Transgenic Research
5, 4 8 7 4 9 1
(1996)
SHORT COMMUNICATION
Efficient targeting of the 11-4 gene in a BALB/c embryonic stem cell line NANCY NOBEN-TRAUTH and B I R G I T L E D E R M A N N
1~, G E O R G E S 2.
KOHLERI"~,
KURT
BURKI 2
1Max Planck Insitut fiir Immunbiologie, Stfibeweg 51, D-79108 Freiburg, Germany ePreclinical Research, Sandoz Pharma Ltd, CH-4002 Basel, Switzerland (Fax: +41 61 324 29 90) Received 25 July 1995; revised 7 September 1995; accepted 8 September 1995
Embryonic stem (ES) cell lines have been derived from the inner cell mass of day 3.5 blastocysts of the inbred mouse strain BALB/cJ. Twenty-three lines were karyotyped and three were selected for injection into C57BL/6J host blastocysts. Two of the three lines, BALB/c-I and BALB/c-IV, produced germ-line chimaeras. The suitability of the BALB/c-I line for gene targeting experiments was tested by transfecting a targeting construct for the interleukin-4 (IL-4) gene. Transfected BALB/c-I cells exhibited efficient homologous recombination of the targeting vector and transmitted the induced mutation through the germline. This newly-characterized BALB/c-ES cell line thus provides an alternative to the traditional 129-derived and the recently described C57BL/6 embryonic stem cell lines, and will be useful in disrupting genes involved in the immune system. Furthermore, the genetically pure BALB/c IL-4 deficient mice will aid in studying the role of IL-4 in several infectious disease models in which the BALB/c mouse is a susceptible strain.
Keywords: BALB/c; embryonic stem cell line; gene targeting; interleuldn-4; homologous recombination
Introduction
Gene targeting in embryonic stem cells has proven to be a powerful method to investigate the function of a particular gene in vivo (Capecchi, 1989). The vast majority of ES cell lines used so far in gene-targeting experiments (D3, CCE, AB1, E14TG2a) have been derived from blastocysts of sublines of the mouse strain 129/Sv. Historically, the 129 mouse was chosen for establishing ES cell lines because of the high incidence of spontaneous testicular teratomas in this strain (Stevens, 1973). Embryonic carcinoma lines were established (Strickland, 1981) paving the way for the derivation of the embryonic stem cells lines used today. The time-consuming and often risky *To whom correspondence should be addressed. :~Present address: The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA. tThis work is dedicated to the memory of Georges Krhler who died during the completion of these studies. 0962-8819 9
1996 Chapman & Hall
endeavor of defining the conditions for growth and evaluating germline competence has deterred the establishment of ES cell lines from other strains. Nonetheless, ES cell lines derived from inbred mouse strains other than 129/Sv have been successfully used, including the C57BL/ 6 derived ES cell lines BL/6-III (Zou et al., 1993; K/igi et al., 1994; Yu et al., 1994), Bruce 4 (Krntgen et al., 1993), and a hybrid C57BL/6 • CBA ES cell line (TT2) (Saga et al., 1992). While most of the deletional mutants have been generated in 129 ES cells, there are disadvantages in using this particular strain to target genes that are specifically expressed in cells of the immune system. In contrast to mutations of developmental or structural genes, which often give apparent phenotypes, immunological knockouts often require antigenic or infectious challenge in order to observe an altered phenotype. Such models of immunization with antigen or tumours, or infection with pathogens have historically been characterized in common inbred mouse strains such as BALB/c or
488
Noben-Trauth et al.
