57BL/6 ES cell injections should be co-isogenic and chimeras preferably identifiable by coat color. There are some C57BL/6 albino strains that are used, but most commonly Balb/C mice, have been reported to provide a suitable environment for participation of ES cells in development and colonization of the germline. Chimeras are identified by a mix of black and white patches. However, Balb/ C mice respond poorly to superovulation and resulting low yields of blastocysts for injection commonly compromise full usage of injection capacities. The increase in numbers of females for superovulation is counterproductive due to higher expense and counteracting the 3Rs animal welfare approach dedicated to the reduction of animal use in research. Available albino C57BL/6 strains have been obtained due to spontaneous mutations in the tyrosinase locus. These provide a suitable alternative but due to the recessive nature of the Tyrc mutation, coat-color detection of ES-cell contribution in germline transmitted G1 offspring requires breeding of chimeras to albino C57BL/6. More importantly, a pure genetic C57BL/6 substrain background may only be maintained by subsequent backcrossing. Furthermore the mutated Tyrc allele
Nils Lindstro¨m1, Elise Cachat2, Katherine Norrby1, Paul Devenney1, Joan Slight1, Weijia Liu2, Jamie Davies2, Nick Hastie1, Peter Hohenstein1 MRC Human Genetics Unit, Edinburgh, UK, 2University of Edinburgh, Edinburgh, UK
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The time-scale and costs that are associated with performing genetic experiments using multiple crosses of transgenic mouse lines are prohibitive for many labs, and lead to a big wastage of those animals that are generated. To circumvent these problems we are taking a three-pronged approach where we aim to (1) increase the number of genetic combinations that can be analysed by combining them in ES-cells; (2) minimise the time taken to obtain samples by eliminating mouse-breeding; (3) maximise the data obtained from of each sample using timelapse microscopy and organ culture techniques. Firstly we have developed a vector system where we can easily produce unlimited different genetic constructs (overexpression and/or knockdown) using the same multi-fragment Gateway cassette vector. This multi-Gateway cassette is designed to be used for uC31-integrase promoted recombinase-mediated cassette exchange (RMCE) to create transgenic ES-cell lines. We call this vector Locus-EXchange in Es cells (pLEXIE). To increase the potential number of alleles we can combine, we are making ES-cells that carry 3 different and independent selectable uC31 integrase RMCE sites (3xuC31RMCE ES-cells). This would allow, for example, the generation of ES cells that carry a tissue-specific expression cassette for any Cre recombinase variant, a ubiquitously expressed loxSTOP-lox controlled miRNA-based knockdown or overexpression construct, and a reporter construct for a pathway under study; all based on the same pLEXIE vector and targeted to
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1166 different RMCE sites. In parallel we are developing an additional construct where pLEXIE is in itself selectable (pSeLEXIE) for piggyBac transposition in vitro. Secondly, to minimise the time taken to arrive at these combinations we will ensure that the 3xuC31-RMCE ES-cells are suitable for efficient F0-generation mouse production using 2i-medium and ES-cell/8-cell embryo aggregations and/or with laser-assisted injections. We are currently comparing the efficiency of these different methods. Efficient F0 protocols would allow direct phenotyping of the combinations of alleles made via pLEXIE without any subsequent breeding. Thirdly, we will use these 3xuC31-RMCE F0-mice to study organ development using time-lapse microscopy and in vitro organ culture. This combination will increase the amount of data that can be obtained from a single embryo. The modular nature of pLEXIE constructs, for instance in the use of specific fluorescent reporters, will maximise the flexibility in adapting alleles for a specific scientific question. LEXIE in 3xuC31-RMCE ES-cells and SeLEXIE with F0generation mice will allow for large-scale genetic combinations using less time and a smaller budget compared to current genetic techniques.
64. Neuronal overexpression of ICER in transgenic rat model; the impact on seisure susceptibility and glucose metabolism Agata Klejman1, Katarzyna Bieganska1, Marcin Wawrzyniak1, Dorota Owczarek1, Marcin Balcerzyk2, Michal Dabrowski1, Pawel Lisowski3, Leszek Kaczmarek1 1 Nencki Institute for Experimental Biology, Polish Academy of Science, Pasteur 3 Street, Warsaw, Poland, 2 Instituto Pluridisciplinar Universidad Compultense de Madrid, Paseo Juan XXIII n. 1 28040 Madrid, Spain, 3 Polish Academy of Sciences Institute of Genetics and Animal Breeding Department of Molecular Biology, Postepu 1, Jastrzebiec, Poland
The cAMP-responsive element binding protein (CREB) pathway is one of the most frequently studied in neuronal cells. CREB is known as a central molecule regulating protection and survival of neurons. Researchers consider CREB also as a critical component of molecular long-lasting memory control. In our study we decided to employee the physiological CREB antagonist ICER (inducible cAMP early repressor), as a tool to downregulate CREB. Therefore we have created the transgenic rat model with overexpression of the ICER II gene under the control of Synapsin I promoter, which limits the overexpression of the transgene only to the brain. Transgenic animals didn’t show any major differences neither in brain anatomy or morphology, nor in the behavioral tests. However we found, that ICER II overexpression diminishes susceptibility to seisures in the PTZ kindling model. To find the mechanism of up normal seizure phenotype we took advantage on the microarray analysis made in normal versus PTZ-evoked seisures rats. We found deregulation of a large number of genes, especially after PTZ treatment. In control conditions we found differences e.g. in genes connected with glucose metabolism between transgenic and control hippocampal mRNA. The results were conformed with [18-F] FDG PET
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Transgenic Res (2011) 20:1139–1189 measurement of glucose metabolism; we found it decreased in tgICER rats in the cerebellum, hippocampus, auditory cortex, amygdala, and medulla. 65. Toxin Receptor Cell Knockout (TRECK) for mouse models of human diseases Hiromichi Yonekawa1, Kunie Matsuoka1, Toyoyuki Takada2, Hiroshi Shitara1, Choji Taya1, Rie Ishii1, Kenji Kohno3, Yoshiaki Kikkawa1 1
Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa Setagaya-ku Tokyo 156-8506, Japan, 2 National Institute of Genetics, 1111 Yata Mishima 411-8540 Shizuoka, Japan, 3Nara Institute of Science and Technology, 8916-5 Takayamacho Ikoma 630-0192 Nara, Japan To generate model mice for human diseases with a new method of gene disruption, we have developed a new technology named the Toxin Receptor Cell Knockout (TRECK) method, for specific ablation of cell lineages (Saito, M. et al., 2001). The principal of this technique is very simple: a minigene is constructed with human diphtheria toxin receptor (hDTR) cDNA and a tissue-specific promoter, such that hDTR is driven by the promoter. Then, the minigene is microinjected into pronucleus-stage eggs of a suitable inbred strain. The resultant transgenic (Tg) mice are sensitive to DT only in the tissue/cell expressing the introduced promoter as a transgene. Sensitivity to DT greatly differed between mice and humans at a ratio of 1:1,000–10,000. After DT administration to the Tg mice, the tissue/cell is disrupted by DT through apoptosis, which does not cause any inflammatory events in or near the disrupted tissue/ cell. We have established several lines of Tg mice with hepatitis (Saito, M. et al., 2001), hyperglycemia, hairless (Takada, T. et al., 2006), and renal tubular nephritis (Sekine, M. et al. 2011), basophil- and eosinophil-less Tg mice. For example, we have established co-isogenic TRECK-Tg mice using the insulinpromoter in the background of C57BL/6 J and BALB/c-scid/ scid. Only 1 founder mouse, the progeny of which exhibited hyperglycemia, was obtained in each genetic background, suggesting that the transgenes are properly expressed or not expressed in the target beta cells in pancreatic islets. However, a few other founder mice showed ectopic expression although the cells have not yet been identified; suggesting that robustness of transgene expression in target cells is not high. The robustness of tissue-specific expression was compared in 2 types of transgenic constructs: (1) direct ligation of the insulin promoter DNA fragment and hDTR cDNA (cDNA-transgenesis) and (2) BAC, which is manipulated in vitro by recombineering (recombination-mediated genetic engineering: BAC-transgenesis). As expected, BAC-transgenesis is superior to cDNA-transgenesis, because all the mice generated by BAC-transgenesis exhibited hyperglycemia. Our most successful examples are the establishment of hairless NC/Nga mice, which is a model for human atopic dermatitis (AD), and will be useful in the development of therapeutic agents as well as to disclose the causative factors for human AD (Takada, T. et al., 2006). Saito, M. et al., Nature Biotechnol. 19, 746–750, 2001. Takada, T. et al., Transgenic Res. 17, 1155–1162, 2008. Sekine, M. et al., Transgenic Res. in press, 2011.
Transgenic Res (2011) 30:1139–1189 66. Recombinase mediated promoter switching enables two different transgene expression levels to be achieved in a single line of targeted transgenic mice Hamid Dolatshad, Rebeca Diaz, Daniel Biggs, Chris Preece, Ben Davies Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK The traditional methodology used for the generation of transgenic models, pronuclear injection, results in the random integration of the transgenic construct at varying copy number within the genome. This uncontrolled integration event can lead to mutation of endogenous sequences and position effects influencing or even extinguishing the transgene expression. Several ‘‘targeted transgenic’’ methodologies have been established to overcome these problems which allow transgenes to be introduced at single copy into defined chromosomal positions known to be permissive for transgene expression. Targeted transgenesis result in dependable and predictable expression but only one level of expression, as defined by the promoter used, can result. In contrast, random integration methodologies can lead to the generation of multiple lines of transgenic mice, each potentially having a different transgene expression level. When performing functional analysis in transgenic mice, it can be useful to obtain a strong overexpressing line together with a weaker expressing line so that the severity of phenotype can then be correlated with the level of transgene expression. We report the development of a novel targeted transgenic methodology in ES cells which combines the advantages of site specific integration with the flexibility of changing the transgene expression level. A PhiC31 integrase cassette exchange system has been adopted to allow the site specific integration of a transgene whose expression is initially silenced. Upon the action of Cre recombinase, the transgene expression becomes linked to the strong promoter, allowing conditional activatable transgene overexpression. The action of Flp recombination substitutes the strong CAG promoter for the weaker Rosa26 promoter allowing the effects of more moderate transgene expression to be investigated. We report preliminary proof-of-concept studies using fluorescent reporters and coat colour markers as transgenes and explore the potential for the system to be used to achieve different levels of knock-down using miR30/shRNA constructs. The technology has been established using JM8F6 cells to enable the models to be generated on a pure C57BL/6 N genetic background.
67. Using the whitten effect in pseudo-pregnant mouse production Natasha Bacarro, Xin Rairdan, Rhonda Wiler
1167 by both our Microinjection Lab and IVF & Cryopreservation Lab. Efficient management of a large embryo-recipient production colony such as this can be complex and labor intensive. We are seeking to streamline the number pseudopregnant females produced each day, as well as minimize the labor applied to identifying each pseudo-pregnant female (plug checking). The synchronizing of estrous by the exposure of females to male pheromones is known as The Whitten Effect. Here, we are testing a simple way to induce this effect by transferring a small amount of bedding from a male cage into the female cage. Results from the first day of plug checks were compared for females that were set-up conventionally and females that received a male pheromone stimulus beginning 2 days prior to set-up. An average of 34.4% of the pheromone-primed females plugged on the first day, a fourfold increase over the 8.5% average first-day plug rate through conventional breeding. These data support the use of The Whitten Effect to efficiently manage pseudo-pregnant mouse production.
68. Comparison of Balb/c and B6-albino mice as blastocyst hosts for injection of C57BL6/n-derived C2 ES cells Tuija Alcantar, Xin Rairdan, Rhonda Wiler Genentech, San Francisco, USA Two commonly used embryo donor strains BALB/cAnNTac and B6(Cg)-Tyrc-2 J/J (B6-albino) were compared, side-byside, to assess their efficiency in generating germline-transmitting chimeras using the C57BL6/N-derived C2 ES cell line through blastocyst injection. A total of 27 constructs consisting of 92 clones were injected into blastocysts of each strain. Resulting chimeras from a total of 62 of the clones injected were set up for germline breeding until the first germline pups were identified from either background strain. Blastocyst yield, birth rate, chimeric rate, high-percentage male chimera rate (above 50%), and germline transmission rates of both clones and all chimeras mated were assessed. Overall chimeric rate was the same for both strains (48%). Balb/c hosts resulted in higher birth rate than B6-albino hosts (35 and 28%) and higher germline transmission rate of all chimeras mated (17 and 14%), respectively. B6-albino hosts resulted in a higher blastocyst yield per donor than Balb/c hosts (3.6 and 2.5), better highpercentage male chimera rate (65 and 51%), and higher germline transmission rate of clones (74 and 63%), respectively. In addition, transfer of 100 injected blastocysts yields an average of 1.5 germline pups using Balb/c donors and 1.2 germline pups using B6-albino donors. We found B6-albino strain to be a more cost-effective blastocyst donor for C2 ES cell injections based on the equivocal results between Balb/c and B6-albino strains, the higher blastocyst yield, and recently lowered cost of B6-albino mice.
Genentech, San Francisco, USA Transgenic mouse creation and cryopreserved embryo resuscitation is highly dependent upon the transfer of embryos into the reproductive tract of a pseudo-pregnant female at the corresponding stage. Our high-throughput, outbred, in-house CD1 colony produces all of the pseudo-pregnant females used
69. Access to novel genetically modified mice: tool strains and models of human disease S. Rockwood, C. Lutz, M. Sasner, L. Donahue The Jackson Laboratory, Bar Harbor, ME, USA
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1168 Genetically modified mice have greatly contributed to our understanding of basic biology and disease processes. Many of these mice are categorized as disease models, and others as ‘tool strains’. Tool strains include mouse lines that offer researchers a wide array of utilitarian functionality such as detection of gene expression, site specific recombinases (often conditional), cell-specific ablation, etc. In order to facilitate access to such mice, the Mouse Repository at The Jackson Laboratory was established to serve as a centralized resource for the archiving and distribution of genetically engineered mice at a high health status. As the number and complexity of alleles increase, so does the importance of providing adequate information to facilitate their selection and use. New mouse strains with applications in a wide range of disciplines continue to be made available to the scientific community through an online database. Most of the 500–600 new mouse lines added to the Repository annually are accompanied by a robust set of supporting data that aid researchers in the selection of appropriate lines for the desired application. Among the new mice available are Batf3 knockout mice which feature impaired development of splenic CD8a+ conventional dendritic cells. Conditional mutants continue to represent a significant portion of the new alleles reposited; a floxed Rptor strain makes possible the examination of a central node of the mammalian pathway that coordinates cell growth with the availability of nutrients, energy and growth factors. Serving as companion strains to floxed models are a wide range of cre recombinase expressing strains; newly added is a transgenic line expressing cre under the direction of a Adipoq promoter, allowing researchers to control cre-mediated recombination effectively in white and brown adipose tissues. Another knock-in model at the Sst locus expresses cre in dendritic inhibitory interneurons. Popular among the new tool strains are a growing number of lines that incorporate channelrhodopsin technology, and ‘‘R26R-Confetti’’ mice which serve as a stochastic multicolor Cre recombinase reporter expressing multiple fluorescent proteins from a single genomic locus. Additionally, diseasespecific resources are included within the Repository that offer a wide range of Alzheimers, Parkinson Type I Diabetes and other models of human disease. Donating a strain to the Repository fulfills the NIH’s requirements for sharing of mice. Researchers wishing to have strains considered for inclusion in the Repository are encouraged to submit their strains at: http://www.jax.org/donate_ a_mouse
70. Understanding the gene function: a global Cyp4 3 1 gene knockout studies in mice Himanshu Kharkwal1, Natali Daw3, Ian Duce1, David Bell3 1 School of Biology,University of Nottingham, Nottingham, UK, 2European Chemical agencies, Helsinki, Finland, 3 Ozgene, Australia, Australia
A novel Cytochrome P450, Cyp4x1 gene that encodes a 507 amino acid polypeptide and share 41–51% identity with members of the CYP4 subfamilies was identified in EST databases by similarity to a conserved region in the C-helix of the CYP4A family. Studies suggest that Cyp4x1 is mainly
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Transgenic Res (2011) 20:1139–1189 expressed in neurons in different regions of the CNS, e.g. brain stem, hippocampus, cortex, and cerebellum as well as in vascular endothelial cells [1]. Recently, it has been found that Cyp4x1 is involved in anandamide metabolism in the brain [2]. Further research in Cyp4x1 showed that, it converts arachidonic acid (AA) to regioisomers of epoxyeicosatrienoic acid (EETs). Endogenous EETs (Cyp4x1 products) have also shown antinociceptive effects in mouse models.To understand the putative role of Cyp4x1 gene, we generated Cyp4x1 global knockout mice using the Cre/lox recombination system. The loss of the Cyp4x1 gene will be correlated with subsequent changes of biological functions in mice. This study aims to understand the effect of knocking out Cyp4x1 gene on the metabolism of endogenous and xenobiotic compounds and postnatal development. Cyp4x1 null mice may also serve as a pain and inflammation model better to understand pain signaling and provide potential in discovering non-narcotic Cyp4x1 based analgesic drugs for pain management. 1. Al-Anizy, M., et al., Cytochrome P450 Cyp4x1 is a major P450 protein in mouse brain. FEBS J, 2006. 273(5): pp. 936–47. 2. Stark, K., M. Dostalek, and F.P. Guengerich, Expression and purification of orphan cytochrome P450 4X1 and oxidation of anandamide. FEBS J, 2008. 275(14): p. 3706–17.
