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Thromb-X, NV, C/O Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
Key Words. Mouse • Inbred • Embryonic stem cell • Germline • Pluripotent
D. Collen, M.D., Ph.D., Thromb-X, NV, C/O Center for Molecular and Vascular Biology, University of Leuven, Campus Gasthuisberg, O & N Herestraat 49, B-3000 Leuven, Belgium. Telephone: 32-16-345772; Fax: 32-16-346001; e-mail: desire.collen{at}med.kuleuven.ac.be
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ABSTRACT |
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INTRODUCTION |
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The efficiency of ES cell derivation is greatly strain dependent. Very few ES cell lines are currently available from inbred strains (mostly C57BL/6) other than the 129 strains, and those have generally been obtained with low success rates. Furthermore, ES cells derived from C57BL/6 blastocysts are more difficult to propagate in vitro, generate chimeras less efficiently, and contribute less frequently to the germline than ES cell lines from the 129 strains [3, 4].
It is generally accepted that it is easier and more efficient to generate gene-targeted mice in hybrid genetic backgrounds, a phenomenon referred to as "hybrid vigor" [5, 6]. Genetic heterozygosity is even presumed to be a crucial parameter for postnatal survival of mice derived from ES cells by nuclear cloning or tetraploid embryo aggregation [6].
In the present study, we surprisingly observed that a rabbit fibroblast cell line transduced with genomic rabbit leukemia inhibitory factor (LIF) allowed the efficient derivation and maintenance in culture of ES cell lines in all of the 10 inbred mouse strains tested, including certain strains that were previously considered to be nonpermissive for ES cell derivation. Germline transmission was confirmed for ES cell lines in all of seven strains tested by blastocyst injection, in all of four strains tested by diploid aggregation, and in all of three strains tested by tetraploid aggregation.
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MATERIALS AND METHODS |
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Mouse Strains
ES cell lines were derived from the following commercially available inbred mouse strains: 129/SvEv (Taconic; Germantown, NY; http://www.taconic.com); C57BL/6NTacfBr (Taconic); BALB/cAnNTacfBr (Taconic); DBA/2NTacfBr (Taconic); FVB/NTacfBr (Taconic); C3H/HeNTacMTVfBr (Taconic); 129/SvJ (The Jackson Laboratory; Bar Harbor, ME; http://www.jax.org); CBA/CaOlaHsd (Harlan; Indianapolis, IN; http://www.harlan.com); DBA/1OlaHsd (Harlan), and C57BL/ 6JOlaHsd (Harlan).
Derivation and Culture of Murine ES Cell Lines
ES cells were derived from 3.5 to 4.5-day-old blastocyst-stage mouse embryos, which were collected and plated individually on a 96-well dish covered with a mitotically arrested mouse embryonic fibroblast feeder monolayer, obtained from 12.5-days-postcoitus mouse embryos as described in detail elsewhere [7]. The blastocysts were allowed to attach to the monolayer, and were re-fed every day with the conditioned cell culture medium. After 5-6 days in culture, the inner cell mass (ICM) outgrowth was selectively removed from the (remaining) trophectoderm and replated after trypsinization with 0.25% trypsin/1mM EDTA (Invitrogen Corp.; Grand Island, NY; http://www.invitrogen.com) on a 96-well dish with a mitomycin-arrested murine fibroblast monolayer. The ES cells were grown to subconfluency and gradually plated on larger culture dishes.
Culture dishes were kept at 39°C in a humidified atmosphere of 5% CO2 in air. The ES cells were passaged every 2-4 days onto freshly prepared feeder layers, and were fed every day with new conditioned cell culture medium.
Derivation of C57BL/6N ES Cells in Different Culture Media
C57/BL6N blastocysts were cultured with different culture media: A) enriched basic medium; B) enriched basic medium with added murine LIF (1,000 IU/ml); C) enriched basic medium with added rabbit LIF (10 ng/ml), and D) basic medium conditioned by the rabbit fibroblast cell line transduced with genomic rabbit LIF. After 10 passages, the number of established ES cell lines was counted for each of the culture media. The basic medium was composed of 500 ml DMEM high glucose, 70 ml fetal bovine serum, 13 ml penicillin/streptomycin, 13 ml glutamine, 6.3 µl ß-mercaptoethanol, and 13 ml nonessential amino acids. Enriched basic medium is basic medium to which another 4% (v:v) fetal bovine serum is added. This addition compensates for the difference in serum concentration between conditioned medium (as described in detail elsewhere [7]) and culture medium to which no LIF, 1,000 IU/ml murine LIF, or 10 ng rabbit LIF is added.
