SEARCH

SEARCH BY CITATION

Keywords:

  • Embryonic stem cell;
  • Mus spretus;
  • Germline transmission;
  • TNF

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Embryonic stem (ES) cells, which can differentiate into almost all types of cells, have been derived from the house mouse Mus musculus, rat, rabbit, humans, and other species. Transmission of the genotype to the offspring of chimeras has been achieved only with M. musculus ES cells, limiting targeted mutagenesis using ES cells to this species. Mus spretus, which exhibits many genetic polymorphisms with M. musculus, displays dominant resistance to cancer and inflammation, making derived inbred strains very useful in positional cloning and interspecies mapping. We show here for the first time the derivation of ES cells from hybrid blastocysts, obtained by the mating of two different species, namely Mus musculus and Mus spretus, and their use for the generation of chimeric mice that transmit the Mus spretus genotype and phenotype to the offspring. These hybrid ES cells allow the genetic manipulation of Mus spretus, as an alternative to Mus musculus.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The Mus musculus species of mice comprises four well-defined subgroups: M. domesticus, M. musculus, M. castaneus, and M. bacterianus. The classical inbred strains are mosaics of these four M. musculus subgroups [1]. The species closest to M. musculus are M. spretus, M. spicilegus, and M. macedonicus. These three species live sympatrically—within overlapping areas—with M. musculus, but interspecific hybrids have not been reported to occur in nature. That means there is a complete barrier to gene flow between the house mouse and each of these aboriginal species. The absence of gene flow between two animal populations that live sympatrically is the clearest indication that they represent different species [2]. Nevertheless, Bon-homme and colleagues were able to produce interspecific F1 hybrids between each of these aboriginal species and M. musculus in the forced, confined environment of the laboratory cage [3,4]. For many biological studies, use of the classical inbred strains is perfectly acceptable. However, in some cases, it obviously makes a difference to use animals with genomes representative of naturally occurring populations. Therefore, major efforts have been exerted to generate new inbred lines directly from wild mice.

Inbred strains recently derived from mice trapped in nature are generally more resistant to carcinogens and to several types of pathogens. A commonly accepted, but not experimentally demonstrated, explanation is that in the classic laboratory strains some alleles that are important for innate or acquired immune responses have been spontaneously replaced by defective mutant alleles, and that the consequences are largely masked by the protected environments in which these mice are kept [5]. M. spretus, a wild mouse species found primarily in South France, Spain, Portugal, and North Africa, diverged from M. musculus about 3 million years ago and developed into a different mouse species. Many genetic polymorphisms can be detected between strains derived from these two species. Several inbred lines have been derived from M. spretus, including SEG, STF, and SPRET/Ei. These mice display very important phenotypes, such as resistance to lung cancer [6], skin cancer [7], and thymic lymphomas [8]. We recently reported that SPRET/Ei mice (compared with M. musculus strains such as C57BL/6) are extremely resistant to the lethal effects of the proinflammatory cytokine tumor necrosis factor (TNF), a cytokine centrally involved in sepsis, arthritis, Crohn's disease, and many other inflammatory pathologies. Interestingly, all of these phenotypes are also observed in F1 hybrids of the M. musculus-derived C57BL/6 laboratory strain and SPRET/Ei, indicating dominance of the SPRET/Ei-derived alleles. The dominant resistance to TNF was found linked to protective loci on chromosomes 2 and 6 [9].

Since many polymorphisms between SPRET/Ei and most laboratory inbred strains are known, SPRET/Ei mice are often used for mapping and positional cloning of genes [5]. In order to functionally test candidate genes by manipulating the SPRET/Ei genome, ES cells are an invaluable tool. Thus far, however, derivation of germline-competent ES cells has been possible only in mouse strains derived from M. musculus. We report here the derivation of germline-competent ES cell lines from (C57BL/6 × SPRET/Ei)F1 hybrid blastocysts, allowing the SPRET/Ei genome to be genetically manipulated.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Mouse Strains

ES cells were derived from the following commercially available mouse strains: C57BL/6J and SPRET/Ei (both from The Jackson Laboratory, Bar Harbor, ME).

Superovulation

Blastocysts were obtained from the matings of C57BL/6J female mice with SPRET/Ei male mice. Superovulation of C57BL/6J mice was induced by injection of 5 IU of pregnant mare serum gonadotropin (Sigma Chemical Corp., St. Louis, MO), followed by injection of 5 IU human chorionic gonadotropin (Pregnyl; Organon, Oss, The Netherlands) after a 48-hour interval.