71. Confirming pregnancy via ultrasound imaging significantly reduces the number of animals required to provide time pregnant genetically altered mice for studies Melanie Domeyer, Ryan Ybbara, Micheal-Anne Sowick, Martin Garcia, Jose Diaz, Vida Asghari, Rhonda Wiler Genentech, South San Francisco, CA, USA In mice, studies that involve the harvest of embryonic tissue at specific developmental time points requires the generation of timed-pregnant females. The traditional way to generate timepregnant females is to check for a copulatory plug in the morning as evidence that mating has occurred. Due to variability in male fertility, approximately 40% of the females observed to have a plug are found not to pregnant at the time of the experiment. This created a major impact to our collaborators’ experimental timelines and wasted difficult to obtain reagents including the complex mutant mice that we provide for these studies. To address this problem, a high throughput procedure for imaging plugged females via ultrasound with E dates of 7.5–12.5 days was developed. The process involved anesthetizing the animals with isoflurane, chemical removal of abdominal fur, imaging the animals on a heated stage, and monitoring for recovery from anaesthesia. We found that an experienced technician can accomplish an imaging session in 6 min with a high degree of accuracy. In addition, the implementation of ultrasound imaging to confirm pregnancy significantly reduced the number of animals used due to the transfer of non-pregnant females and provides a more reliable value added service.
Transgenic Res (2011) 30:1139–1189 72. The development of a device for fluorescent protein imaging and screening Hsiao-Hui Joyce Chang1, Stephen Yang2, Si-Tse Jiang1, Chi-Kuang Leo Wang1 1
National Laboratory Animal Center, National Applied Research Laboratories, Taipei, Taiwan, 2Lumos Technology Co., Ltd., Taipei, Taiwan Reporter genes are widely used in current animal models in research and analysis. Among the reporter genes, fluorescent proteins are most commonly used due to their availability, stability and diverse color of chose. However, observation and data keeping often become a difficult task due to the pricy equipment, suboptimal photographic quality, and the lack of flexibility for imaging of different fluorescent wavelengths. Not to mention the difficulties in simultaneously observation object with multiple fluorescent wavelengths. To overcome such obstacles, an imaging device was designed using macro ring light with switchable twin light sources. The macro ring light is compatible with most high-end digital cameras and some internal focusing macro DSLR lenses. To accommodate diverse needs for different fluorescent wavelengths, both light sources can be customized to specific excitation wavelengths. The device is powered by battery/AC duel design to provide flexibility and mobility. For the needs of frequent commute between barriers, the device is also designed to withstand repetitive sterilization. In addition to the application in fluorescent protein imaging, the device can also be used to detect body fluid in forensic investigation. 73. Overcoming difficulties in the generation of functional knockin/knockout humanized mouse models John Wiseman1, Pernilla Gregersson1, Meint Schuelke1, Anna Ramne1, Mohammad Bohlooly1, Fernanda Pilataxi2, Chris Morehouse2, Philip Brohawn2, Patrick Strout3, Song Cho3 1
AstraZeneca R&D, ATCG, Mo¨lndal, Sweden, MedImmune, Department of Translational Sciences, Gaithersburg, USA, 3MedImmune, Department of Preclinical Oncology, Gaithersburg, USA
1169 Our standard method for generating these mice is to place the human cDNA downstream of the mouse promoter elements (ATG fusion). When adopting this strategy there is a possibility that the mouse promoter fails to express the human form in a manner that compensates for loss of the mouse form. Thus to understand more around the biology of our KIKO’s we have developed a new set of models focussed on DLL4, a target where we have previously failed to generate a humanized form. DLL4 plays a central role in embryonic angiogenesis and at sites of pathological angiogenesis in the adult. DLL4 regulates vascular development and antagonists have been shown to inhibit tumor growth in preclinical rodent models of cancer. Thus an antibody specifically targeting DLL4 would be anticipated to inhibit tumour growth. New models include: (a) Expressing a chimeric cDNA consisting of mouse transmembrane/intracellular and human extracellular domains; (b) Expressing the chimeric cDNA from a strong, constitutively active promoter; (c) Random integration of a full length human Dll4 BAC on a DLL4 KIKO homozygote background; (d) A human genomic DLL4 replacement containing 15 kb of human promoter driving the complete exonic/intronic and UTR regions of human DLL4. By adopting a broader biologic strategy, we have successfully generated humanized DLL4 KIKO models using two of the above strategies. This has led to a greater understanding of critical biological aspects to consider when developing humanized KIKO models.
74. Mitochondrial imaging in tissues by using mtDsRed2-Tg mouse strain Junya Yamaguchi1,2, Satoshi Nishiyama1,2, Midori Shimanuki1, Tomio Ono1, Akitsugu Sato2, Kazuto Nakada2, Jun-Ichi Hayashi2, Hiromichi Yonekawa1,2, Hiroshi Shitara1,2 1 Laboratory for Transgenic Technology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan, 2 Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki, Japan
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Knockin/knockout (KIKO) models to humanize mice by insertion of a human gene to replace an endogenous mouse gene are widely used to support biopharmaceutical research. In cancer research, where one aim is to develop antagonistic antibodies against targets that can block tumour growth by control of tumour vascularisation, this approach has been key to generate in vivo proof-of-concept efficacy data. However, such studies regularly require mouse models where only the human version of the target molecule is present. As many KO mice in this area of research are homozygous lethal, viable KIKO models are dependent upon the generation of homozygous mice expressing only the human molecule, compensating for loss of the mouse form. However, in a number of cases this strategy has failed to generate homozygous mice even though the human form is expressed and the endogenous mouse locus is correctly targeted. Different biological explanations for this will be discussed.
Mitochondria are important organelles that play important roles in cell functions such as ATP production, calcium storage, and apoptosis. In living cells, mitochondria frequently change in terms of their morphology, fusion, fission, and transportation, and these dynamics are probably associated with their functions. Abnormalities in mitochondrial dynamics give rise to pathogenic phenotypes. Therefore, mitochondrial imaging of inherent cells of tissues can be a useful technique. For mitochondrial imaging, we established a transgenic (Tg) mouse strain, mtDsRed2-Tg, in which red fluorescent protein DsRed2 was localized in the mitochondria. The mitochondria of mtDsRed2-Tg mice in various tissues exhibited strong fluorescence intensity under a confocal laser microscope and fluorescence microscope. We found that mitochondrial morphologies differ largely among tissues; for example, mitochondria in the acinar cells of pancreas were larger than those in the islets of Langerhans. To examine the localization pattern of mitochondria and the mitochondrial transcription factor A (TFAM), mtDsRed2-Tg mice were mated with another Tg strain that expresses the Tfam-EGFP fusion gene.
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1170 Consequently, red and green fluorescence were simultaneously detected in all examined tissues of the double Tg mice. Recently, several types of Tg mouse strains expressing the EGFP fusion gene have been established. By crossing a mouse strain that expresses the EGFP fusion gene with our Tg mice, we were able to analyze the interaction between mitochondria and the EGFP fusion proteins. Therefore, we conclude that mtDsRed2-Tg mice are useful tools for further studies on mitochondrial function.
75. 3i method is effective for improving germline differentiating potency in mouse embryonic stem cells Hiroshi Kiyonari1, Mari Kaneko1, Kazuki Nakao1,2, Shinichi Aizawa1 1 RIKEN CDB, Kobe, Japan, 2The University of Tokyo, Tokyo, Japan
The C57BL/6 mouse is the most standard strain used in mouse genetics. Recently, we reported that the 3i medium developed by Austin Smith’s group (2008) was extremely instrumental in establishing and culturing germline-competent ES cells in the C57BL/6 N strain (Kiyonari et al., 2010). Here we show that some of the less germline-competent ES cells established with other media showed higher germline-competency after they were cultured with 3i medium at least for 3 days. This result indicates that the 3i medium is effective not only for establishing ES cells, but also for obtaining germline chimeras from less germline-competent ES cells established.
76. Lentiviral vectors mediated transgenesis Sonia Verp, Michelle Blom, Marc Friedli, Saskia Delpretti, Isabelle Barde, Denis Duboule, Xavier Warot, Didier Trono EPFL, Lausanne, Switzerland One of the most interesting recent developments in the applications of lentiviral vectors is their use for generating transgenic animals as an alternative to the standard DNA pronuclear injection. We are one of the very few platforms that offer this fast and efficient method to generate transgenic animals. Lentiviral vectors can mediate the integration and stable expression of transgenes. They have now been improved to enable broad or tissue specific, constitutive or externally controllable transgene expression, as well as RNAi-mediated gene knockdown. The main advantage of lentiviral vector mediated transgenesis is that it is simple and highly efficient, with an average of 67% of the animals carrying the provirus (based on approximately 10,000 injections we have performed). The platform provides an all-round service: starting from production of high quality lentiviral vectors, generation of transgenic mice using perivitelline injection of oocytes, to genotyping of animals allowing the delivery of only positive progenies. The complete procedure is extremely rapid, as it can be performed in around 10 weeks. In the platform, we have successfully used the method to carry out various studies such as transgene or promoter evaluation, miRNA profiling and enhancer screening. The latter application will be presented, along with detailed characteristics of lentiviral transgenesis and
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Transgenic Res (2011) 20:1139–1189 compared to the same type of projects performed through classical pronuclear injection.
77. Comparison of FACS sorted and cultured spermatogonia derived from STRA8-GFP transgenic rats Ilya Chuykin1, Fatimunnisa Qadri1, Elena Popova1, Kaomei Guan2, Frieder Wolf2, Wolfgang Engel3, Stephan Wolf3, Michael Bader1, Natalia Alenina1 1 Max-Delbruck Center for Molecular Medicine, Berlin, Germany, 2Department of Cardiology and Pneumology, Heart Center, Georg-August-University of Go¨ttingen, Goettingen, Germany, 3Institute of Human Genetics, Georg-August-University of Go¨ttingen, Goettingen, Germany
Derivation of Spermatogonial Stem Cells (SSCs) in culture have been achieved and allowed to study the signaling pathways governing self-renewal of SSCs. However, the question to what extent the in vitro cultured SSCs are similar to undifferentiated spermatogonia in vivo remains the most intriguing. We have generated a transgenic rat strain expressing GFP under the promoter of Stimulated by Retinoic Acid 8 (STRA8), a germ cell-specific gene. By immunohistochemistry GFP was expressed in a wide range of male germ cells of STRA8-GFP transgenic rats. The pattern of GFP expression was similar to those of the endogenous Stra8 gene, confirming the specificity of the transgene expression. FACS analysis of cells obtained from testis of 3–5 weeks old transgenic rats showed, that GFP-positive cells were mostly distributed into 2 populations differing in the forward size scatter parameter (size). RT-PCR analysis revealed, that the expression of SSC markers such as GFRa1, PLZF, and E-cadherin is prevalent in smaller cells, whereas bigger GFP-positive cells expressed PCSK4—a marker of meiotic cells. Therefore, smaller GFP-positive cells represent are enriched in SSCs. We have also established a stable SSC line from testis of STRA8-GFP rats. These cells maintained the expression of SSC marker genes and were able to repopulate the seminiferous tubules of busulfan treated rats. Interestingly, the STRA8-GFP transgene was silenced in in vitro cultured SSCs. For comparison of spermatogonia in vivo and in vitro we investigated the transcriptome of spermatogonial populations derived by FACS sorting and of the cultured SSCs before and after stimulation with retinoic acid (RA). We have identified clusters containing genes, characteristic for SSCs, for instance, PLZF, GFRa1, EpCAM, Ddit4, CyclinD2, E-cadherin. The microarray data were confirmed by real-time PCR. In contrast to sorted STRA8-GFP positive cells, STRA-8 and c-Kit expression was downregulated in cultured SSCs. However, stimulation with RA induced the expression of c-Kit and STRA8, as well as led to the reactivation of the STRA8-GFP transgene, whereas the expression of PLZF and E-cadherin was downregulated and self-renewal was blocked in the presence of RA. Thus, we used a STRA8-GFP transgenic rat model to compare in vitro cultured SSCs and FACS sorted spermatogonia. Interestingly, in vivo undifferentiated spermatogonia
Transgenic Res (2011) 30:1139–1189 express STRA8-GFP; however switch it off in cell culture. This observation points to additional mechanisms balancing RA signaling in vivo in a single spermatogonia allowing to keep the undifferentiated state.
78. Transgenic overexpression of the Nfe2l2 in the spontaneously hypertensive rat ameliorates oxidative stress and features of the metabolic syndrome Vladimir Landa1, Vaclav Zidek1, Ludmila Kazdova2, Jiaming Wang3, Jefferson Chan3, Theodore W. Kurtz3, Michal Pravenec1 1 Institute of Physiology Academy of Sciences of the Czech Republic, Prague, Czech Republic, 2Institute for Clinical and Experimental Medicine, Prague, Czech Republic, 3 University of California, Department of Laboratory Medicine, San Francisco, California, USA
Insulin resistance, a central feature of metabolic syndrome, is associated with oxidative stress. Nuclear factor-erythroid 2-related factor-2 (Nfe2l2) is a key transcription factor that plays an important role in cellular defense against oxidative stress. To test the role of this transcription factor in the pathogenesis of metabolic disturbances, we derived a transgenic line of the spontaneously hypertensive rat (SHR) by microinjecting zygotes with the mix of the Sleeping Beauty (SB) construct containing Nfe2l2 mouse cDNA under control of the SV universal promoter and the SB100X transposase. The Nfe2l2 transgene was significantly expressed in multiple tissues which was associated with significantly increased activities of antioxidant enzymes and reduced oxidative stress when TBARS levels in the liver and kidney were significantly reduced compared to SHR controls (0.96 ± 0.10 vs. 1.64 ± 0.17 nM/mg and 0.55 ± 0.04 vs. 0.69 ± 0.05 nM/mg, P \ 0.05, respectively). Transgenic rats exhibited increased insulin stimulated lipogenesis measured as incorporation of radioactively labeled glucose into epididymal fat triglycerides (90 ± 17 vs. 43 ± 6 nmol gl/mg prot./2 h, P \ 0.05) and increased adrenalin stimulated lipolysis (5.2 ± 0.7 vs. 3.2 ± 0.6 mmol/g NEFA, P \ 0.05) which suggests higher metabolic activity of adipose tissue. In addition, insulin stimulated incorporation of glucose into diaphragm was significantly increased in transgenic rats compared to controls (313 ± 48 vs. 151 ± 35 nmol gl/g/2 h, P \ 0.05). These findings provide evidence that overexpression of the Nfe2l2 transgene in the SHR ameliorates oxidative stress and several components of metabolic syndrome. 79. Identification of germline competent chimaeras by copulatory plug genotyping Steve Wilson1, Steven Sheardown2, 1, Joanne Doran2 GlaxoSmithKline, Stevenage, UK, 2Takeda Cambridge Limited, Cambridge, UK 1
Here we describe the development of a novel and straightforward method for selecting germline competent chimaeras by copulatory plug genotyping (1). The method is designed to determine whether the chimaera sperm carries the mutated allele and is therefore potentially germline competent.