Germline Transmission of ES Cell Genomes
The ability of the ES cell lines to colonize the germline of a host embryo was tested by injection of these ES cell lines after 10 or more passages into host blastocysts, or by their aggregation with morula-stage diploid embryos or eight-cell tetraploid embryos, and implantation of these chimeric embryos into pseudopregnant foster mothers using standard procedures [8, 9]. In order to allow easy estimation of the percentage chimerism (i.e., contribution of the ES cell genome to the chimeric offspring), ES cell lines from mouse strains with colored coats (129/SvEv, C57BL/6N, C57BL/6JOla, CBA/CaOla, DBA/2N, DBA/1Ola) were injected in blastocysts or aggregated with diploid morulas of albino Swiss Webster mice, whereas ES cell lines from mice with whiteor cream-colored furs (129/SvJ, BALB/c, FVB/N) were chimerized with black C57BL/6N blastocysts or diploid aggregates. Germline transmission of the ES cell genome was then tested by crossing high-percentage male chimeras with female Swiss Webster or C57BL/6N mice, as appropriate, to establish the ES-cell-line-derived coat color in F1 offspring.
Blastocyst injection was carried out using day-3.5 blastocysts collected from the uteri of superovulated females by flushing with M2 medium (Eurogentec; Seraing, Belgium; http://www.eurogentec.be/hp/hp.htm). Superovulation was induced by injection of 7.5 IU pregnant mare serum gonadotropin (Sigma; St. Louis, MO; http://www.sigmaaldrich.com) followed by injection of 7.5 IU human chorionic gonadotropin (Pregnyl; Organon; Oss, The Netherlands; http://www.organon.com) after a 48-hour interval. The collected blastocysts were washed with and cultivated in M16 medium (Eurogentec) under 5% CO2 in air at 39°C.
ES cell lines were passaged 2 days before microinjection on bare gelatinized dishes. At the day of microinjection, these dishes were trypsinized with 0.25% trypsin/1mM EDTA for approximately 2 minutes at 39°C. The new conditioned cell culture medium was added and the suspension was pipetted to produce a single-cell suspension. After centrifugation (1,100 rpm/min for 5 minutes), the ES cells were resuspended in the new conditioned cell culture medium and kept in the incubator at 39°C.
Blastocyst injection was carried out by injecting 15-20 ES cells of mouse strains with colored coats into host blastocysts of albino Swiss Webster mice, or ES cells of mice with white- or cream-colored coats into host blastocysts of black C57BL/6N mice. After injection, blastocysts were reimplanted (7-8 blastocysts in each horn of the uterus) into 2.5-day pseudopregnant Swiss Webster females, previously mated to vasectomized males.
Diploid aggregation was carried out using ES cells derived from three mouse strains with colored coats and morulas from albino Swiss Webster mice, or ES cells derived from FVB/N mice with morulas from C57BL/6N mice. Therefore, oviducts of superovulated females were flushed 2.5 days after copulation to collect late eight-cell-stage diploid embryos. Zonae pellucidae of these eight-cell-stage diploid embryos were removed by treatment with acid Tyrode’s buffer. Aggregation was performed in M16 medium (Eurogentec) between one eight-cell-stage diploid embryo and a clump of ES cells. The aggregates were cultured in micro-drops of M16 medium until the blastocyst stage and were then reimplanted into the uteri of 2.5-day pseudopregnant Swiss Webster recipient females. The percentage of chimerism of the offspring (portion of the new-born pup presumed originating from the ES cell lines) was visually estimated from the relative coat color determined by the ES cell genome, compared with the coat color originating from the recipient embryo.