Embryonic Stem Cell Derivation

The 3.5-day-old blastocysts were collected by flushing uteri with M2 medium, and then they were plated individually on a 96-well dish covered with a mitotically arrested mouse embryonic fibroblast feeder monolayer, obtained from 12.5 days–postcoitum embryos. The blastocysts were allowed to attach to the monolayer, and were re-fed daily with TX-ES medium (Thromb-X N.V., Leuven, Belgium) [10]. The production method for this ES cell culture medium is described in detail elsewhere [11]. Briefly, it comprises standard Dulbecco's modified Eagle's medium (DMEM), nonessential amino acids, glutamine, β-mercaptoethanol, and fetal calf serum, and is conditioned by an immortalized rabbit fibroblast cell line transduced with genomic leukemia inhibitory factor (LIF). Conditioning of the medium by the transduced fibroblast cell line was carried out until the LIF concentration in the medium reached 15 ng/ml, as determined by enzyme-linked immunosorbent assay (ELISA) for human LIF (R&D Systems, Minneapolis, MN), which cross-reacts with rabbit LIF. After 5–6 days of culture, the inner cell mass outgrowth was selectively removed from the remaining trophectoderm with a micropipette and then was replated after trypsinization with 0.25% trypsin per 1 mM ethylenediaminetetraacetic acid (EDTA; Invitrogen Corp., Grand Island, NY) on a 96-well dish with a mitomycin-arrested murine fibroblast monolayer. ES cells were grown to subconfluency and gradually plated on larger culture dishes, which were kept at 38.5°C–39°C in a humidified atmosphere of 5% CO2 in air. ES cells were passaged every 2–4 days onto freshly prepared feeder layers and were fed every day with TX-ES medium.

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 six passages into host blastocysts and implantation of these chimeric embryos into pseudopregnant foster mothers using standard procedures [12,13]. In order to allow easy estimation of the degree of chimerism (percentage contribution of the ES cell genome to the chimeric offspring), the F1 ES cell lines (derived from mice with an agouti coat color) were injected into blastocysts of albino SWISS mice. Germline transmission of the ES cell genome was then tested by crossing high-percentage chimeras with SWISS mice, to establish the ES cell line–derived coat color in some of the N2 offspring. Blastocyst injection was carried out using 3.5-day blastocysts, collected from the uteri of superovulated females by flushing with M2 medium (Eurogentec, Seraing, Belgium). ES cell lines were passaged on bare gelatinized dishes 2 days before microinjection. On the day of microinjection, these dishes were trypsinized with 0.25% trypsin per 1 mM EDTA (Life Technologies, Paisley, U.K.) for approximately 2 minutes at 39°C. Conditioned cell culture medium (TX-ES medium; Thromb-X, Leuven, Belgium) was added, and the suspension was pipetted to produce a single-cell suspension [10]. After centrifugation at 1,100 rpm for 5 minutes, ES cells were resuspended in the conditioned cell culture medium and kept in an incubator at 39°C. Blastocyst injection was carried out by injecting 15–20 ES cells into host blastocysts of SWISS mice. Blastocysts were then reimplanted (7–8 blastocysts in each horn of the uterus) into 2.5-day pseudopregnant SWISS females, which had previously mated with vasectomized males.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

ES Cell Derivation of (C57BL/6 × SPRET/Ei)F1 Blastocysts

Because of the dominant nature of SPRET/Ei phenotypes, we decided to derive ES cells from (C57BL/6 × SPRET/Ei)F1 blastocysts. We used the highly efficient conditioned medium TX-ES [10], and derived ES cells from 3.5- to 4.5-day-old blastocyst stage mouse embryos, obtained after natural mating or superovulation. From 27 blastocysts, 16 ES cell lines were derived (Table 1), which represents an efficiency of 59%. The undifferentiated character of the established ES cell lines was originally determined by immunochemical staining for the presence of alkaline phosphatase (Vector Laboratories, Burlingame, CA), or for the absence of vimentin and cytokeratin (DAKO A/S, Copenhagen, Denmark). Only ES cell lines that consist of more than 90% of undifferentiated cells were maintained in culture.

Table Table 1.. Establishment of ES cells from (C57BL/6× SPRET/Ei)F1 blastocysts
  • a

    aNumber of blastocysts obtained from one mouse.

  • b

    bStable for 10 or more passages.

  • c

    cPercentage of established stable ES cell lines compared to the number of blastocysts used.

  • d

    Abbreviation: ES, embryonic stem.

Mating procedureCultured blastocystsaEstablished ES cell linesbEfficiencyc
Natural mating3266
Superovulation241458
Total271659

Germline Transmission of (C57BL/6 × SPRET/Ei)F1 ES Cells

Due to male hybrid sterility of F1 animals [14], the female ES cells were our primary interest. The ES cell lines were genotyped using a Y-specific probe [15]. Of the 16 F1 ES cell lines, nine were male and seven were female (data not shown).