1171 Modification of the mouse genome by gene targeting is extensively applied across multiple biological disciplines. Fundamental to the technology is the ability to transmit genetically modified embryonic stem cells (ES cells) through the mouse germline. The use of ES cell lines from strains other than 129, in particular C57BL/6, is increasing. However C57BL/6 ES cell lines often transmit inefficiently through the germline and, in our experience, the degree of coat colour contribution is a poor indicator of germline competence. To address this issue we have developed an efficient and rapid method for DNA extraction from copulatory plugs, these contain substantial quantities of sperm and it is straightforward to assay for the presence of sperm derived from the targeted ES cell line. Thus far we have observed a perfect correlation between the copulatory plug genotype and chimaera germline competence. From 54 chimaeras we identified 13 whose sperm showed presence of the targeted allele; all subsequently produced ES cell clone derived offspring containing the targeted allele. The implications for animal welfare are profound. Taking the small number of mice utilised in our study as an example, using only the plug positive chimaeras would have reduced animal usage at this stage by 80%. This reliable method for pre-selecting the best chimaeras has the potential to save many thousands of mice. 1. Wilson S and Sheardown SA (2010) Identification of germline competent chimaeras by copulatory plug genotyping. Transgenic Research DOI 10.1007/s11248-0109413-6
80. Investigation of the intractable epilepsy by a knockin mouse model Chun-Yu Chen1, Ming-Shian Tsai1, I-Shing Yu2, You-Tzung Chen3, Po-Yuen Wu2, Li-ping Tseng2, Horng-Huei Liou4, Shu-Wha Lin1 1 Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei, Taiwan, 2Transgenic Mouse Model Core Facility of the National Research Program for Genomic Medicine, Taipei, Taiwan, 3Institute of Genomic and Proteomic Medicine, National Taiwan University College of Medicine, Taipei, Taiwan, 4Department of Pharmacology, National Taiwan University College of Medicine, Taipei, Taiwan
Background: Dravet syndrome is an autosomal dominant disease caused by de novo mutations in the voltage-gated Sodium channel Ia subunit (SCN1A) gene. Dravet patients suffered from severe epilepsy started from their infancy and they are refractory to current medical treatments. The variations in both disease symptoms and drug responses are, at least partly, related to patient’s genetic backgrounds. Objective: To identify genetic heterogeneity which influence the severity and mortality of the disease. Method: We first generated an Asianspecific point mutation allele of Scn1a in the R1 mouse ES cell using a recombineering approach. Germline chimeras were mated with 129/J females to generate mutant mice in a pure 129 genetic background. Some of the resulting heterozygous
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1172 mutant males were outcrossed to C57BL/6 females to generate F1 hybrids that mutant progenies in the heterogeneous genetic backgrounds with various degrees of phenotypic presentations can be obtained by F1 intercrosses. Result: In the pure 129 genetic background, heterozygous mutant mice were resistant to the disease. However, the homozygous mutant pups became malnourished and showed a significant reduction in their body weight and sizes within 2 weeks. They were also found to have an unstable gait and developed spontaneous seizures. Mild limb tremors, side-to-side swaying and inabilities to maintain posture gradually developed. All of them died within the third postnatal week. In contrast, when the mutant allele was brought to a heterogeneous 129 and C57BL/6 mixed genetic background, sporadic sudden death was observed in some heterozygous animals after the third postnatal week. The survival rate of the adult heterozygotes was reduced to 60% at 7 weeks. These results suggested that some modifier loci in the two different genetic backgrounds have different effects on the mutant phenotypes. Conclusion: We successfully generated a mouse model for Dravet syndrome. The fact that Scn1a mutant seizure severity and gross lethality differ in 129 and C57BL/6 genetic backgrounds indicates an invaluable opportunity to identify modifier genes using these mutant strains.
81. Characterization of mitochondrial morphology in various tissues of and primary adherent cells from transgenic mice expressing mitochondrially targeted EGFP Midori Shimanuki1, Hiroshi Shitara1,2, Jun-Ichi Hayashi2, Hiromichi Yonekawa1,2 1 Laboratory For Transgenic Technology, Tokyo Metropolitan Institute Of Medical Science, Tokyo, Japan, 2 Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki, Japan
Defects in mitochondrial structure have been frequently observed in association with various diseases, and the functions of mitochondria are believed to be directly and closely related to their morphology. Mitochondria are commonly understood to be static oval forms, as observed in electron micrographs. However, the morphology and location of mitochondria change drastically and continuously, and mitochondria show dynamic alterations such as fusion, fission, and transportation in vivo. These morphological changes are thought to be important for living organisms with respect to maintaining biological functions, such as respiratory activity, by exchanging mitochondrial contents (i.e., the so-called mitochondrial complementation). The most common and classic approaches for analyzing mitochondrial morphology are immunostaining and mitochondria-specific fluorescent dye staining, which have been used for optical microscopy analysis of mitochondria. However, these techniques have several drawbacks: (1) they require complicated and time-consuming experimental procedures; (2) many steps in the procedures require operators with excellent skills and experience; and (3) despite these efforts, satisfactory images are not always obtained. Consequently, these drawbacks present obstacles for analyzing and observing mitochondrial in vivo images.
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Transgenic Res (2011) 20:1139–1189 In this study, we propose a new approach for characterizing mitochondrial morphology by using transgenic (Tg) mice, the mitochondria of which are exclusively labeled with enhanced green fluorescent protein (EGFP). We named the Tg mice as mtGFP-Tg mice; in these mice, the EGFP was localized in the mitochondrial matrix. This Tg strain enables us dramatically easy observation of mitochondria in tissues and primary cultured cells. Recently, many mouse models for human disease have been established. Therefore, it will be possible to examine the morphological in vivo changes in mitochondria under disease conditions by using the hybrids between mtGFPTg mice and the model mice.
82. Therapeutic effect of Tfam over-expression in a mouse model of mitochondrial disease Hiroshi Shitara1,2, Satoshi Nishiyama1,2, Kazuto Nakada2, Tomio Ono1, Akitsugu Sato1,2, Hidenori Suzuki1, Tetsuhiro Ogawa3, Haruhiko Masaki3, Jun-Ichi Hayashi2, Hiromichi Yonekawa1,2 1 Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan, 2University of Tsukuba, Ibaraki, Japan, 3The University of Tokyo, Tokyo, Japan
Mammalian mitochondria contain multiple copies of about 16 kbp double-stranded circular DNA, which is called mitochondrial DNA (mtDNA). Generally, 1,000–10,000 copies of mtDNA molecules exist in a single somatic cell, and the mutation rate of mtDNA is 10 times higher than that of nuclear DNA. The pathogenic mutations in mtDNA, such as point or deletion mutations, can cause mitochondrial dysfunction and then lead to the development of mitochondrial diseases (myopathy, encephalopathy, etc.). In mitochondrial diseases, it has been generally observed that at least 2 types of mtDNA exist in the same cell, and this genetic state is called as heteroplasmy. The phenotypes of mitochondrial diseases have been considered to be regulated by the proportion of wild-type and pathogenically mutated mtDNAs in the cell. That is, mitochondrial dysfunction is caused only when the proportion of the pathogenic mtDNAs exceeds a certain threshold level (threshold effect). Several treatment options for mitochondrial diseases focusing on a decrease in the proportion of pathogenic mtDNA have been developed. However, effective and versatile therapeutic procedures have not yet been established. To develop a therapeutic procedure for mitochondrial diseases, we analyzed the therapeutic effect of the mitochondrial transcription factor A (Tfam), which has been known to regulate replication, transcription, and stabilization of mtDNA, and found that the over-expression of Tfam caused an increase in the mtDNA copy number. We developed transgenic mice that over-express the Tfam/EGFP fusion gene, and we mated these mice with the mitochondrial disease model mice (mito-mice) that had a mutant mtDNA with a large deletion (DmtDNA) (Tg-mito-mice). We found that the mtDNA content in Tg-mitomice was higher than that in mito-mice. The following symptomatic improvements were observed in the Tg-mitomice: partial recovery of cytochrome c oxidase activity, extension of life-span, and improvement of kidney disorder. Therefore, over-expression of Tfam could be a possible therapeutic option for mitochondrial diseases.
Transgenic Res (2011) 30:1139–1189 83. Large animal models of serious human diseases Tatiana Flisikowska1, Claudia Merkl1, Simon Leuchs1, Nousin Rezaei1, Alexander Kind1, Martina Landmann1, Alexander Tschukes1, Mayuko Kurome2, Barbara Kessler2, Eckhard Wolf2, Angelika Schnieke1 1 Chair of Livestock Biotechnology, TU Muenchen, Freising, Germany, 2Chair of Molecular Animal Breeding and Biotechnology, LMU Muenchen, Munich, Germany
Recent decades have seen dynamic developments in biomedical research in which genetically modified animals have played an important role. Overwhelmingly the most common species used are rodents, particularly mice. However, mice are often of limited value in predicting the clinical efficacy of any drug or evaluating new medical techniques. Our objective is to provide genetically defined large animals, notably rabbit and pig, for a variety of biomedical applications, such as models of human disease, disease predisposition and transplantation studies. To achieve this we are continuously refining transgenic technologies, particularly gene targeting in primary cells, including stem cells such as mesenchymal stem cells (MSCs) that can be used for nuclear transfer. Now, novel techniques such as Zinc finger nuclease (ZFN)-mediated gene targeting directly in fertilised oocytes offer to simplify precise genetic engineering in mammals. Our work in pig focuses on cancers of the gastrointestinal tract and associated organs, particularly colon, pancreatic and gastric cancer. Each of these is a major source of mortality and morbidity with a serious unmet clinical need, particularly techniques for early diagnosis. We are using gene targeting in fibroblasts and MSCs and nuclear transfer to introduce into pigs a number of defined mutations in tumour suppressor genes and proto-oncogenes (APC, TP53 and Kras). We have achieved efficiencies ranging from 5 to 77% of total stable transfected cell clones undergoing a targeting event. Nuclear transfer animals have been produced that are potentially predisposed to colorectal cancer. All positive cell clones and nuclear transfer animals were verified for correct targeting by PCR, RT-PCR, sequencing and Southern hybridization. Nuclear transfer in rabbits is notoriously difficult. To enable the production of targeted rabbits and streamline the production of gene targeted animals in other large mammals we investigated ZFN-mediated targeted gene replacement directly in rabbit oocytes. ZFN mRNA was co-injected into fertilised oocytes with linear targeting vector DNA and embryos transferred to foster mothers. Analysis of late stage rabbit fetuses showed that ZFN-mediated gene targeting by homologous recombination could be achieved with 17% efficiency. Again, correct targeting was confirmed by PCR, DNA sequencing and Southern hybridization.
84. A novel approach to selectively target neuronal subpopulations Dieter Engelkamp1,2, Karsten Benzing2, Stefanie Flunkert2, Martina Do¨hler1, Andreas Schedl3 1
University Erlangen-Nuremberg, Erlangen, Germany, Max Planck Institute for Brain Research, Frankfurt, Germany, 3INSERM U636, Nice, France
2
1173 In mammals, many genes execute a unique set of distinctive and common functions in different cell types. Strategies to address these individual roles often involve the generation of series of transgenic animals. Here, we present a novel approach which combines a single transgenic mouse line with tissuespecific transfection protocols and organotypic cultures to enable the quick analysis of numerous genes in a cell-specific manner. We adopted the Tet binary system and generated YAC (yeast artificial chromosome) transgenic mice which expressed the tetracycline dependent transactivator (tTA) in all Pax6 positive cells. As a proof of principle, we analyzed the function of transcription factors in tangentially migrating neurons of the developing vertebrate hindbrain. We identified a temporary halt in migration as a novel mechanism for neurons to decide whether to cross or not the midline. Our model may serve as a general tool to quickly study axonal pathfinding, neuronal cell migration or patterning processes in a well defined population of neurons.
85. Simple method for determining homozygosity employing a rapid genomic DNA extraction protocol Marta Garcı´a-Flores, Vero´nica Domı´nguez, Alfredo Serrano, Bele´n Pintado CNB-CBMSO Transgenesis Unit -CSIC-UAM, Madrid, Spain Development of molecular methods to determine homozygosity in transgenic mice is a major improvement when establishing a genetically modified line based on additive transgenesis. These analyses allow a significant reduction in the number of animals used, avoiding the necessity of performing the classical homozygous test and speed up the establishment of a homozygous colony. SYBR Green Realtime PCR has been previously demonstrated to be a suitable method for this proposal, as long as every reaction has been previously validated with an specific primer set that must be capable to provide an efficient amplification of the transgene, together with a primer set for the reference gene to normalize the results. It is essential to combine this technique with a rapid method for isolating genomic DNA, but the most common protocols take two days. Although it had been described the employment of simple protocols to obtain genomic DNA preparation, they are based on the use of lysis buffers that contains a mix of several chemicals which could interfere with the PCR reaction. Our aim has been to study the feasibility of the ‘‘hot-shot’’ procedure as an alternative for this task. Hotshot procedure needs only two reagents (NaOH and Tris buffer) and a 30 min incubation at 95°C, yielding enough and good quality genomic DNA starting from little tail snips (2 mm). From our results, we can conclude that samples extracted with this method remain stable after several months allowing reproducible assays, in case it would be needed to check the results. This method is valid regardless the number of transgene copies, allowing to discern heterozygous from homozygous even in single copy insertions, which is the case of the reference gene. In conclusion, this method contributes to simplify the procedure to analyze mice homozygosity and represents a technical improvement when establishing a mouse transgenic line in homozygosis.
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1174 86. Establishment and application of an inducible transgenic rat platform Hsiao-Hui Joyce Chang, Wei-Lun Huang, Hsin-Chih Hsu, Pei-Hua Wu, Hsian-Jean Chin, Chi-Kuang Leo Wang National Laboratory Animal Center, Taipei, Taiwan Due to the difficulties in rat embryo manipulation and superovulation, transgenic rat services are far behind the tremendous demand. Current progress in both ES cell manipulation and gene-targeting in rat has reopened the window for a promising future. Consequently, increasing demand for rat Cre strains are expected. In this proof-of-principle study, we generated a duel reporter/inducible Cre transgenic rat system. Our goal is to establish a systematic pipeline to generate inducible transgenic Cre rats to further facilitate the usage of rat GM models in both transgenic and gene targeting experiments. Our data showed that specific reporter gene expression can be detected before or after Cre-recombination according to the design of transgenic construct. The efficiency of Cremediated recombination was tested using different methodologies both in vitro and in vivo. The effectiveness of tested methodologies was compared in depth to serve as references for future applications. Our data suggest this conditional platform not only allows temporal-spatial regulation of transgene expression but also offer proper mechanism for researcher to choose in switching transgene expression on or off. The result from our study can serve as a reference in experiment design for optimal outcome. 87. An efficient method for the rederivation of mouse lines from small breeding populations Xin-an Pu1, Ming Lu1, H. Michael Kubisch2, Vincenzo Coppola1, Anthony P. Young1 1
The Ohio State University, Columbus, OH 43210, USA, Tulane National Primate Research Center, Covington, Louisiana 70433, USA
Transgenic Res (2011) 20:1139–1189 the same day from the first group of donors at 2.5 dpc and from the 2nd group at 0.5 dpc. Finally, fertilized embryos are transferred into the oviduct of pseudopregnant recipients. Using this strategy, we have rederived 10 genetically modified mouse lines, using as few as 1–3 stud males. Each line is usually completed within 9 to 11 weeks. The same strategy can also be useful for the rederivation of lines from which it is difficult to obtain sufficient embryos in one superovulation attempt. For example, this may happen in cases either with larger numbers of stud males of reduced fertility or when embryo donors are poor superovulators. Because this method does not require any prior expansion of small breeding groups it saves time, which in turn reduces space requirements and per diem cost, and eventually speeds up the research progress.
88. Efficient production of germ line rat chimeras Charles-Etienne Dumeau, Harry Leitch, Kathryn Blair, William Mansfield, Austin Smith. Centre for Stem Cell Research, University of Cambridge, Cambridge, UK The first published germ-line competent rat ES cells were reported by Austin Smith and Qi-Long Ying in December 2008. Almost two years later in September 2010 Qi- Long Ying published a paper describing homologous recombination in rat ES cells to produce a p53 knockout rat. In all of these papers efficiency was low with only one ES cell line transmitting through the germline, either XX or XY. This was thought to be because of chromosomal instability and the combination of unsuitable ES cell and host blastocyst strain. Using a combination of DA (Dark Agouti) and SD (Spague Dawley) rats we show multiple ES cell and EG cell lines both XX and XY producing germline transmission. Here we will detail the method used to produce these chimaeras.