Tetraploid aggregation, to obtain entirely ES-cell-derived embryos, was carried out using ES cell lines from three colored coat strains and tetraploid ROSA26 embryos, which express LacZ ubiquitously throughout their entire development [10]. Therefore, oviducts of superovulated ROSA26 mice were flushed 44-46 hours after mating to collect late two-cell-stage embryos. These embryos were electrically fused, and subsequently aggregated as eight-cell tetraploid embryos with the established ES cell lines to form chimeric embryos, which were then implanted in 2.5-day pseudopregnant Swiss Webster recipients, and male offspring were mated with Swiss Webster females to verify their germline transmission capability. The ES cells (almost) exclusively contributed to the development of the embryo proper, and the tetraploid cells to that of the extra-embryonic membranes.
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RESULTS |
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). The efficiency of ES cell line derivation ranged from 5%-66%. In two 129/Sv strains, 61% (in 129/SvEv) and 58% (in 129/SvJ) of the explanted blastocysts resulted in ES cell lines that survived 10 or more passages. In two C57BL/6 backgrounds, the efficiency of ES cell line derivation was above 40%. Surprisingly, a high efficiency of ES cell line derivation was obtained from CBA/CaOla mice, a strain previously believed to be nonpermissive for ES cell derivation [11]. In two series of experiments with the BALB/c strain, established ES cell lines were obtained from 5% and 41% of blastocysts. A success rate of 11% was obtained in the DBA/1Ola strain. Established ES cell lines were obtained from the DBA/2N, the C3H/HeN, and the FVB/N strains with efficiencies of 37%, 31%, and 22%-50%, respectively.
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). These chimeras had the capability to pass the ES cell genome to the next generation, as judged from offspring with the coat color of the ES cell strain after mating with relevant recipient females. On average, more than 20% of reimplanted blastocysts resulted in live offspring from which, on average, more than 60% displayed coat-color chimerism. Most of the tested chimeras transmitted the ES cell line genome to their offspring (Table 2).
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). On average, 30% of reimplanted embryos resulted in live offspring, of which in excess of 35% had a significant ES cell chimerism as estimated from the coat color. Chimeric mice born after diploid aggregation of ES cells from 129/SvEv, C57BL/6N with embryos of the Swiss Webster strain, and FVB/N ES cells with C57BL/6N morulas, all had 100% chimerism. Fifty percent of all chimeric animals born after diploid aggregation of CBA/CaOla ES cells with Swiss Webster morulas showed 100% chimerism.
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). From 3%-30% of the reimplanted embryos of 129/SvEv-derived ES cell lines produced live offspring. All but one of the males tested were germline transmitters. Seven pups (12% of all reimplanted embryos) were born after tetraploid aggregation of a selected C57BL/6 ES cell line at passage 12, and two of the males, randomly selected out of the seven, showed germline transmission. ES cells with germline transmission capability were also obtained after tetraploid aggregation with ES cells from CBA/CaOla mice, a strain previously believed to be nonpermissive to ES cell derivation [11].
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). ICM outgrowth rapidly differentiated before or during the first passage. When either murine or rabbit LIF was added to the enriched medium, the efficiency of ES derivation increased to 34% and 38%, respectively. The efficiency of ES cell derivation increased to 61% when medium conditioned by the rabbit fibroblast cell line transduced with genomic rabbit LIF was used. After one passage, a clear difference was observed in the morphology of the ES cell colonies in the different culture media. Enriched basic medium with added murine or rabbit LIF gave rise to rather flat ES cell colonies, while the use of medium conditioned by the rabbit fibroblast cell line transduced with genomic rabbit LIF resulted in three-dimensional ES cell colonies. When basic medium alone was used, all cells were differentiated after one passage.
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DISCUSSION |
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ES cells used for homologous recombination are commonly derived from strain 129, for which a large number of substrains exist [12]. These 129 substrains exhibit poor reproductive performance, which has prompted inbreeding of chimeras to other strains such as C57BL/6. This approach introduces genetic heterogeneity that precludes generation of gene-targeted mice and isogenic controls (i.e., controls that are genetically identical except for the targeted locus). Indeed, whereas breeding of 129-derived chimeras into C57BL/6 generates hybrid F1 mice that are genetically identical because they inherit one chromosomal complement each from the 129 and C57BL/6 strains, intercrossing of these F1 mice to generate mice homozygous for the target locus produces F2 offspring with a random mix of 129 or C57BL/6 chromosomal DNA throughout the genome. Furthermore, continuous inbreeding of these F2 mice may select for phenotypic changes, such as survival advantage, that are independent of the targeted gene.