C57BL/6. With the emergence o f targeted immunological mutants, however, it has become necessary to define the immune responses of the 129 mouse in these systems, or to initiate time-consuming backcrossings o f the 129 knockout to well-characterized strains. The 129 strain poses the additional complication o f being genetically resistant to infections with parasites such as Leishmania major (DeTolla et al., 1981) and Schistosoma (Coulson and Wilson, 1989), and to the murine AIDS (MAIDS) virus (Morse et al., 1992). The inherent resistance of the 129 strain has contributed to the difficulties in analysing the response of targeted mutant strains to these infections. For example, the resistance of 129/Sv and 129/Ola strains to infection with the virus causing MAIDS has caused discrepancies in assessing the role of IL-4 in MAIDS infection. Although I 1 - 4 - / - mice which have been crossed to the MAIDSsusceptible C57BL/6 strain are resistant while the control IL-4+/+ littermates die from the infection (Kanagawa et al., 1993), the phenomenon may be due to an additional genetic factor present in the 129 • C57BL/6 segregating background, and not to the absence o f IL-4 alone (Morawetz et al., 1994). Establishing embryonic stem cell lines from immunologically well-characterized mouse strains would eliminate the need for backcrossing and circumvent segregating genetic backgrotmds. In contrast to the 129 strain, the BALB/c strain is genetically susceptible to a number o f infectious organisms ranging from the baeterial parasite Salmonella typhimurium, and the intracellular protozoa Leishmania spp. to intestinal nematodes such as Nippostrongylus brasiliensis. In many of these infection models, a polarized cytokine response dominated by interleukin-4 is thought to be responsible for the susceptibility to the disease. Evidence for the role of IL-4 in disease exacerbation in these models has been primarily tested by injections o f neutralizing antibody to IL-4 in vivo. The
generation o f an IL-4 deficient strain in BALB/c ES cells will provide a powerful tool to confirm the role o f IL-4 in these infections. Materials and methods, results and discussion The results o f the stem cell isolation experiments are summarized in Table 1. Blastocysts were derived from hormone-treated BALB/cJ females and outgrown in conditioned medium on a layer o f 5637 bladder carcinoma feeder cells. The primary isolation o f ES cells on a feeder layer o f 5637 cells was previously shown to increase the frequency of isolating C57BL/6 ES cell lines by 6-fold when compared to growth on primary embryonic fibroblasts (Ledermann and Bfirki, 1991). After disaggregation of the inner cell mass, the cells were seeded onto primary embryonic fibroblast feeder (EF) cells. Morphologically typical embryonic stem cell colonies were picked, expanded, and analysed regarding karyotype and sex as described (Ledermann and Bfirki, 1991). O f the colonies analysed 74% (17/23) exhibited a modal number o f 40 chromosomes (Table 1). O f those, 16 exhibited Y-specific signals upon Southern analysis. Three o f the male BALB/ cJ lines were injected into day 3.5 C57BL/6J host blastocysts. As summarized in Table 2, the lines BALB/ c-I and BALB/c-IV were both transmitted through the germ-line. The line BALB/c-IV had a lower percentage
Table 1. Isolation of BALB/cJ embryonic stem cell lines on human bladder carcinoma cells (5637) No. of embryos
ES cell lines
%
124
23
18.5 22
Y
40 chromosomes
Germline
17
2*
*3 lines of 22 were tested for ability to colonize the germ-line (see Table
2).
Table 2. Generation of chimaeras with BALB/cJ ES cells
Line
BALB/c-I p9b BALB/c-IV p13 BALB/c-VI.4 p12
2n Karyotype a
Blastocysts injected
Animals born (%)
Chimaeras (%) Male
69%
16l
24(15)
16(67)
50%
60%
6(10)
2(33)
40%
92
0
0
Female
Males tested
Germline chimaeras partial
100%
ll c
2
11d
1
7
1
1
1
1
0
.
.
.
.
.
a36 metaphases scored per cell line bp9 = passage 9 Ccoat colour chimaerism 5-100% dthree males did not transmit BALB/c derived sperm Ten to 20 BALB/cJES cells were injectedinto day 3.5 C57BL/6Jhost blastocysts.After injection,the embryoswere transferredinto day 2.5 pregnantNMRI foster mothers. Chimaeras identifiedby eye and coat pigmentationwere bred to BALB/cJfemales at the age of about 8 weeks. Germlinetransmissionof the ES genome was indicated by albino offspring.
Efficient targeting o f the IL-4 gene in a BALB/c embryonic stem cell line
normal karyotype (40%) which may account for the lack of germ-line transmission. The BALB/c-I line in particular showed a high transmission frequency and therefore was chosen to test its suitability for gene-targeting purposes. BALB/c-I ES cells were transfected with the IL-4 targeting vector p6910 (kindly provided by Manfred Kopf, Basel Institute for Immunology) (Fig. l a). Briefly, the X h o I - S a l I fragment of pMClneo (Stratagene) was inserted into an Xho I site created at a previous Sac I site in exon 3 of the 7.5-kb genomic E c o R I fragment of IL-4. The transcriptional orientation of the neo gene is parallel to the gene and differs from the previously published targeting construct p5420 (Kopf et al., 1993). The vector was linearized with SalI and 25 ~g of DNA was electorporated into approximately 107 BALB/c-I ES cells (passage 16) at 250V and 500 ~F (BioRad). The cells were plated at 5 • 106 cells per 64-cm 2 dishes containing EF feeder cells. Four individual transfections were performed. Selection medium containing LIF and G418 (Gibco, 0.4 mg -1 ml) was added the following day. Neo-resistant colonies were isolated 12 days after addition of selection media and screened for correct homologous recombination events by nested-primer PCR (Nitschke et al., 1993).