2
Embryo transfer is the most widely used method for rederiving mouse lines to establish specific pathogen free (SPF) colonies. Infectious pathogens or agents, such as MPV, MHV, Helicobacter spp, pinworm, to mention a few, can be successfully eliminated through this approach. However, a number of factors can restrict the efficiency of rederivation including the limited availability of suitable stud males, a poor superovulatory response of embryo donors resulting in too few suitable embryos for transfer, or the limited success rate of establishing pregnancies among recipients. Here we describe an effective strategy that has allowed us to successfully rederive every mouse line in our facility for over a decade. This approach is particularly suited for rederivation of lines that originate with fewer than three, and sometimes even a single male transgenic mouse because it does not require prior expansion of the breeding population. The method relies on at least one male of proven fertility, as well as two groups of sequentially superovulated donors. PMSG is administered on day 1 to the first group and day 3 to the second group of donors, and hCG is correspondingly given on day 3 to group 1, and day 5 to group 2. The male is placed overnight with each group following hCG injections and the embryos are then collected on
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89. A comparison of exogenous promoter activity at the ROSA26 locus using a PhiC31 integrase mediated cassette exchange approach in mouse ES cells Daniel Biggs, Chiann-mun Chen, Jon Krohn, Shoumo Bhattacharya1, Ben Davies Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK Comparative analysis of transgenic constructs is complicated by the high variation in expression resulting from uncontrolled copy number and chromosomal position effects when using random insertion based transgenic approaches. Targeted integration in mouse embryonic stem cells overcomes these problems and facilitates the direct comparison of variant transgenic constructs from within the same genomic context. The ROSA26 locus has become a standard docking site, with transgene insertions frequently taking advantage of the endogenous ubiquitous ROSA26 promoter. However, little data is available to assess the suitability of this site for the insertion of exogenous promoter driven transgenes. Here we report the use of a PhiC31 integrase mediated cassette exchange approach for the efficient insertion of
Transgenic Res (2011) 30:1139–1189 multiple variant transgenes within the ROSA26 locus. This allowed us to systematically perform a quantitative analysis of the strengths of eight ubiquitous promoters (CAGGS, CMV, CMVd1, human UbC, EF1a, PGK, chicken b-actin and MC1), stably integrated into the ROSA26 locus in both sense and antisense orientations, in comparison with the endogenous ROSA26 promoter. The behaviour of these exogenous promoters reveals a large variation in their absolute activity with some promoters, but not all, showing increased activity in the sense orientation as a consequence of integration.
1175 about the untimely expression of genes and make the abnormal implantation of SCNT embryo. As a conclusion, taken together, the mir-19b is one of the key factors which can control the blastocysts quality. And transgenic somatic cells which overexpressing the miR-19b in the special period may enjoy a higher successful rate. 91. TARGATTTM: a fast and site-specific technology for the generation of transgenic mice Henrike Siemen1, Junfeng Wu1, Richard Xu1, Esther Tang1, Ruhong Jiang1, Yanry Chen-Tsai1
90. More miR-19b may bring a higher successful rate to somatic cell nuclear transfer (SCNT) cloned embryos Rui Hao, Ning Li The State Key Laboratory for Agrobiotechnology, Beijing, China MicroRNAs (miRNAs) are a class of small noncoding RNAs (&22 nucleotides) that regulate gene expression at a posttranscription level. They have been reported to be involved in multiple biological processes, including epigenetic modification, cell proliferation, stem cell pluripotency maintain and initiation of cancer. However, microRNAs in livestock, like bovine, early embryo development remains elusive. Our study investigated the difference of miRNA expression profiles between the AI(Artificial Insemination), IVF(in Vitro Fertilization), PA(Parthenogenesis)and NT(Nuclear Transfer)blastocysts. We demonstrated that the invitro manipulation will affect the micrRNA expression patterns. The NT blastocysts have the smallest microRNAs Transcriptome, and lots of these micrRNAs show low expression level. The cluster mir-17-92 is an outstanding one. The cluster expression pattern investigation, during the period from the bovine zygot to the blatocyst, proved that the miR19b is the main component in that cluster which appears just from the zygotic gene activation, during the 8–16 cell stage of bovine embryo. Mir-19b overexpression resulted in the change of both the quality and quantity of mature blastocysts, through the modification of the H3K27me3. Recent studies show that trimethylation of H3K27 (H3K27me3) contributes to the maintenance of embryonic stem cell pluripotency because the differentiation genes are always occupied by nucleosomes trimethylated at H3K27, which represses gene expression. We demonstrated that the differential H3K27me3 modification exists between normal fertilized blastocysts and somatic cell nuclear transfer (SCNT) cloned blastocysts, which is consistent with the results in mouse. H3K27me3 was specifically found in cells of the inner cell mass (ICM) of normal blastocysts, whereas no in the ICM of cloned blastocysts. MiR-19b overexpression adjusted the ICM H3K27me3 modification. It rescued the SCNT to the normal level, the similar modification of H3K27me3, but reduced the blastocyst rate. Inhibition was opposite. However, the blastocysts survived showed better quality. Our results suggest that manipulation of nuclear transfer may result in insufficient expression of miR-19b when the zygotic gene activated in the cloned embryos, and that lead to defective modification of H3K27me3 to the differentiation-related genes in ICM cells. This will bring
Applied StemCell Inc., Menlo Park, CA, USA, 2Stanford Transgenic Facility, Stanford University School of Medicine, Stanford, CA, USA 1
For decades, pronuclear DNA injection into zygotes has been widely used as a method to generate transgenic animal models. Although this method has been successful, there are several disadvantages. One of them, referred to as ‘‘position effect’’ is due to the random insertion of a transgene and can result in substantial variations in expression levels depending on the transgene’s integration site. Additionally, the transgene can be completely turned off by the host genome. Another outcome of random insertion is unpredictable phenotypes caused by the interruption of endogenous genes by the injected DNA. A further disadvantage of conventional transgenesis is that the copy number of the transgene is not controlled, leading to results that are either hard to interpret or reproduce. Taken together, these disadvantages significantly limit the application of transgenic animal models as tools in both basic research and the pharmaceutical industry. Here we report a new transgenic method: the TARGATTTM technology. This technology uses the prokaryotic enzyme Integrase to catalyze site-specific DNA integration or recombination-mediated cassette exchange (RMCE) between two different substrate sites. Any gene of interest can be efficiently integrated into the mouse genome that has been pre-engineered with a specific docking site. TARGATTTM improves several aspects in the generation of transgenic animals. (1) High gene integration efficiency reduces time and costs. (2) Site-specific integration at a proprietary locus ensures that no endogenous genes are interrupted and guarantees high gene expression of the transgene. (3) Single copy integration enables a precise comparison of the effects of the transgene among different lines. Using the TARGATTTM system, we can efficiently generate transgenic mouse models in 3 months. Tissue-specific expression models are also compatible with our TARGATTTM system. The overall benefits of the TARGATTTM system are lower cost to generate transgenic mice, a shorter production time and the precise integration of a single transgene copy in a defined, transcriptionally active locus. 92. Novel C57BL/6NTac ES cells carrying Hprtb-m3 for single copy site-specific knock-in Sonia F. Black1, Andrea J. McLeod1, Kathleen G. Banks1, Russell J. Bonaguro1, Kristi Hatakka1, Tom W. Johnson1, Elizabeth M. Simpson1,2 1 Centre for Molecular Medicine and Therapeutics, Child & Family Research Institute, University of British Columbia,
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1176 Vancouver, BC, Canada, 2Department of Medical Genetics and Department of Psychiatry, University of British Columbia, Vancouver, BC, Canada The mapped deletion break point at the hypoxanthine guanine phosphoribosyl transferase (Hprtb-m3) gene facilitates singlecopy knock-ins at this selectable locus in mouse embryonic stem cells (ESCs) and has been used in the Pleiades Promoter Project, which developed 188 novel mouse strains. This technology is an improvement over random multi-copy insertions, which can have undesirable phenotypic and genomic effects. Unfortunately, the Hprtb-m3 allele has only been available on the 129S1/SvImJ (129) and B6129F1 backgrounds; however, the world standard genetic background is C57BL/6 (B6). Using ESCs from the B6 background allows germline pups to be used for experiments without additional backcrossing, thereby reducing the use of animals, time, and money, thus creating an important need to derive novel B6 ESCs carrying the Hprtb-m3 deletion. C57BL/6 J and N are the two main substrains of C57BL/6 mice, J being the original Jackson Laboratory mouse strain derived from Little’s 1921 C57BL, and N being a subline originating at NIH in 1951. We set out to derive novel C57BL/6 J and C57BL/6NTac ESCs carrying the Hprtb-m3 allele. Single nucleotide polymorphism (SNP) studies demonstrate these two substrains are highly related. We tested both B6 substrains with the purpose of finding one that would generate high quality Hprtb-m3 ESCs. During characterization of these lines, chimeras were generated by blastocyst microinjection, and germline ability determined. Using similar techniques, the success rate for deriving new ESCs was visibly higher for the NTac ESCs when compared to the J ESCs. Of 97 blasts collected for C57BL/6 J, only 21 ES cell lines were successfully frozen (22%); of 60 blasts collected for C57BL/6NTac, 32 ES cell lines were successfully frozen (53%) (P \ 0.005). Blast injections for 3 C57BL/6 J ES cell lines resulted in only 4 chimeras from 2 ES cell lines, with only 1 chimera and cell line resulting in germline transmission. Blast injections for 3 C57BL/6NTac ES cell lines resulted in 11 chimeras with eight going germline, comprising of all 3 ES cell lines. This resulted in an overall success rate of 25% of J chimeras going germline and 73% of N chimeras going germline, despite similar coat colour chimerism. Additional injections showed similar data with the N lines consistently providing greater numbers of chimeras with greater germline ability. We are confident these new C57BL/6NTac ES cell lines will allow for better chimeras than their J alternatives and will shorten the time between injection and new experiments by eliminating subsequent backcrossing.
Transgenic Res (2011) 20:1139–1189 integrity and copy number of the transgene cannot be controlled. The insertion of DNA into different chromosomal loci at random could disrupt the function of endogenous genes. Moreover, it subjects the transgenes to the local chromatin environment (position effect) that can lead to transgene silencing or ectopic expression. An additional concern is that the transgenic DNA concatemerized into a large array is subject to repeat-induced gene silencing. Single-copy transgenesis in mice can be achieved with retroviruses and transposons, but these approaches integrate transgenes throughout the genome. They also subject transgenes to the local chromatin environment and can cause endogenous gene disruption, although depending on the purpose of transgenesis, the mutagenic properties of transposons are sometimes desirable. These problems can be overcome by targeting the transgene to a specific chromosomal locus via homologous recombination in embryonic stem (ES) cells. However, this method is significantly more laborious and time-consuming, as it involves creation of modified ES cells and mouse chimeras, as well as eventual germline transmission of the transgene. Integrase enzymes have been used to catalyze integration of transgenes in heterologous systems. Integrases catalyze irreversible recombination between appropriate attB and attP sites, one in the circular DNA and the other one in the genome. In mice, PhiC31 integrase from a Streptomyces phage has previously been used to catalyze integration of circular DNA into pseudo-attP sites in the genome for gene-therapy, and for low-efficiency transgenesis. Moreover, it has been used as a recombinase in mouse ES cells for cassette exchange, and for removal of undesirable transgene portions or reporter activation. Here, we describe a new phiC31 integrase-mediated highly efficient method for site-specific transgenesis in mice via pronuclear microinjection, with integration efficiencies of up to 40%. The system depends on a pre-targeted attP site into the mouse genome and injection of a DNA/RNA mix into mouse zygotes. In our system, phiC31 integrase catalyzes recombination between an attB site from a circular recombinant DNA, and an attP site that we previously inserted into a specific locus in the mouse genome. We also show that the expression from integrated transgenes is affected by plasmid’s bacterial backbone (BB) and a nearby transgenic tissue-specific promoter, and that the removal of these elements results in robust expression of the transgene from a ubiquitous promoter.
94. Production of transgenic bovine cloned embryos using piggyBac transposition 93. Site-specific integrase-mediated transgenesis in mice via pronuclear microinjection Bosiljka Tasic1, Simon Hippenmeyer1, Charlene Wang1, Matthew Gamboa1, Hui Zong2, Yanru Chen-Tsai1, Liqun Luo1
Goo Jang1, SuJin Kim1, Saadeldin Islam1, WooJae Choi1, SongJeon Lee1, WonWu Lee1, ByecongChun Lee1, JongKi Cho1,2, JoonHo Moon1 1
1
Stanford University, Stanford, CA, USA, 2University of Oregon, Eugene, Oregan, USA
College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea, 2College of Veterinary Medicine, ChungNam National University, DaeJeon, Republic of Korea
Microinjection of DNA into zygotic pronuclei is the predominant method used to produce transgenic mice. However, this method has several important limitations: the insertion site,
Transgenic research on cattle embryos has been developed to date using viral or plasmid DNA delivery systems. In this study, a different gene delivery system, piggybac transposition, was
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Transgenic Res (2011) 30:1139–1189 employed to investigate if it can be applied for producing transgenic cattle embryos. Green or red fluorescent proteins (GFP or RFP) were transfected into donor fibroblasts, and then transfected donor cells were reprogrammed in enucleated oocytes through SCNT and developed into pre-implantation stage embryos. GFP was expressed in donor cells and in cloned embryos without any mosaicism. Induction of RFP expression was regulated by doxycycline treatment in donor fibroblasts and pre-implantational stage embryos. In conclusion, this study demonstrated that piggybac transposition could be a mean to deliver genes into bovine somatic cells or embryos for transgenic research.
95. Effect of euthanasia method on the production and viability of embryos obtained from in vitro fertilization procedures Alfredo Serrano, Marta Garcia-Flores, Veronica Dominguez, Belen Pintado CNB-CBMSO Transgenesis Unit CSIC-UAM, Madrid, Spain The effect of euthanasia system: CO2 inhalation vs. cervical dislocation on embryo production and viability after in vitro fertilization has been studied. Cervical dislocation allows in situ slaughter of donor females and fast recovery of oocytes meanwhile CO2 chambers might be restricted to animal facilities, which implies a certain delay in oocyte collection. Cervical dislocation is not considered the method of choice for rodent euthanasia and for this reason some ethical committees do not allow it. A consistent recovery procedure that minimizes loss of viability may enhance embryo production and viability allowing a reduction in the number of animals used. We compared both systems: CO2 and cervical dislocation, in 2 different backgrounds: C57Bl/6 J vs. hybrids (B6D2F1), and two recovery times: collection immediately after death confirmation and collection 5 min after death confirmation. We used the standard IVF protocol of our Unit. For each replicate the same sperm sample was employed to inseminate all experimental groups. Sperm capacitation and sperm-oocyte coculture was performed in hTF, followed by in vitro culture in KSOM. Our results show a clear detrimental effect when delaying oocyte collection for 5 min regardless the euthanasia system employed (p \ 0.01), or the genetic background. However the effect was more evident with B6 donors after CO2 euthanasia, where percentage of 2-cell embryos obtained, decreased more than twofolds. In conclusion, cervical dislocation should be recommended when the use of CO2 implies a certain delay in oocyte recovery. In that case not only there is a significant decrease in the percentage of 2-cell embryos obtained, but also viability is compromised, since a significantly lower number of 2 cell embryos reach the blastocyst stage after in vitro culture
96. Feasibility of B6-derived ES cells with a modified 2i system to achieve homologous recombination Veronica Domı´nguez, Alfredo Serrano, Marta Garcı´a-Flores, Bele´n Pintado CNB-CBMSO Transgenesis Unit, Madrid, Spain
1177 The generation of genetically modified mouse lines by gene targeting embryonic stem cells (ES) is one the major tools for manipulating the mouse genome and for elucidating the function of mammalian genes. Most of these modification have been performed in ES cells derived from the 129 strain (Hughes et al. 2007) due to their permissiveness for derivation and culture, their genetic stability and the high frequency of germ line transmission. However, genetic and phenotypic differences have been described in gene targeted mice derived from 129 cells due to the genetic complexity of this strain (Linder et al. 2006).The use of C57Bl/6 ES cells avoids the need of backcrossing to a defined background, therefore this has been the option chosen by the international large-scale mouse mutagenesis projects (Skarnes et al. 2011). C57Bl/6 ES cell show genomic instability, a greater tendency to lose their pluripotency and they are more difficult to work with (Auerbach et al. 2000). In general, for some transgenic facilities, it is more difficult to obtain good chimeras, able to go germ line, with these consortium-targeted or trapped clones, in contrast with 129 or hybrid clones. The 2i system described in 2008 by Nichols and Ying, combined with different versions of conditioned media, has been successfully applied to obtain efficient germ line transmitting chimeras from the suboptimal C57Bl/6 ES cells more often used by International Consortiums (Gertsenstein et al., 2011). Our aim was to check if the use of a defined medium without serum and with 2i from derivation to cell injection, improved the quality of C57Bl/6 ES cell lines and their targeted clones compared to existing lines. Using a defined media with 2i and a serum replacement, we have established new C57Bl/6 ES cell lines with an efficiency of 83% and have selected the best one based on sex, euploidy, undifferentiated state and growing studies. We used this line for homologous recombination (HR) in gene targeting projects, comparing the results with the hybrid G4 ES line we usually work with. To date, 6 different vectors (same preparation) have been electroporated into both lines. We have not found statistical differences regarding recombination frequency of targeted clones obtained (p [ 0.05). The first karyotype data do not suggest that there are differences in percentage of euploidy between positives HR clones of both lines. Data will be presented according ES cell lines background, %HR, euploidy, generation of chimeras and germ line transmission.