These limitations may be partially overcome by continuous inbreeding into a common inbred strain, which generates congenic mice. Six generations of backcross breeding (taking about 2 years) are required before the genetic backgrounds are statistically (>99%) homogeneous. It is becoming standard practice for leading scientific journals to request that phenotypes in genetically modified mice be compared with those of congenic controls. Still, congenic strains contain some parental genomic DNA upstream and downstream from the genetically altered locus. This occurs because, as the modification is bred into the inbred strain, the genetically modified locus, and therefore DNA in the vicinity of (or linked to) the locus, will be selected in each backcross generation. The exact location of the break point between the original and the inbred genomic DNA upstream and downstream from the target locus will depend on where recombination between the two genomes occurs. Therefore, nontargeted littermates and wild-type inbred mice will lack not only the targeted locus but also the closely linked original genomic DNA, which may contain modifier genes.
Ideally, control mice for gene-targeting experiments should be genetically identical or isogenic to the experimental mice. Such isogenic strains, differing only in the presence or absence of the targeted locus, constitute the "gold standard;" in practice, this is only obtainable if inbred ES cell lines of specific genetic backgrounds are available for gene targeting.
Here, a novel culture medium was developed that allowed derivation of germline-competent ES cell lines from nine inbred mouse strains tested, including some strains that were previously considered nonpermissive for ES cell derivation. Germline transmission of ES cells after 10 or more passages was demonstrated after blastocyst injection in each of seven strains, diploid aggregation in each of four strains, and tetraploid aggregation in each of three strains. The present study was confined to the demonstration of germline transmission of established ES lines to the hybrid F1 generation with either albino Swiss Webster (for colored-coat strains) or black C57BL/6N (for albino or light-colored fur strains). Future breeding of isogenic gene-targeted and control mice from genetically altered ES cell lines will require mating of chimeras with the original strain from which the ES cell lines were derived to obtain inbred F1 offspring, combined with genotyping to identify isogenic gene-targeted and control offspring. Such breeding will unveil phenotypic differences between inbred strains, which may have been masked in the present hybrid F1 generation.
Data from the C57BL/6N ES cell derivation in different culture media using our derivation method showed that enriched basic medium did not support ES cell derivation, even when murine fetal fibroblast cells were used as a feeder layer. The addition of LIF alone to the enriched basic medium was sufficient to support ES cell derivation. As shown by others [13], ES cells maintained in the presence of LIF do not only retain their full developmental capacity, but they can also be established de novo by direct culture of early embryos in medium supplemented with LIF [14, 15]. Three other cytokines, oncostatin, ciliary neurotrophic factor, and interleukin-6 in combination with its soluble receptor, have thus far been found to support ES cell derivation [16]. However, the efficiency of ES cell derivation from C57BL/6N mice almost doubled when the medium conditioned by the rabbit fibroblast cell line transduced with genomic rabbit LIF was used. In addition, the morphology of the ES cell colonies in conditioned medium was superior to ES cell colonies derived in enriched basic medium with added murine or rabbit LIF.
Basic medium conditioned by the rabbit fibroblast cell line transduced with genomic rabbit LIF allowed the derivation of ES cells with germline transmission capability from nine different inbred mouse strains. Murine fibroblasts supplemented with LIF are apparently not capable of replacing this unique rabbit fibroblast cell line transduced with genomic rabbit LIF. Although worldwide ES cells with germline transmission capability can readily be established from mouse embryos of strain 129, and with less success from strain C57BL/6, the isolation of ES cells from other strains of mice remains problematic [3].
The factor or factors responsible for the unusual and unexpected properties of this conditioned medium still remain to be identified. No measurable levels of stem cell factor, interleukin-6, ciliary neurotrophic factor, soluble gp-130, fibroblast growth factor, G-CSF, vascular endothelial growth factor, or placenta growth factor-2 were detected in the medium conditioned by the rabbit fibroblast cell line transduced with genomic rabbit LIF using available ELISA-based kits against the human and/or murine factors. However, since there are presently no specific assays against the rabbit homologues of these proteins, an absence of cross-reactivity with the murine or human antibodies and a subsequent false-negative reaction cannot be excluded. Only interleukin-1 and interleukin-11 were found to be secreted in the medium during the conditioning process (data not shown).
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ACKNOWLEDGMENT |
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REFERENCES |
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