A
From 572 clones analysed, 15 were positive for correct recombination by PCR. DNA from four of these clones was analysed by Southern blotting, three of which showed a correct targeting event (Fig. lb). Each of the Southern-blot-positive clones was injected into C57BL/6 blastocysts. Chimaeric male offspring which showed over 40% chimaerism were bred to BALB/c females. Of the nine chimaeras mated, six transmitted via the germline. Clones 1.7D and 5.6D each produced germ-line chimaeric males. The presence of the mutated allele was confirmed by PCR analysis of tail DNA. Positive mice from clone 5.6D were interbred to obtain homozygous knockout ( I L - 4 - / - ) and wild-type litter mates (IL-4+/and IL-4+/+). The functional loss of IL-4 in the BALB/c-targeted mice was assayed by in vivo treatment with anti-CD3 monoclonal antibody as described (Yoshimoto and Paul, 1994). Anti-CD3 treatment in vivo has been shown to activate T cells, inducing high levels of IL-2, IL-4, and IFN-y (Flamand et al., 1990; Scott et al., 1990; Yoshimoto and Paul, 1994). Purified monoclonal antiCD3 antibody 145-2Cll (Leo et al., 1987), (kindly provided by Matyas Sandor, University of Iowa), was injected intravenously into 6-7-week-old female BALB/c
~-
BamHI
HSV-tk
BH 1 2
L_~I
P:6910SI neE ~ 3H . ,~
H
SHH ,,~
E E , ~#,,
S
E S BH 4
H
SHH E
E
HindIlI
Barn H1 HindIII 19 5.5
B ,
19kb
BH 12
489
B
6,6
,.
6,6
noo
probe
A P1 ~P2 1.3 kb PCR
1 kb
in vivo anti-CD3 IFN-?
<8.0
IL-4
]
control
<1.3
+/-~ -/-
<8.0
0
5
10 15 20 25 30 oo/ml
0
2
4 6 Units/ml
8
10
6.6kb
D
5.5kb
-"
Fig. 1. Targeting of the IL-4 gene. (A) The IL-4 targeting vector p6910 is shown along with the wild-type and disrupted IL-4 locus. Clones were screened for correct homologousrecombinationby the presence of a 1.3 kb PCR product followed by Southern blot analysis using Probe A. (B) Southern blot analysis of IL-4-targeted clones. Membranes containing DNA from PCR-positive clones were probed with a 2.5 kb EcoRI-Stu I fragment from the IL-4 genomic clone (Probe A). Digestion of the DNA with Bam HI reveals a wild-type fragment of tgkb and a disrupted allele of 6.6 kb, while digestion with Hin dIII gives a wild-type fragment of 5.5kb and a 6.6kb fragment for the disrupted allele. (C) Assessment of the fimctional lack of IL-4 activity in vivo. The loss of IL-4 was measured by in vivo challenge with anti-CD3 antibody as described (Yoshimoto and Paul, 1994). Control IL-4+/- mice were injected with a hamster monoclonal IgG antibody. Supernatants from splenic single-cell suspensions were assayed for levels of IL-4 and IFN-y by ELISA (Endogen).