97. Adaptable and universal construct for expression of multiple cDNAs from BAC constructs or endogenous promoter in vivo Melanie Lawrence, Karamjit Singh-Dolt, Eve Miller-Hodges, Peter Hohenstein MRC Human Genetics Unit, Edinburgh, UK Transgenic expression of genes such as reporters, miRNAs and Cre-recombinase are useful tools for investigating gene expression and function. Development of such expression constructs can be lengthy and complex. Here, we describe a universal and highly adaptable construct for gene expression from a BAC or endogenous promoter, using a BAC cloning platform and multi-site Gateway technology. The construct contains four attB-flanked cassettes allowing for easy removal
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1178 or insertion of cDNAs and other functional elements (IRES, polyA, etc.), as well as a Neo/Kan cassette under mammalian and bacterial promoter control flanked by F3 sites for ES cell and bacterial selection. Multiple cloning sites at the 50 and 30 sides of the cassettes allow insertion of homologous arms unique to any gene of interest, making this construct universal provided a BAC containing the gene of interest is available. As expression is driven by the promoter available on the BAC, this construct is readily adaptable for the development of transgenic animals used for a variety of analyses such as gene expression during development, tissue-specific knockdown and tissuespecific inducible gene expression. Alternatively, standard recombineering tools can be used to retrieve part of the BAC into a plasmid to use as target vector for the endogenous locus. We have used this construct to create promoter reporters for five genes—Agr2, Meckelin, Nanog, Six2 and Podocin. Expression of GFP, mCherry or mKate2 is driven by the BAC, leading to reporter expression in all cells that also express the gene of interest. Insertion of a GFP-Cre fusion protein in the case of Agr2 and Six2 or GFP-CreERT2 in the case of podocin will be used to provide lineage tracing analysis and allow tissue-specific Cre driver mice for generating tissuespecific conditional mouse strains. Using multi-site Gateway technology combined with recombineering, the universal nature of the construct makes it a useful tool for highthroughput genetic analysis, and fast and easy development of transgenic constructs. Work is under way to adapt the system to an ES-based recombinase-mediated cassette exchange system (see abstract from Nils Lindstro¨m).
98. Optimizing the superovulation protocol for 129S1/ SVImj mouse stain Kuzhilani Vasudevan, Jorge Sztein ARTiC/CMB/NIAID/NIH, Rockville, Maryland, USA For practical reasons rodent superovulation has been standardized as a generic prescription for all strains. However, not all strains respond similarly to same treatment. Harvesting large number of oocytes often do not correlate with acceptable quality. The mouse strain 129S1/SVImJ has been commonly used in genetic engineering studies. Even though these mice respond well to superovulation treatment the IVF fertility rate is low. In this study we investigated different parameters to optimize the superovulation regimen for 129S1/SVImJ strain in order to improve the quality of oocytes and fertility rate of IVF. Females were divided into four groups based on the hormone and timing of injection. Group 1 received 5 IU PMSG (Gonadotrophin from Pregnant Mare Serum) and 48 h later 5 IU of hCG (Human Chorionic Gonadotrophin), group 2 received hCG 52 h post PMSG, group 3 55 h post PMSG and group 4 received 20 mg/kg of Buserelin (Gonadotrophin releasing hormone agonist—GnRH), PMSG 24 h post Buserelin and HCG 55 h post PMSG. In vitro fertilization was performed using 129S1/SVImJ oocytes and sperm; C57BL/6 N sperm was used as a fertility control. The IVF fertility rate for group 1 and 2 was 1%, group 3 was 30% and group 4 was 59% for 129 oocytes fertilized with 129 sperm. For 129 oocytes fertilized with C57BL/6 sperm the fertility rate was 5% for group 1, 10% for group 2, 40% for group 3 and 60% for group
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Transgenic Res (2011) 20:1139–1189 4. These results suggest that extending the interval between PMSG and hCG and giving GnRH in addition to PMSG and hCG can improve the IVF fertility rate of 129S1/SvIMJ strain significantly. This research was supported by the Intramural Research Program of the NIH, CMB/NIAID.
99. The small animal modeling and imaging core facility at the Moffitt Cancer Center in Tampa, Florida Noreen Luetteke, Hartmut Berns, Gary Martinez, Epi Ruiz, Robert Gillies H Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA The Small Animal Modeling and Imaging (SAMI) Core Facility at the Moffitt Cancer Center is an integrated shared resource to provide proficient generation and examination of rodent models for both basic and translational cancer research. SAMI offers specialized consultation and services for the derivation of genetically engineered mice and a library of tumor cell lines for xenograft and syngeneic transplant mouse models. Cancer development and treatment are evaluated using a portfolio of modern state of the art imaging techniques that permit longitudinal noninvasive interrogation of tumor anatomy, growth, physiology, metabolism, and gene expression. SAMI provides no-cost consultation on selection of animal models and imaging applications, preparation of IACUC protocols and grant applications, and design of transgenic and targeting vectors. Specific services for competitive rate fees include gene-targeting in murine embryonic stem (ES) cells; karyotyping and microinjection of targeted ES clones into blastocysts to generate chimeric mice; microinjection of DNA into zygotes to derive transgenic founders; hybridizationbased genotyping (slot or Southern blots) of mouse DNAs; in vitro fertilization, rederivation, cryopreservation and revival of mouse strains; luciferase and fluorescent tagged tumor cell lines for transplant models; bioluminescent and biofluorescent imaging (Caliper IVIS-100 and -200); tomographic fluorescence lifetime imaging (ART Optix MX3); beta particle imaging; high resolution ultrasound (VisualSonics Vevo 2100); and magnetic resonance imaging (Varian 7T ASR310, Oxford HyperSense). A trimodal PET/SPECT/CT scanner (Siemens Inveon) will soon be added to the animal imaging suite in the vivarium. The SAMI core is currently targeting murine ES cells to allow Cre Recombinase Mediated Cassette Exchange (RMCE) at the Rosa26 locus as an improved method for derivation of transgenic mice with more reliable expression. The Zinc Finger Nuclease (ZFN) technique of mutagenesis in the rat is also under development. The SAMI Core can thus serve as a regional resource for rodent research in central and south Florida.
100. Derivation of iMEF from pMEF by serial passage Amy Handy, Wenhao Xu University of Virginia, Charlottesville, VA, USA Described here is a straightforward procedure in which primary mouse embryonic fibroblasts (pMEF) from a single
Transgenic Res (2011) 30:1139–1189 embryo of three individual lines (two mutants: PTK-/- and PTK7 Cherry/Cherry ; one wildtype: PTK7+/+) are immortalized by serial passaging in tissue culture. The immortalized mouse embryonic fibroblasts (iMEF) are thus generated without the addition of exogenous transforming factors such as the commonly used SV40 large T-antigen. During the natural selection process, the pMEF undergo initial exponential proliferation and then crisis, followed by the recovery phase to permanently bypass the cellular senescence. The PTK7-/-, PTK7Cherry/Cherry and PTK7+/+ lines are recovered after 25, 28 and 31 passages respectively. It is our opinion that most pMEF lines can be spontaneously immortalized this way, provided a critical concentration of cells is maintained through the crisis stage. It is also possible that the cells could recover more quickly if several embryos of like type are pooled. The methodology for deriving iMEF from pMEF by serial passage could be easily adapted by most transgenic facilities to expand their service portfolios.
101. EMMA: The European mouse mutant archive Julia Fernandez1, Sabine Fessele2, Lluis Montoliu1, Glauco Tocchini-Valentini3, Yann He´rault4,10, Steve Brown5, Urban Lendahl6, Jocelyne Demengeot7, Ewan Birney8, Ramiro Ramı´rez-Solis9, George Kollias11, Radislav Sedlacek12, Raija Soininen13, Thomas Ru¨licke14, Martin Hrabe´ De Angelis2
1179 Monterotondo, Italy holds the core structure. The EMMA network is directed by Professor Martin Hrabe´ de Angelis who also heads the HMGU/IEG in Munich. EMMA’s primary objective is to establish and manage a unified repository for maintaining biomedically relevant mouse mutants and making them available to the scientific community. Therefore, EMMA archives mutant strains and distributes them to requesting researchers. At present, EMMA holds more than 2600 mouse strains, corresponding to transgenic mice, different type of mutants, gene-traps, knock-ins, knock-outs and also including some targeted alleles from Deltagen, Lexicon and EUCOMM projects. EMMA’s technology development programme is focusing on improving sperm cryopreservation methods, the implementation of laser-assisted IVF and ICSI protocols, and has explored different techniques to allow the shipment of unfrozen mouse embryos. EMMA also hosts cryopreservation courses, to promote the use and dissemination of frozen embryos and spermatozoa. Dissemination of knowledge is further fostered by a dedicated resource database (Wilkinson et al. Nucleic Acids Res. 2010, 38:D570-6). All EMMA procedures and all required information to deposit or request mouse lines from EMMA are easily available through the EMMA web site at: www.emmanet.org. EMMA is supported by the partner institutions, national research programmes and by the EC’s FP7 Capacities Specific Programme.
1
Centro Nacional de Biotecnologia (CNB-CSIC), Madrid, Spain, 2Helmholtz Zentrum Mu¨nchen, Institute of Experimental Genetics (HMGU-IEG), Munich, Germany, 3 CNR Campus A. Buzzati-Traverso, Monterotondo/Rome, Italy, 4Centre National de la Recherche Scientifique, Transge´ne`se et Archivage d’Animaux Mode`les (CNRSTAAM), Orleans, France, 5Medical Research Council, Mammalian Genetics Unit (MRC-MGU), Harwell, UK, 6 Karolinska Institutet, Department of Cell and Molecular Biology (KI-CMB), Stockholm, Sweden, 7Fundac¸ao˜ Calouste Gulbenkian, Instituto Gulbenkian de Cieˆncia, Oeiras, Portugal, 8European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Hinxton, UK, 9Genome Research Limited, Wellcome Trust Sanger Institute (WTSI), Hinxton, UK, 10GIE-Centre Europe´en de Recherche en Biologie et en Me´decine, Institut Clinique de la Souris (GIE-CERBM-ICS), Illkirch/Strasbourg, France, 11 B.S.R.C. Alexander Fleming, Vari/Athens, Greece, 12 Institute of Molecular Genetics (IMG), Prague, Czech Republic, 13Biocenter Oulu, Oulu, Finland, 14Biomodels Austria (BIAT), Vienna, Austria The European Mouse Mutant Archive (EMMA) is a nonprofit repository for the collection, archiving (via cryopreservation) and distribution of relevant mutant strains essential for basic biomedical research. The laboratory mouse is the most important mammalian model for studying genetic and multi-factorial diseases in man. Thus the work of EMMA will play a crucial role in exploiting the tremendous potential benefits to human health presented by the current research in mammalian genetics. The EMMA network is a partnership of 14 laboratories and other institutions throughout Europe. The current membership includes 14 nodes in eleven different countries. The CNR Campus ‘‘A. Buzzati-Traverso’’ in
102. MENDEL: a simple solution to compare the observed and expected distributions of genotypes/ phenotypes in transgenic and knockout mouse crosses involving up to three unlinked loci by means of a chisquare test Lluis Montoliu Centro Nacional de Biotecnologia (CNB-CSIC), Madrid, Spain The analysis of transgenic and knockout mice always involves the establishment of matings with individuals carrying different loci, segregating independently, whose presence is expected among the progeny, according to a Mendelian distribution. The appearance of distorted inheritance ratios suggests the existence of unexpected lethal or sub-lethal phenotypes associated with some genotypes. These situations are common in a number of cases, including: testing transgenic founder mice for germ-line transmission of their transgenes; setting up heterozygous crosses to obtain homozygous individuals, both for transgenic and knockout mice; establishing matings between floxed mouse lines and suitable cre transgenic mouse lines, etc. The chi-square test can be used to assess the significance of the observed frequencies of genotypes/phenotypes in relation to the expected values, in order to determine whether the observed cases fit the expected distribution. MENDEL is a simple solution, created within an Excel workbook, to compare the observed and expected distributions of genotypes/phenotypes in transgenic and knockout mouse crosses involving up to three unlinked loci by means of a chi-square test. The file is freely available for download from the laboratory’s web page at: http://www.cnb.csic.es/*montoliu/Mendel.xls
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1180 103. Factors affecting the efficiency in the generation of transgenic mice with yeast artificial chromosomes by standard pronuclear microinjection Almudena Ferna´ndez1,2, Diego Mun˜oz1,3, Patricia Giraldo1,4, Marta Cantero1, Julia Ferna´ndez1, Soledad Montalba´n1, Alfredo Serrano5, Bele´n Pintado5, Lluis Montoliu1,2 1
Centro Nacional de Biotecnologia (CNB-CSIC), Madrid, Spain, 2Centro de Investigacio´n Biome´dica en Red de Enfermedades Raras (CIBERER), ISCIII, Madrid, Spain, 3 Centro de Biologı´a Molecular -Severo Ochoa- (CBMSOUAM/CSIC), Madrid, Spain, 4Departamento de Biotecnologı´a, ETS Ing. Agro´nomos, UPM, Madrid, Spain, 5 CNB-CBMSO Transgenesis Unit -CSIC-UAM, Madrid, Spain The use of genomic-type DNA constructs normally ensures optimal transgene expression, once inserted into the host genome, because their large size includes most if not all the regulatory elements, in cis, that have been evolutionary and functionally conserved, and hence are needed for the correct expression of the locus. Very large heterologous fragments of genomic DNA can be easily maintained and modified by homologous recombination techniques in bacterial or yeast cells, through the use of Bacterial Artificial Chromosomes (BACs) or Yeast Artificial Chromosomes (YACs), respectively. Although BACs have become the most popular vector for genomic-type transgenic approaches, YACs are unique vectors because they allow the manipulation of larger DNA molecules, in excess of 1 Mb (1,000 kb). Some mammalian loci (i.e. the APP locus, *450 kb, associated with Alzheimer’s disease) greatly exceed the maximum size for inserts that can be accommodated into BACs (*300 kb). Therefore YACs are currently the only available robust and reliable solution for working with these large genes. The isolation and purification of intact and clean YAC DNA molecules is a challenge for standard molecular biology laboratories, where bacterial procedures are routine but yeast protocols might not be common. Special care and attention must be paid during the preparation of YAC DNA for microinjection into the pronucleus of fertilized mouse oocytes. Several factors are of paramount relevance for preparing YAC DNA samples of the highest quality, suitable for the generation of transgenic mice by standard microinjection. These factors include: the right amount of yeast cells embedded in agarose-plugs, the use of optimal enzymes for digesting the yeast cell wall, the addition of polyamines to protect the YAC DNA, the appropriate dilution factor applied to YAC DNA stock samples, to titrate-off any remaining co-purified toxic agents and the use of automated microinjection devices that can withstand the pressure to avoid breaking the fragile YAC DNA molecules. In this work we will present examples of several successful YAC transgenic mouse projects carried out in the laboratory with genomic constructs ranging from 200 to 600 kb. The transgenic efficiencies achieved and the associated germ-line transmission features observed will be discussed.
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Transgenic Res (2011) 20:1139–1189 104. Genetics of coat colour markers used in mouse transgenesis Lluis Montoliu Centro Nacional de Biotecnologia (CNB-CSIC), Madrid, Spain Mice are probably the best mammalian species to be used for animal experimentation. In particular, mice can be used to generate most adequate animal models for many human diseases. The impressive mouse genetics toolbox allows us to manipulate their genome at will. We can generate, virtually, any desired genetic modification, by inserting, removing or altering any sequence of their genome. Furthermore, the existence of many genetically different inbred mouse strains, with diverse phenotypical properties, provides an enormous amount of variability and combinations to work with. Among the genetic variability, identifying each mouse strain, there are some genes encoding for proteins impacting on the coat colour, involved in the production and/or distribution of pigment in the body. These colour traits have been extremely useful to mouse geneticists, to identify mouse strains and, specifically, to follow their derived cellular lineages in the generation of mouse chimeras. Currently, there are about 400 loci in the mouse genome encoding colour genes. More than half of them still remain to be cloned (are only known as mouse mutants, i.e. patchwork) and hence, they have not been yet assigned to any previously known gene. The Coat color genes web site, maintained by the European Society for Pigment Cell Research (http://www.espcr.org/micemut/) holds a table, regularly updated, from all known cloned and not-cloned mouse genes involved in coat colour formation, and their counterpart homologues in the human and zebrafish genomes. Among all these hundred of loci only a few are relevant for mouse transgenesis. This Poster will highlight the list of mouse loci that have been applied for the generation of mouse chimeras or are useful for the identification of common mouse inbred strains used in mouse transgenesis. The list of these genes include: Tyr (tyrosinase/albino/color), A (agouti, Asp), Tyrp1 (tyrosinase-related protein 1/brown), Oca2 (pinkeyed-dilution), Myo5a (dilute) and a few other loci. Illustrative examples, using mouse pictures, will be presented showing the interactions between these loci and the resulting effect in the coat colour of mice.
105. Strategies of superovulation for the different mouse strains Charlie Luo1, Juliana Zun˜iga2, Earnessa Edison1, Shana Palla1, Wenli Dong1, Jan Parker-Thornburg1 1 Texas MD Anderson Cancer Center, Houston, Texas, USA, 2Baylor College of Medicine, Houston, Texas, USA
Superovulation has been used in the production of transgenic mice to reduce the number of animals used. The oocyte yields in response to superovulation are variable among different
Transgenic Res (2011) 30:1139–1189 mouse strains possibly due to different genetic background. Therefore, the superovulation protocol needs to be optimized for different mouse strains. We examined different body weight ranges and hormone dosages used to determine superovulation protocols for 6 female mouse strains commonly used in genetic engineering: C57BL/6NHsd, B6(Cg)-Tyrc-2 J/J, B6D2F1/Hsd, FVB/NHsd, BALB/cAnNCr, and Crl: CD1(ICR). Mice from each strain were divided into groups based on body weight roughly corresponding to those of 3-, 4-, 5-, and 6-wk-old mice. Mice were treated with 5 IU pregnant mare serum gonadotropin (PMSG) and 5 IU human chorionic gonadotropin (HCG). The body weights of mice that produced maximal numbers of oocytes in response to these doses were 14.2 g or less for C57BL/6NHsd, 13.7 g or less for B6 (Cg)Tyrc-2 J/J, 6.0 to 9.9 g for B6D2F1/Hsd, 14.5 to 16.4 g for FVB/NHsd, 14.8 g or less for BALB/cAnNCr, and 23.5 g or more for Crl: CD1 (ICR). We then compared PMSG dosages of 5 and 2.5 IU per mouse and determined whether 2 doses of PMSG (5 or 2.5 IU, depending on prior results) administered 1 wk apart, followed by the standard HCG injection, would produce more oocytes when compared to a single dose of PMSG. FVB, B6D2F1, BALB/c, and CD1 mice responded best to a single dose of 5 IU PMSG, whereas B6 (Cg)-Tyrc-2 J/J mice produced more oocytes after 2.5 IU PMSG. Although C57BL/6 mice given the standard dose produced good numbers of oocytes, the number was higher after 2 doses of PMSG at 5 IU per dose. We conclude that response to superovulation can be optimized based on mouse strain, weight, and the dose of hormone injection. Our analysis demonstrates that for our facility and for several commonly used strains, the weight of the mice and dosage of PMSG are key factors in maximizing superovulation rates.
106. A ‘‘Lymphoreporter’’ mouse for in vivo imaging of lymphangiogenesis in development, inflammation and tumour metastasis Ine´s Martı´nez-Corral1, David Olmeda1, Rodrigo Die´guez-Hurtado1, Tuomas Tammela2, Kari Alitalo2 , Sagrario Ortega1 1 Spanish National Cancer Research Center (CNIO), Madrid, Spain, 2University of Helsinki, Helsinki, Finland
The lymphatic vascular network includes the lymphatic vessels and the lymph nodes and plays very important roles in tissue drainage and homeostasis, immune surveillance and dietary fat absorption. It is also involved in different pathologies such as lymphedema, inflammation and tumour cell dissemination and metastasis. Despite of its relevance still much remains to be known about the molecular mechanisms that control the formation of new lymphatic vessels in development and disease. Moreover the therapeutic impact of targeting this system is not sufficiently explored yet. Here we present the first mouse model for non invasive in vivo imaging of lymphangiogenesis (growth of new lymphatic vessels). We have called it the ‘‘Lymphoreporter’’ mouse. In this model an IRES-EGFP-luciferase cassette has been introduced, by homologous recombination, into the 30 -UTR (untranslated region) of the Vegfr-3 gene, one of the best molecular markers of lymphatic endothelium. This bicistronic
1181 reporter leads to the concomitant expression of the traceable EGFP-luciferase fusion and the VEGFR-3 protein under the endogenous transcriptional regulation of the Vegfr-3 gene. Luciferase and EGFP expression can be independently monitored by luminescence and fluorescence emission using different imaging techniques and they faithfully recapitulate endogenous Vegfr-3 expression. We show the use of the Lymphoreporter model for imaging of lymphatic vessel development during embryogenesis. In the adult, the Lymphoreporter mouse allows for tracking of lymphangiogenesis during wound healing and in contact hypersensitivity (CHS)-induced inflammation. We have also used the model to show the impact of anti-inflammatory drugs in lymphangiogenesis in these processes. Furthermore this is the first mouse model that allows for in vivo tracking of tumor-induced pro-lymphangiogenic signals both at the periphery of the tumour and also, very importantly, at the lymph nodes, preceding tumor metastasis. This ‘‘seed and soil’’ hypothesis of tumour dissemination is an active area of research in the field, but it has not has not yet been properly monitored in real time. Therefore, our model represents also a very attractive platform for anti-metastatic drug discovery.
107. Extraction of DNA with a novel method for the evaluation of genetic quality of NMRI outbred mice through analysis of two molecular markers Nadia Milani1, Melcenia Moreno2, Juana Andrade-Lopez1, Rosa De Jesus3 1
Bioterio, Instituto de Biomedicina, Universidad Central de Venezuela, Caracas, Venezuela, 2Laboratorio de Biologı´a Celular, Instituto de Biomedicina, UCV, MPPS, Caracas, Venezuela, 3Bioterio Central, Universidad de los Andes, Merida, Venezuela The NMRI mice are used principally in pharmacological and toxicological experiments, which require the use of animals with high genetic variability, similar to that of human populations. However, genetic variability is reduced in animals which have been isolated for long time, and this fact makes necessary the genetic monitoring of the NMRI mice, evaluating its genetic variability for guarantee the appropriate animal for researchers. Genetic markers (microsatellites) are a useful tool for supervising genetic characteristics in mice without consanguinity. This study aimed to evaluate the genetic quality through microsatellites analysis in stocks of NMRI outbred mice from the mouse room of the Instituto de Biomedicina of the Universidad Central de Venezuela and Ministerio del Poder Popular para la Salud (UCV-MPPS). The studied sample consisted of 20 mice, 10 of each sex of stock included in the analysis. Genomic DNA was isolated through the novel Ananase technique using two millimeters of tissue from the ear of each mouse. This technique is based on the action of the bromelain enzyme (20 mg/ml). There was verified the integrity and measured the size of the isolated DNA in agarose gel (0.9%), concentration and purity were determined by spectrophotometry. Finally, the microsatellites D1Mit17 and D11Mit2 were amplified by PCR using specific primers; amplified products were visualized in agarose gels (2.5%). The isolated DNA had good quality (23 kb), with an average concentration
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1182 of 1,200 ng/ll, which are larger than those reported previously. Results suggest the heterozigous condition of the microsatellite D1Mit17, because the fragment showed two alleles with 180 and 200 pb approximately. There is presumably homozygocity for the microsatellite D11mit2, because it presented just one allele with 100pb approximately; however in some samples, the absence of amplified would show a genetic modification in the evaluated locus, it prevents the amplification of the genomic region tested, and also would indicate genetic variation between individuals of the colony. The Size of each fragment is similar to the reported in the database Mouse Genome Informatics for other inbred strains: 1Mit17 with 176 pb (BALB/cJ and LP/J) and 190 pb (CAST/EiJ); 11Mit2 with 109 pb (SPRET/EiJ). We suggest to analyze a higher number of microsatellites for characterize better the stocks of these experimental animals.
108. Mmrn2 null mice are hypertensive: dissecting the role of VSMCs and ECs Paola Braghetta1, Dario Bizzotto1, Nicola Facchinello1, Francesco Da Ros1, Daniela Carnevale2, Dino Volpin1, Giuseppe Lembo2, Giorgio Maria Bressan1 1
Dept. of Histology, Microbiology and Medical Biotechnologies, University of Padova, Padova, Italy, 2 Dept. of Angiocardioneurology, Neuromed Institute, Pozzilli (IS), Italy Multimerin-2 is an extracellular matrix glycoprotein of the Emilin/Multimerin family produced exclusively by the endothelium and endocardium, from early stages of embryonic development to adulthood. Mmrn2 null mice, obtained by homologous recombination in embryonic stem cells, display increased systolic and diastolic blood pressure. Similarly to what described for knockout mice of another member of the same protein family, Emilin-1 (Zacchigna et al., 2006), the hypertensive phenotype could be rescued in vivo by inactivation of one Tgfb1 allele. Unexpectedly increased levels of phospho-Smad2 positive nuclei were found both in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) of mesenteric arteries from null mice. These data suggest a pathogenetic role of TGF-b1 in hypertension of Mmrn2-/mice, involving both multimerin-2 expressing (ECs) and nonexpressing (VSMCs) cells. In vitro analyses in which Mmrn2 and proTGF-b1 were transfected into MEK293 cells showed that Multimerin-2 reduces TGF-b1 signalling through the inhibition of proTGFb1 processing into the LAP/TGF-b complex by the formation of a supramolecular association with proTGF-beta. Mmrn2 knockdown in human umbilical vein endothelial cells (HUVEC) specifically increased Smad3, but not Smad1/5/8, phosphorylation, suggesting that Multimerin-2 affects TGFbeta signalling through ALK5, but not through ALK1 receptors. Important experiments using co-coltures of VSMCs from normal resistance arteries and HUVEC expressing control or Mmrn2 specific interfering siRNAs revealed that Multimerin-2 expressed by ECs regulates paracrine TGF-beta signalling in VSMCs, as shown by the increase of P-Smad2 phosphorilation. Ex vivo studies comparing constriction of normal and mutant resistance arteries to phenylephrine, angiotensin II and
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Transgenic Res (2011) 20:1139–1189 Endothelin-1 disclosed that Mmrn2 null arteries developed higher tension compared to controls, suggesting an increased reactivity of resistance vessels to different G-protein coupled receptor agonists. Moreover, treatment of Mmrn2 knockout mice with the a1-adrenergic receptor antagonist prazosin normalized blood pressure. These results suggest that Mmrn2 regulates the processing of proTGF-b1 in ECs and that the mature TGF-b1 produced by these cells contributes to the global signalling of TGF-beta in neighbour VSMCs leading to increased contraction to physiological stimuli such as catecholamines and Angiotensin II and hypertension.
109. Effect of cell quantity in obtaining mouse embryonic chimeras by demi-blastocysts aggregation Caio P. Godoi, Pablo D. Moc¸o, Bruno Cazari, Patrı´cia T. Mihara, Patrı´cia V. Silva, Isabele P. Emanuelli, Marcelo F. Gouveia Nogueira Dept. of Biological Sciences, FCL, Sao Paulo State University (UNESP), Assis, Sao Paulo, Brazil Eight-cell-stage to pre-compaction morula are the most used embryonic stages to aggregation, because the embryos, in these early stages, synthesize cell adhesion molecules that increase the aggregation chances among them (Vestweber et al., 1987; Developmental Biology 124:451–6). Although post-compaction embryos produce reduced aggregation rates, they are not refractory to this process (Nogueira et al., 2010; Transgenic Research 19:344–5). Based on this evidences, the aim of this study was to evaluate, in mice, the influence of cell quantity in the chimerism rate of split blastocysts. Embryos, with preferentially different phenotypes, were obtained from C57BL/6/ EGFP and/or Swiss Webster strains. Females ranging from 21 to 45 days old were superstimulated and mated according to Mancini et al. (2008; Transgenic Research 17:1015). Eightcell-stage embryos (8C) and pre-compaction morula (PCM) were recovered (2 to 2.5 days post coitum) and had their zona pellucida removed using pronase treatment (2 mg/mL for 15 min), while blastocysts (recovered 3.5 dpc) were split with microblade controlled by micromanipulator in an inverted microscope (NK2, Eppendorf, Germany and Eclipse Ti, Nikon, Japan, respectively). The aggregation groups were: a control (C) with two pre-compaction whole embryos 8C and/or PCM and two experimental with post-compaction embryos, i.e., two (2DB) or four (4DB) demi-blastocysts. The structures (2 or 4) of the groups were sticked each other with the use of phytohemagglutinin (1 mg/mL) and cultured in vitro by 24 h (37°C, 5% CO2 and saturated humidity). After culture, the presence of chimeric embryos was verified by detection of a single, cohesive cell mass or a structure in ‘‘8 shape’’ with more than half of its total diameter aggregated. For the 4DB group, a successful aggregation was considered when, at least, two of four demi-blastocysts have aggregated. The results were analyzed using Chi-square test, Fisher’s exact test and Kruskal–Wallis (to comparison among groups, between groups and among median of group replicates, respectively) and significance was considered when P \ 0.05. The aggregation rates for the groups C, 2DB and 4DB were, respectively, 77.3a; 8.3b e 36.4%c (P \ 0.001). The increasing of the aggregation
Transgenic Res (2011) 30:1139–1189 technique efficacy, in post-compaction stages, would be particularly interesting in farm animals (e.g., bovine species), where it is not feasible to obtain, in vivo, pre-compaction stages embryos (as eight cells) and when only trophectoderm aggregation is aimed. It was concluded that cell increasing (from 2 to 4 demi-blastocysts) improved the chimerism rate, but not enough to be similar to control group. Financial support: FAPESP of Brazil.
110. Chimeric mouse blastocysts reconstructed by inner cell mass and trophectoderm aggregation Isabele P. Emanuelli, Caio P. Godoi, Bruna C. S. Campanha, Pablo D. Moc¸o, Bruno Cazari, Patrı´cia V. Silva, Marcelo F. Gouveia Nogueira Dept. of Biological Science, FCL, Sao Paulo State University (UNESP), Assis, Sao Paulo, Brazil The efficiency of embryonic chimerism tends to decrease when embryos in advanced stages of development, i.e. post-compaction, such as morula and blastocysts, are used1. To perform the inner cell mass (ICM) transfer to a recipient trophectoderm (TE) it is essential to use embryos at an advanced stage of development and with well-established blastocoels. In mice, few studies have tried the aggregation between ICM and TE and, so far, only microinjection methods have been used for such reconstructions2, 3. The aim of this study was to validate, in mice, the technique of blastocyst reconstruction using the method of ICM and TE aggregation. Swiss Webster females aged between 21 and 45 day, were superstimulated and placed to mate according to Mancini et al. (2008; Transgenic Research 17:1015). At 3.5 days post coitum (dpc), expanded blastocysts were recovered by uterine flush and were sectioned with microblade assisted by micromanipulators (NK2, Eppendorf, Germany) and mounted on an inverted microscope (Eclipse Ti, Nikon, Japan) in order to isolate ICM and TE. The section was designed to be tangential to ICM and to produce two fragments, one with just TE and another with the whole ICM and a minimum amount of the TE. The joining and subsequent aggregation were tested between pairs ICM + TE (n = 28) from different blastocysts, i.e. the ICM and TE of the same pair did not come from the same blastocyst. After joining, pairs were cultured in vitro for 24 h (37°C, 5% CO2 and saturated humidity). The parameter used to detect a chimera in the postculture structure was the image of a single and cohesive cell mass or a strongly aggregated mass spannning more than half of structure total diameter. The viability of the 56 sectioned structures was observed after 24 h of culture by the parameter of cellular reorganization and blastocoels re-expansion. The aggregation rate of the reconstructed blastocysts (chimerism rate) was 25% (7/28) and the viability of sectioned structures (ICM and/or TE) was 84% (47/56). Despite of low adhesion potential of embryonic cells after compaction, the proposed aggregation method for blastocyst reconstruction technique was considered feasible to the chimera production. (1) NOGUEIRA, M.F.G. et al.; 2010. Transgenic Research, 19:344–5 (2) ZHENG, Y.L. et al.; 2005. Zygote, 13:73–7 (3) SOTOMARU, Y. et al.; 1997. Theriogenology, 48:977–84. Financial support: FAPESP, Brazil.