490 IL-4 +/- and IL-4 -/- at 12 ~g per mouse. Control IL-4 +/mice were injected with the hamster monoclonal IgG antibody HH16 (kindly provided by Carol Kinzer, Laboratory of Immunology, NIAID, NIH, USA) at 12 ~tg per mouse: Spleens from injected mice were removed 90 rain later and made into single-cell suspensions. Cells were washed twice in HBSS and resuspended in RPMI-Glutamax media (Gibco) containing 10% FCS (Sigma), penicillin (100 U -I ml), streptomycin (100 txg-1 ml), sodium pyruvate (1 mM) and 2-ME (50 mM). 5 • 106 cells per well were cultured in 24well plates and incubated for 1 h at 37 ~ 10% CO2. Supernatants were removed and assayed for levels of IL-4 and IFN-y by ELISA (Endogen). While high amounts of IL-4 were observed in heterozygous mice after anti-CD3 treatment, there were no detectable levels of IL-4 in the homozygous I L - 4 = / mice. The levels of IFN-y were strongly upregulated by anti-CD3 treatment in vivo in both the IL-4+/- and IL4 - / - littermates, confirming the activity of the anti-CD3 antibody. The loss of IL-4 transcripts in the IL-4-mutant mice was also verified by RT-PCR from the spleens of anti-CD3 injected mice (1995, data not shown). With the availability of C57BL/6 and BALB/c-derived ES cells, it is now possible to induce a mutation on the genetic background of choice and to analyse the induced mutation in a different background without laborious and time-consuming breeding. Furthermore, the BALB/cJderived ES cells may be used to generate transgenic mice since it is highly inefficient to produce transgenic mice on this background via pronuclear microinjection due to the overall reduced efficiency in inbred strain backgrounds (Brinster et al., 1985). In a broader scope, the BALB/c ES cell line may also be useful in general gene targeting applications. In situations where a genomic clone was isolated from BALB/c DNA, the isogenic BALB/c ES cell line would be the most appropriate line for obtaining high frequencies of homologous recombination (te Riele et al., 1992; van Deursen and Wieringa, 1992) and would bypass the additional step of isolating a 129-strain genomic clone. The generation of a genetically pure BALB/c IL-4deficient mouse will clearly expediate the analysis of the role of IL-4 in infections with Mycobacterium leprae, Trypanosoma cruzi, and the filarial worm Brugia malayi. In each of these, the BALB/c mouse has been termed an 'IL-4' responding mouse strain, producing high levels of IL-4 upon infection (Huygen et al., 1992; Holt et al., 1993; Pearlman et al., 1993). The cytokine IL-4 has also been shown to be a key factor in determining the outcome of infection with the flagellated protozoan Leishmania major. BALB/c mice produce high levels of IL-4 upon infection and eventually succumb to the disease, while the C57BL/6 mice have
Noben-Trauth et al. lower amounts of IL-4, elevated levels of IFN-y, and are able to resolve the infection (Heinzel "et al., 1989, 1991). Injection of monoclonal antibody to IL-4 at the time of infection induces healing in the normally susceptible BALB/c strain (Sadick et al., 1990; Chatelain et al., 1992). The previously published 129 I L - 4 - / strains (Kilhn et al., 1991; Kopf et al., 1993) are genetically resistant to Leishmania major, and must be backcrossed to the proto-type Leishmania-sensitive BALB/c strain in order to study the infection. Using IL-4 targeted BALB/c mice we will verify and expand on the role of IL-4 in leishmaniasis. The usefulness of a BALB/c ES cell extends beyond infection models to genetic diseases such as diabetes (Leiter, 1985), atherosclerosis (Nishina et al., 1993), and tumour biology. The role of the inflammatory cytokine IL-6 in tumorigenesis, for example, would be more easily studied in the BALB/c mouse strain which can be induced to develop plasmacytomas (Anderson and Potter, 1969). The genetic background may affect the phenotype of the induced mutation. For example, such a straindependent phenotype was noted in the case of the IL-2 mutated gene. IL-2 deficient mice on a 129/ Ola • C57BL/6 segregating background reveal a phenotype of ulcerative colitis (Sdhorle et al., 1991; Sadlack et al., 1993), while on a BALB/c genetic background develop a haemolytic anaemia (I. Horak, 1994, personal communication). Disruption of the same gene in ES cells derived from different strains may expose a range of phenotypic alterations and thus provide a clearer function of the gene in vivo.
Acknowledgements This work was partially supported by the American Cancer Society IM707 and USPHS T32-CA-09217 and the National Cancer Institute CA-66331 (N. NobenTrauth) and the German-Israeli Foundation I 0260.162.02/92 (G. K6hler). We would like to thank Matthias Kunz for excellent technical assistance, Ingrid FiNer and Cathrin Westphal for their excellent microinjection services, Pascale Kropf for measurement of IL-4 and IFN-y, and Barbara Knowles and Ingrid Mtiller for critical review of the manuscript.
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Efficient targeting o f the IL-4 g e n e in a B A L B / c embryonic stem cell line
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