1183 111. Use of tolfenamic acid in recipient mice given at the moment of surgical embryo transfer Geraldine Schlapp1, Lucı´a Goyeneche1, Gabriel Ferna´ndez1, Alejo Menchaca2, Martina Crispo1 1
Unidad de Animales Transge´nicos y de Experimentacio´n. Institut Pasteur Montevideo., Montevideo, Uruguay, 2 Instituto de Reproduccio´n Animal Uruguay, Montevideo, Uruguay Administration of non-steroidal anti-inflammatory drugs (NSAIDs) to embryo recipient mice is a common practice in transgenic facilities, optimizing postoperative care and wellbeing of animals. The mechanism of action of NSAIDs includes anti-inflammatory, analgesic and antipyretic effects, but may also have a positive effect on the maintenance of pregnancy since they suppress cyclooxigenase enzymes inhibiting the synthesis of prostaglandin F2alpha, the main hormone that triggers luteolysis. Use of flunixin meglumine and carprofen in mice has already been reported, but tolfenamic acid, a NSAID commonly used in farm animals, has not been described in mice yet. The objective was to evaluate the effect of tolfenamic acid administration on pregnancy and birth rates after embryo transfer surgery in recipient mice. A total of 73 Swiss or B6D2 foster females at 0.5 dpc were transferred by oviduct with 1233 B6SJL F2 zygotes (13–20 per female) that were previously microinjected or re-derived, and assigned to three experimental groups to be subcutaneously injected with 1 mg/kg tolfenamic acid (Tolfedine, Vetoquinol, France; TA group, n = 27 recipients and 434 embryos), or 2.5 mg/kg flunixin meglumine (Niglumine, Calier, Spain; FM group, n = 27 recipients and 510 embryos), or bi-distilled water (control group, n = 19 recipients and 289 embryos). Pregnancy rate (pregnant/transferred recipients) and birth rate (number of pups born per transferred embryos) were analyzed by logistic regression. Pregnancy rate was higher for TA treated females (18/27, 66.7%) than for FM (8/27, 29.6%; P \ 0.05) and showed a tendency versus control group (8/19, 42.1%; P \ 0.1). Birth rate was higher for TA (69/434, 15.9%) than for FM (49/510, 9.6%) or control group (16/289, 5.5%) (P \ 0.05). We suggest that tolfenamic acid administration at the moment of surgery for embryo transfer, improves pregnancy and birth rates in recipient mice. We thank Dr. S. Kmaid from Universal Lab for kindly providing Tolfedine.
112. Use of C57BL/6/EGFP mouse testicular cells to validate the technique of microinjection in embryonic chimera production Daniela M. de Souza, Hugo Fernandes, Patrı´cia V. Silva, Bruno Cazari, Pablo D. Moc¸o, Bruna C. S. Campanha, Isabele P. Emanuelli, Marcelo F. Gouveia Nogueira Dept. of Biological Sciences, FCL, Sao Paulo State University (UNESP), Assis, Sao Paulo, Brazil Among the techniques to produce chimeras, microinjection (MI) of embryonic stem cells (ESC) into blastocysts—or in the perivitelline space (PVS) of the embryos with 4–8 cells—is one of most popular. A well-established training model for this technique could be very useful when ESC were not available
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1184 and that was able to identify the injected cellular components and their subsequent aggregation within the embryo. Hence, we aim to validate, in mice, a training model for MI in embryos (Swiss Webster, SW) using a pool of EGFP cells derived from testis of C57BL/6/EGFP strain. Embryos were recovered from prepubertal females SW (n = 20) superstimulated and mated according to Mancini et al. (2008; Transgenic Res 17:1015). As an alternative to the classical MI in blastocysts (Nagy et al., 2003; Manipulating the Mouse Embryo 3rd ed., CSHL), in this study were used 4 to 8 cells embryos (collected at 2.5 dpc). Embryos from the same female were randomly allocated to three groups: control (C, n = 17), embryos not subjected to MI; perforated (P, n = 15), embryos submitted to perforation by micropipette, without cell injection; and microinjected (MI, n = 32), embryos perforated and submitted to PVS injection with 6 to 8 cells from EGFP testis. After manipulation, embryos from all groups underwent 24 h of in vitro culture (37°C, 5% CO2 and saturated humidity). The viability and quality of the embryos (according to the general and specific criteria; IETS Manual, 1998 and Nagy et al., 2003, respectively) and, in group MI, the fluorescence of testicular cells, were evaluated pre and post-culture. The results were analyzed by X2 test (total frequency observed) and ANOVA (considering the four replicates) with significance being considered when p \ 0.05. There was no difference among mortality rates of the groups (5.9, 26.7 and 25.0% for C, P and MI, respectively). The percentage of embryos that have retained the quality, after 24 h of culture, was different (p \ 0.01) among groups C, P and MI (94.1, 73.3 and 43.8%, respectively). It was obtained one chimeric blastocyst in the MI group (3.1%, 1/32). Considering the proposed conditions, this model for training of MI of EGFP testicular cells in the PVS was feasible and practical to acquire skills, when ESC are not available. Moreover, the method allows easy identification of injected and, eventually, aggregated cellular components. Finally, the source for EGFP testicular cells could be obese or senile EGFP males (scheduled to be sacrificed). Financial support: FAPESP of Brazil.
113. Novel embryonic stem cell resources for the laboratory mouse Laura Reinholdt, Anne Czechanski, Stephanie Dion, John Kulik, Judy Morgan, Stephen Murray, Gareth Howell, Simon John, Leah Rae Donahue The Jackson Laboratory, Bar Harbor, Maine, USA Embryonic stem cells from mice of or related to 129 Sv/ImJ were among the first ES cell lines to be derived and used for the production of genetically engineered mice. These 129 ES cell lines, along with recently available lines from C57BL/6 have proven to be the most robust ES cell lines available for gene targeting. Unfortunately, the availability of embryonic stem cell lines from other inbred strains lags behind. Significant strain dependent differences exist in the efficiency of ES cell line derivation and in the stability of newly established lines. Many strains, like NOD, CBA and DBA, are considered ‘recalcitrant’ because repeated derivation attempts failed to yield targetable, germ line competent ES cell lines. To overcome strain recalcitrance, we aimed to develop derivation
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Transgenic Res (2011) 20:1139–1189 protocols that take advantage of recently published innovations in culture conditions that maximize pluripotency and selfrenewal, including the growth of emergent ES cell lines in the presence of inhibitors of GSK3 and FGFR/MEK and ERK signaling pathways. To define the most efficient protocol for the derivation of ES cell lines from recalcitrant strains, we asked whether derivation and culture in serum free media containing 3 inhibitors ?LIF (3i ? LIF) would provide improved results over our previously established derivation (ES cell media with knockout serum replacement ?1i ?LIF) and growth (standard ES cell media with serum ?1i ?LIF) conditions. By systematically deriving a large number of ES cell lines from the recalcitrant strain, DBA/2 J, we found that derivation and culture of ES cell lines in serum free media with 3 inhibitors results in ES cell lines with robust expression of NANOG and higher frequencies of chimerism following injection into C57BL/6 J host embryos. Moreover, we show, for the first time, genet targeting and robust germ line contribution of targeted clones from the recalcitrant inbred strain DBA/2 J. Based in part on these data, the Genetic Resources Sciences group at the Jackson Laboratory has now established an ES cell derivation program. With funding from the Mutant Mouse Regional Resource Center at The Jackson Laboratory, as well as funding from NICHD and the ALS alliance, the focus of the program is to derive new ES or iPS cell lines from inbred and genetically engineered strains of mice.
114. Establishing Cre strain resources for conditional mutagenesis Stephen Murray, Caleb Heffner, C. Herb Pratt, Randall Babiuk, Michael Sasner, Cathleen Lutz, Stephen Rockwood, James Kadin, Martin Ringwald, Janan Eppig, Leah Rae Donahue The Jackson Laboratory, Bar Harbor, ME, USA Large-scale mouse gene targeting projects, such as KOMP, EUCOMM, NorCOMM, and TIGM (collectively, the IKMC), promise to deliver a vast number of conditional-ready loxPflanked alleles to the scientific community. To capitalize on this IKMC resource will require that a large, diverse set of well-characterized Cre driver lines are available to researchers around the world. Unfortunately, at present, most existing Cre driver mouse strains are not available from public repositories and until recently, there was no single database that proposed to house comprehensive information about the functionality of Cre driver strains available to the scientific community. Moreover, despite the best efforts of those developing new Cre lines, the fidelity of Cre activity is not always ideal. Many difficulties have been reported in various Cre lines, including mosaic or incomplete deletion in a target tissue/cell type, inconsistent activity, expression in non-target tissues, and/or Cre-related toxicity. In many cases, however, these data are not reported or available to the potential user. To address these issues, The Jackson Laboratory (JAX) has committed to increasing the number of Cre lines available to the scientific community. The JAX Cre Repository currently houses and distributes more than 200 lines, including 156 that are currently distributed as live colonies. In addition, we
Transgenic Res (2011) 30:1139–1189 initiated a project to add value to these strains by comprehensively characterizing Repository Cre lines. We have developed a comprehensive pipeline for the characterization of Cre driver strains using a LacZ reporter strain in a wide range of tissues and at multiple time points, including both target and nontarget tissues. Thus far we have completed the characterization of 45 strains, and an additional 44 are underway. Our results indicate the vast majority of Cre driver strains exhibit unexpected recombinase activity in a number of tissue types, highlighting the need for extended analysis. In addition, we have also developed the Recombinase (Cre) Portal (www.creportal.org) to provide critical data about recombinase constructs, the driver/promoter contained, whether they are inducible (and by what), availability through public repositories, and publications describing the construct and conditional mutagenesis work done using them. The database provides annotations and images of tissues, anatomical structures, and ages assayed that define the specificity of the Cre allele. Together, these resources provide the user with essential information to make informed judgments about the suitability of a particular strain for their experiments and easy access to these strains, enhancing the impact of large-scale mouse mutagenesis projects.
115. Production of transgenic sheep by inhibin a shRNA Xiangyang Miao, Ruijie Zhang Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China RNA interference (RNAi) has emerged as a powerful method directed towards regulating gene expression in animals. This method uses gene specific double-stranded (ds) RNA to knock down gene expression at the level of mRNA. Here, we describe the first application of this method to regulate an endogenous gene in sheep. This study investigated the use of RNA interference (RNAi) to knockdown the inhibin alpha subunit (INHA) expression in Small Tail Han sheep to produce transgenic sheep with high fecundity using a gonad-injection transgenic method. Four lentiviral vectors, two negative control vectors, and one expression vector for the INHA of Small Tail Han sheep were constructed. The four shRNA vectors and two negative control vectors were co-transfected with the INHA expression vectors into 293T cells to evaluate the knockdown effect of the four shRNA vectors by real-time PCR (rtPCR) and Western Blot. Lentiviral particles, including those expressing INHA shRNA which had the largest knockdown effect, were used to treat forty six female Small Tail Han sheep for integration into genome of germ cells. Semen was collected from male Small Tail Han sheep and used to inseminate female Small Tail Han sheep. Blood samples were collected from 4 to 5-month old lambs produced from the artificial insemination procedure. Analysis of the blood samples was performed by rtPCR and Western Blot for determining if the offspring were transgenic. Subsequently, 41 lambs were produced. Nine of which, 45 months old, were detected. Four were proved to be transgenic. Transgenic-positive Small Tail Han sheep were obtained by a gonad-injection transgenic method and suggests that
1185 transgenic manipulation of Small Tail Han sheep through an RNAi approach using gonad-injection is feasible. With this technology, transgenic Small Tail Han sheep with higher fecundity can be produced. This method is a promising approach for efficient production of transgenic animals. This work was supported by a grant from The Major Science and Technology Project of New Variety Breeding of Genetically Modified Organisms(2009ZX08008-004B),the National High Technology Research Development Program of China (863 Program) (2008AA10Z140), the National Natural Science Foundation of China(30571339) and the Innovation Research Foundation of CAAS(No.2004-CAAS-1).
116. Study the therapeutic effect of the high activity FIX variants using hemophilia B mouse model Yu-Chen Hsu1, Chung-Yang Kao1, Shu-Jhu Yang1, Chia-Ni Lin1, I-Shing Yu1, Mi-Hua Tao2, Hua-Lin Wu3, Guey-Yueh Shi3, Po-Ku Chen3, Shu-Rung Lin8, Yung-Li Yang4,5, Ming-Ching Shen6,7, Shu-Wha Lin1,4 1 Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan, 2Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, 3Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, 4 Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan, 5Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan, 6Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan, 7Department of Internal Medicine, Changhua Christian Hospital, Changhua, Taiwan, 8Department of Bioscience Technology, College of Science, Chung Yuan Christian University, Taoyuan, Taiwan
The deficiency or dysfunction of coagulation factor IX (FIX) results in the X-linked bleeding disorder, Hemophilia B. The conventional treatment of hemophilia B patients involves intravenous infusion of plasma-derived or recombinant FIX. In previously study, we generated the FIX variants containing three point mutations (V86A/E277A/R338A, FIX-Triple) with 13-fold higher specific clotting activity compared with FIX wild-type (FIX-WT). The higher specific clotting activity FIX variants can reduce FIX protein usage in protein replacement therapy and viral dose in gene therapy. In this study, we generated a novel FIX variant with three replacements at residues 86, 277, and 338 (V86A/E277A/R338L, FIX-TripleL) in which the leucine residue 338 we included was reported with fivefold to tenfold higher specific clotting activity than FIXWT. In specific clotting activity assay, the purified FIX-R338L, FIX-Triple, and FIX-TripleL proteins respectively revealed 8, 11, and 15-fold higher than FIX-WT. In protein replacement therapy for hemophilia B mice, FIX-R338L, FIX-Triple, and FIX-TripleL respectively resulted in fourfold to eightfold greater clotting activity than FIX-WT. Additionally, hemophilia B mice treated with serotype 8 recombinant adenoassociated vectors expressing either FIX-R338L, FIX-Triple, or FIX-TripleL exhibited 7, 9, and 14-fold higher specific clotting activity than FIX-WT, respectively. Our results indicate
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1186 that the FIX-TripleL variant exhibits significantly enhanced clotting activity relative to FIX-WT and the other gain-offunction FIX variants.
117. Generation of mice chimeras by a new tool for piezo-assisted micromanipulation Marco Schneider TaconicArtemis GmbH, Koeln, Germany Piezo-assisted micromanipulation is a powerful and proven technology for the generation of transgenic mice. The method, however, is very sensitive with regard to reproducibility and efficiency. Reproducible set-up and parameter setting have a great influence on efficiency rates. Here we evaluate a new device for piezo-assisted micromanipulation, the PiezoXpert whereby Balb/cJBomTac mouse blastocysts were injected with C57BL/6- ES cells to generate chimeras. Our results show that the new device leads to a 5% higher rate of liveborn pups (per injected blastocysts) and to a 4% higher rate of chimeras (per injected blastocysts) in comparison to the previously used setup. In addition we visually observed a better recovery of injected blastocysts within 2 h after injection of ES cells. These results indicate that micromanipulations with the PiezoXpert is a less harmful procedure for injection of ES cells. The results will be discussed in detail and an overview of the features of the device will be given.
Transgenic Res (2011) 20:1139–1189 recombination. Point mutations and other cassettes can also be introduced into genes in the genomic context provided by the BAC by recombineering. The consequences of these changes are studied by transfecting BAC DNA into cultured cells or by preparing BAC transgenic mice. Human disease genes caused by point mutations can be introduced to develop relevant in vivo disease models. Easily detected reporter genes allowing for straightforward detection of gene expression in cells and transgenic mice. A series of specialized plasmids were developed to prepare transgenes and targeting vectors exclusively homologous recombination with PCR amplified DNA fragments or plasmids and BACs. Simple procedures, such as reporter knockins can be prepared and verified by pulsed field gel restriction mapping and insert sequences in a few weeks. Recombineering was used to knockin eGFP or DsRed-MST reporters in-frame in genes of interest, insert single point mutations at userdefined locations, build targeting vectors for the manipulation of the mouse genome. A series of genetic modifications and subcloning of genomic fragments of various sizes were prepared to analysis genetic control elements in animal models. BAC transgenic mice prepared from these BACs are used for lineage tracing in developmental models, to inactivate conditional alleles of genes in a tissue specific manner, and to model human disease.
119. Evaluation of Sequence Specific Length Polimorphic (SSLP) regions of commonly used mouse strains for marker assisted speed congenics
118. Recombineering bacterial artificial chromosome transgenes
Channabasavaiah Gurumurthy, Scott Kurz, Donald Harms
Michael Zeidler1, Thom Saunders2
University of Nebraska Medical Center, Omaha, USA
1
Since the advent of genetic engineering techniques for modifying the mouse genome, mutant (transgenic, knockout and knock-in) mice have become invaluable tools in basic and biomedical research. Several thousand mutant mice have been generated in the last three decades that have advanced our understanding of gene function. It is well accepted that the genetic background greatly influence phenotypes and that particular strains of mice are better suited for a specific research purpose. Traditionally, mutant mice are generated using strains that have shown exceptional performance in terms of their suitability for production of transgenic or knockout mice lines. For example, historically pro-nuclei from FVB mice are commonly used for transgenic mice production, while ES cells from the 129 strain are used for knockout mice production. However, a given mutation under a particular strain background may limit its use for a specific research purpose. In such a situation the mutation has to be transferred into a strain background of choice through a process called backcrossing-ten generations of successive breeding into a recipient strain of choice to achieve 99.99% congenic for that strain. The availability of sequence information on various inbred mouse strains has facilitated the characterization of ‘microsatellite markers’- small sequence differences between mouse strains that are useful tools in detecting the chromosome regions of origin when two inbred strains of mice are crossed with each other. This lead to the development of ‘‘Marker
Transgenic Animal Model Core, Ann Arbor, USA, University of Michigan Medical School, Ann Arbor, USA
2
Bacterial artificial chromosomes (BACs) contain large segments of chromosomal DNA (average size is 197,000 bp). Transgenic mouse technology provides scientists with a powerful tool to investigate gene function in a small mammal whose physiology resembles that of humans in many ways. The use of large BAC DNA transgenes provides more information to cells and tissues in the mouse so that gene expression occurs at physiological levels in the appropriate cell types while recapitulating normal developmental expression timing. Genomic libraries prepared in BACs for the mouse and human genome sequencing projects are a ready source of large DNA transgenes. BAC recombineering technology is used to modify BAC genomic clones to (1) express proteins resulting from point mutations, (2) mark specific cell populations with fluorescent protein reporters or (3) express exogenous proteins in a cell specific fashion expression. The extensive DNA sequence contained in BACs confers significant advantages in understanding gene expression. Shorter DNA fragments often do not contain enough gene expression information to completely reproduce normal gene expression patterns. The large size of BACs precludes their facile manipulation and mutagenesis by standard molecular biology techniques. Recombineering methods permit the precise insertion of reporter cassettes in BACs by homologous
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Transgenic Res (2011) 30:1139–1189 assisted speed congenics’’ technique that has reduced the backcrossing time from 10 generations to about 5 generations. Many microsatellite markers have been identified for inbred strains and characterized by various researchers. However, the information about the suitability of such markers for speed congenics has not been tested sufficiently and is not readily available. In this study, we tested and standardized 6–10 markers per chromosome for their suitability in marker assisted speed congenics using genomic DNA from common mice strains. The protocols are streamlined such that most markers could be resolved using conventional agarose gel electrophoresis with simple and common equipment available in a standard laboratory. This translates to cheaper costs in comparison to the recently developed SNP based methods that require expensive equipment. We anticipate that the protocols we have established will serve as valuable tools to the research community in their choice of microsatellite markers for speed congenics or linkage analysis.
120. A role for the mitochondrial phospholipid cardiolipin in the development of male germ line Douglas Strathdee, Laurence Cadalbert, Farah Naz Ghaffar, Sheila Bryson, David Stevenson Beatson Institute for Cancer Research, Glasgow, UK Barth syndrome (BTHS) is an X-linked genetic disorder which is caused by mutation of the Tafazzin (Taz) gene. BTHS is characterised by growth delay, cardiomyopathy, muscle weakness. Tafazzin is a transacylase which is important for remodelling of the mitochondrial phospholipid cardiolipin. Maintenance of the normal levels of cardiolipin is critical for mitochondrial function and metabolism. In order to better understand the biology of this disease we set out to generate a mouse model. Using conventional gene targeting we inserted a selectable marker cassette into the endogenous locus. High percentage chimeras carrying the Taz mutant allele appeared to be of a similar size and weight, exhibiting no phenotypes similar to BTHS. However the high percentage chimeras did not have any offspring. Low percentage chimeras only transmitted the host genotype. Subsequent analyses of the ES cells showed that the insertion of the selectable cassette disrupted the normal expression of the Taz gene. Examination of the testes of the high percentage male chimeras showed that they were considerably smaller than wild-type and in addition contained no mature sperm. Histological examination of the mutant testes showed a severe disruption of the normal organisation of cells in the seminiferous epithelium and also an increase in the number of giant multinucleate cells. This suggests a role for a critical role for mitochondrial cardiolipin in male ferility.
121. Humanized mouse models for pharmaceutical R&D: genomic swapping as a viable humanization strategy Wenning Qin, Jeffrey Stock, William Blake, Melanie Allen, Kenneth Miller, Rosalba Sacca Pfizer Inc, X, USA To enable the use or increase the translational value of a mouse model, it is often necessary to replace a murine gene with its
1187 human orthologue. Due to technical constraints, most humanization was historically accomplished with the cDNA sequence of a gene. Much progress has been made in recent years such that genomic swapping can be achieved readily, which offers an attractive option as it preserves the exon/intron structure of a gene and thus, is thought to be less disruptive of the regulatory mechanism governing expression of the gene. We present several mouse models that we created within Pfizer that have been humanized to a varied extent, from a few exons to the entire gene. One example is the thrombopoietin receptor (TPOR) knock in mouse model. Thrombopoietin is a cytokine produced in liver and exerts its effect by binding to the thrombopoietin receptor (TPOR) expressed on CD34? hematopoietic progenitors and the megakaryocytic lineage cells to regulate thrombopoiesis. Orally active, nonpeptidyl agonists of TPOR have been reported by several groups, including those from Pfizer, and all these compounds only bind to TPOR from humans and chimpanzees. To develop a small rodent model for our program, we created a mouse model carrying a chimeric TPOR gene in which the leucine codon at position 490 of the murine gene had been replaced with the histidine codon at position 499 of the human gene. When administered the Pfizer lead TPOR agonist, PF-02542376, our humanized TPOR mouse model showed an increase in platelet counts and the effect is dose dependent. Thus, we conclude that humanization of only exons 8–10, which includes the codon coding for the critical histidine at 499 of the human gene, is sufficient to confer compound binding to the murine TPOR. Furthermore, limited extent of humanization may even be desirable, as it preserves those murine sequences, including particularly the intracellular domain, to maximize interactions with murine proteins for proper signal transduction.
122. A simple method for removal of yeast contamination from ES cell cultures Jennifer Dunlap, Erin McDermott, Carlisle Landel Thomas Jefferson University, Philadelphia, USA Despite best efforts, cell cultures can become contaminated. To some extent, bacterial contamination can be removed by treatment with antibiotics; fungal contamination, on the other hand, can be difficult to eradicate, especially with ES cell cultures that are killed by fungizone, the common anti-mycotic drug. We were faced with yeast contamination of a valuable targeted ES cell clone, and we describe a simple method combining treatment with Fungin and the manual selection of uncontaminated cells to rescue yeast-free cultures of ES cells.
123. Validation of a commercial zing finger nuclease targeting kit for the ROSA26 locus in mice Jennifer Dunlap, Curtis Kugel III, Adam Snook, Laurence Eisenlohr, Carlisle Landel Thomas Jefferson University, Philadelphia, USA Zinc finger nucleases (ZFN) provide a new methodology for gene targeting. We were provided with a commercial kit for targeting the ROSA26 locus in mice by Sigma, and have
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1188 evaluated it in both ES cells in cultures and by direct application to mouse embryos. ZFN targeting represents a simple and direct method for producing knock-ins in this commonly-targeted locus.
Transgenic Res (2011) 20:1139–1189 utility to aid in chimera production as well as maintaining the welfare balance of animals sampled for genotyping. Here we describe the enhancement and development of a commonly available background strain to act as a complimentary donor embryo strain for the use with non-agouti pigmented embryonic stem cells.
124. Management of a large-scale murine genetics facility using integrated data management systems Rhonda Wiler, Vida Asghari, Erik Bierwagen Genentech, South San Fransisco, USA Data management systems are an essential tool for the management of large, complex genetically engineered mouse model (GEMM) resources. Yet, ‘‘off-the-shelf’’ software packages are seldom adaptable to substantially complicated GEMM production operations. While a custom approach is warranted, these often fail to achieve the desired objectives. Here, we present the successful design, development, and implementation of an innovative, scalable, and data-rich solution while maintaining data-integrity and avoiding the pitfalls of over-requirement and scope creep. To set this stage, we provide an overview of the operation of a centralized largescale mouse genetics facility capable of managing over 1,000 active breeding colonies and over 400 individual users. Then, we describe the approach taken to design, develop, and implement a novel colony management system which utilizes data collection via wireless technology. Finally, we exhibit how this colony management system saves over one-million dollars per year in operating costs and allowed for a seamless transition to a new facility located 80 miles from the main campus.
125. The development of a complimentary embryo donor colony for C57BL/6 derived stem cells James Bussell, Richard Houghton, Edward Ryder, Jennifer Salisbury, Ramiro Ramirez-Solis, William Skarnes Wellcome Trust Sanger Institute, Cambridge, UK The use of coat colour differentiation in chimera and F1 progeny has been an important component of establishing genetically modified knockout mouse colonies since the first Embryonic Stem Cells (ESC) for gene manipulation were identified in the early 1980’s. These ESC’s were propagated in 129 backgrounds such as the 129SvEv and 129P2/Ola which derive their pigmentation from the agouti coat colour locus. As the technology developed and stable pluripotent stems cells became available the ability to inject the ESC’s into a contrasting donor embryo such as the C57BL/6 substrains ensured that the resulting chimeras could be quickly assessed for both initial contribution and the subsequent level of chimerism. Matings to obtain germ line transmission could then use the colour differentiation that ESC derived germ cells would provide when mated to a complimentary background where the agouti allele would remain dominant. With the advent of ESC derived from a C57BL/6 origin and the subsequent need to microinject into albino embryos the use of coat colour differentiation has become limited. The ability to use the coat colour differentiation is important both from as a
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126. New rapid and improved methods for genetically modified rat production: custom knockout, knock-in and transgenic rats Eric Ostertag1, Joseph Ruiz1, Derek Jantz2, Michael Nicholson2 1
Transposagen Biopharmaceuticals, Inc., Lexington, USA, Precision BioSciences, Durham, USA
2
Due to their size, physiology, and toxicology, rats are often superior to mice as model organisms. Until recently, access to custom genetically modified rats has been limited due to high production costs, long timelines, restrictive licensing terms, and the possibility of unwanted mutations associated with previous technologies, such as zinc finger nucleases (ZFNs). In order to generate custom rat models that mitigate or eliminate these issues, Transposagen and Precision BioSciences have combined rat spermatogonial stem cell (SSC) technology with meganuclease (MN)-based Directed Nuclease Editor (DNETM) technology for the creation of knockout and knock-in rats and with the piggyBacTM Genetic Modification System technology for the creation of transgenic rats. We demonstrate the use of DNE technology to create mutations in the rat Rag1 gene in rat stem cells at a frequency of 4.7%. Multiple deletions were identified at the MN binding site, ranging in length from 16 to 40 base pairs. These results indicate that engineered meganucleases can be used to produce animal models and should prove to be an attractive alternative to ZFNs. We also demonstrate the creation of transgenic rats using piggyBacTM technology, offering significant advantages over pronuclear injection methods. Importantly, rat SSC-based methods have significant advantages over methods using embryonic stem cells or embryos, such as the avoidance of chimera production, thereby offering major reductions in timelines for the production of genetically modified rats. Importantly, these technologies should allow facile genetic modification of any mammalian animal model.
127. New transgenic mouse models for familial and sporadic Alzheimer’s disease Diego Mun˜oz1,2, Almudena Ferna´ndez1,3, Marta Cantero1,3, Carmen Go´mez4, Javier Martı´n4, Sagrario Ortega4, Marı´a Jesu´s Bullido2, Fernando Valdivieso2,5, Lluis Montoliu1,3 1
Centro Nacional de Biotecnologı´a (CNB-CSIC), Madrid, Spain, 2Centro de Biologı´a Molecular ‘‘Severo Ochoa’’ (CBMSO-CSIC/UAM), Madrid, Spain, 3Ciber de Enfermedades Raras (CIBERER-ISCIII), Madrid, Spain, 4 Centro Nacional de Investigaciones Oncolo´gicas (CNIO), Madrid, Spain, 5Neuron Biopharma SA, Armilla, Granada, Spain
Transgenic Res (2011) 30:1139–1189 Most animal models generated in mice, to date, for the study of the Alzheimer’s disease (AD) are of limited use for the sporadic form of AD, accounting for 99% of the cases of this devastating disease. In particular, in most cases the corresponding gene constructs fail to be expressed at the correct endogenous levels of loci whose expression are aimed to be investigated, thereby rendering these models not adequately suited for studying the ethiology of this neurodegenerative disease. In addition, other animal models of AD, focused in the familial or genetic forms, require several mutations and/or the overexpression of up to three independent loci resulting in multiple genetically modified mice that do not correspond to human cases. With these limitations in mind, we set up to establish new transgenic mice that could be useful for our understanding of AD, both familial and sporadic forms. We decided to express the human APP locus correctly in mice, in their endogenous tissues, according to their genomic regulatory elements, as included within a yeast artificial chromosome (YAC). A new YAC was prepared by homologous recombination in yeast cells, removing neighboring genes from previously-used constructs, genes whose expression could interfere with that of the APP locus. Also, from this modified YAC we further decided to include some forms of the genetic AD and, with this aim, prepared mutated versions of the locus, also by homologous recombination in yeast. The corresponding YAC transgenic mice carrying these two genomic-type constructs were obtained by two independent methods, namely: direct microinjection of YAC DNA into mouse fertilized oocytes and the lipofection of YAC DNA into ES cells and the subsequent derivation of the corresponding transgenic mouse lines from the germ-line transmitting chimeras that could be obtained. We plan to expose and challenge these transgenic mice to different enhancer factors that have been associated with the risk or the development of AD. We envisage that these animal models could help shedding some light on the causes and factors that lead to AD, and therefore could help us to understand what else could be possibly done to prevent and treat this life-threatening disease. We will report the current status of our studies.
128. High, stable and heritable production of human lactoferrin in milk of transgenic animals Elena R. Sadchikova1,2, S.G. Georgieva1,2, A.N. Krasnov1,3, A.V. Deikin1,3, I.L. Goldman1,2 1 Department of Transgenesis, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia, 2 Transgenepharm LLC Skolkovo Innovation HUB Development Foundation, Moscow, Russia, 3 Transgenebank, Moscow, Russia
1189 Past experience in obtaining transgenic animals that produce milk with biologically active human proteins convincingly shows that level of expression of the protein of interest substantially depends on the transgene design, its site of integration in the genome, the transgene copy number, the period of lactation, and the type of animals. Scientists of the Institute of Gene Biology Russian Academy of Sciences have created transgenes that produce high levels of human lactoferrin in the milk of transgenic animals. These constructs were tested in mice, obtained via pronuclear microinjection of DNA. In total there were 100 founder transgenic mice with human lactoferrin researched as well as up to 5,000 offspring from those founders. The following factors were analyzed: frequency of transgene integration; number of copies integrated; tissue specificity of transgene expression; human lactoferrin concentration in the milk of transgenic animals; germline transmission of transgene and protein concentration dynamics over several generations; lactoferrin concentration dynamics though the lactation period; physical, chemical and biological consistency of recombinant human lactoferrin with natural lactoferrin; state of health and breeding potential of transgenic animals. The percentage of microinjected zygotes resulting in transgenic founders varied from 0.3 to 4.5% and the percentage of live pups that were transgenic varied from 4.2 to 46.2%. The transgene copy number varied from 1 to 230. Human lactoferrin expression was tissue specific. Its level of expression depended on the number of transgene copies. It was stable through the lactation period. Concentration of human lactoferrin in the milk varied from 0.2 to 40 g/l depending on type of transgene and individual animal. The best transgene produced stable and economically meaningful concentrations of human lactoferrin in several generations of transgenic mice with average level of 16.7 g/l. Maximum concentration of recombinant human lactoferrin of one particular family was up to 160 g/l in F1 and remained high during three lactation periods (160, 95, and 105 g/l, respectively). Concentration was also high in offspring’s milk (over 80 g/l). Via microinjections the first transgenic male goats were produced carrying the best constructs. Their daughters produced milk containing 10.8 g/l of human lactoferrin. Chemical, physical and biological characteristics of such lactoferrin were identical to natural lactoferrin. These transgenic goats are being actively cultivated. State of health and breeding potential of all transgenic animals were normal. The conclusion is that such transgene constructs will produce transgenic goats that can be used as commercial producers of recombinant human lactoferrin.
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