To test whether the female ES cells were germline competent, ES cells were injected into SWISS blastocysts and transferred to SWISS pseudopregnant females. In all litters, chimeric mice were born. Coat-color chimerism varied from 5%–100% (i.e., entirely agouti) (Table 2). High-percentage chimeras were crossed with SWISS or C57BL/6 mice, and the progeny were tested for the presence of SPRET/Ei or C57BL/6 alleles by evaluating coat colors. An 80% chimera derived from the female ES cell line B6/SPRET#3 delivered three white, one black, and four agouti offspring (Fig. 1). Microsatellite typing of germline offspring from another chimeric mouse (derived from the female ES cell line B6/SPRET#18) showed that all mice contained SPRET/Ei-specific markers (Table 3). These N2 mice theoretically contain 25% SPRET/Ei, 25% C57BL/6, and 50% SWISS genome (Fig. 2).

Table Table 2.. Germline transmission of (C57BL/6 × SPRET/Ei)F1 embryonic stem cells
  1. a

    #2, #3, and #18 are the numbers of different B6/SPRET ES cell lines.

ES cell lineSexPassage (n)Chimeras/Total offspringChimerism (%)High % chimera mated for germline offspring
B6/SPRET#2M76/215–25No data
B6/SPRET#2M92/4100100% chimeric male × C57BL/6 female: no germline offspring
B6/SPRET#3F620/275–8080% chimeric female × SWISS male: germline offspring
B6/SPRET#3F82/5100100% chimeric female × C57BL/6 male: germline offspring
     100% chimeric male × C57BL/6 female: no germline offspring
B6/SPRET#18F63/820–5050% chimeric female × SWISS male: germline offspring
Table Table 3.. SPRET/Ei-specific markers in chimeric offspring derived from embryonic stem cell line B6/SPRET#18
  • a

    + indicates presence of SPRET/Ei-specific marker; – indicates absence of SPRET/Ei-specific marker.

  • a

    aSample error.

  • c

    Abbreviations: A, agouti; B, black; nd, no data; W, white.

 1 (B)2 (W)3 (A)4 (A)5 (B)6 (W)7 (W)8 (A)9 (A)a
D2mit32++++nd
D2mit417++++nd
D6mit104++nd
D6mit150++++++nd
thumbnail image

Figure Figure 1.. Female chimera and offspring after crossing with a SWISS male. An 80% chimera was obtained after injection of the embryonic stem cell line B6/SPRET#3 into SWISS blastocysts and their subsequent transfer to a pseudopregnant SWISS mouse. The chimera had a litter of eight offspring, including three white, one black, and four agouti mice.

Download figure to PowerPoint

thumbnail image

Figure Figure 2.. Family tree of 50% chimera and offspring derived from the ES cell line B6/SPRET#18. Parental mice (P), used for harvesting blastocysts for ES cell derivation and to make chimeric mice, contain 0% or 100% of SPRET/Ei genomes. ES cells and the chimeric offspring contain 50% SPRET/Ei genomes in a portion of their cells and are therefore called F1. The color or percentage of chimerism of the offspring of the foster mother is indicated. After crossing the 50% chimera with SWISS, nine offspring were born. These mice are called N2 and contain on average 25% of SPRET/Ei genomes. These offspring (and also the two chimeras) were injected with 500 μg tumor necrosis factor (TNF). All mice, except one of the germline offspring, died from the challenge. This surviving mouse was crossed with several SWISS, which resulted in five nests with 8, 6, 9, 10, and 11 offspring. These mice were called N3 and contain on average 12.5% of SPRET/Ei genome. N3 mice were injected with 250 μg TNF. Four offspring survived this challenge, one from the first nest and three from the second nest. Since none of the mice of the other nests survived the challenge (gray triangles), the individual offspring are not shown. Finally, the surviving N3 mice were crossed with SWISS, resulting in N4 offspring, containing on average 6.125% of SPRET/Ei genome. Of the five nests, one survived a challenge of 250 μg TNF. Labels: circles, females; squares, males; pentagons, blastocysts; triangles, complete nest died; gray, dead mice; white, surviving mice.

Download figure to PowerPoint

Germline Transmission of SPRET/Ei-Derived TNF-Resistance Phenotype

Most M. musculus–derived laboratory strains (including inbred C57BL/6 or outbred SWISS) develop a lethal inflammatory shock associated with hypothermia upon challenge with TNF, and die from a dose of 25–50 μg. In contrast, SPRET/Ei or (C57BL/6 x SPRET/Ei)F1 mice are resistant to doses of at least 250 μg. To assess the functional significance of the inherited SPRET/Ei alleles, the nine offspring N2 mice were injected with 500 μg of recombinant mouse TNF. Only 1 out of 9 N2 mice was completely refractory to the injected TNF (mouse 8 in Table 3). Interestingly, this TNF-resistant female was the only one that had inherited the SPRET/Ei-protective loci on chromosomes 2 (D2mit417) and 6 (D6mit104), both of which are necessary for TNF resistance [9]. After crossing this mouse with SWISS mice, the N3 progeny were injected with 250 μg mouse TNF at the age of 8 weeks. Again, in contrast to C57BL/6 and SWISS controls, 4 out of 47 N3 mice survived the challenge, and the survivors were the only ones positive for the markers on chromosomes 2 and 6. These N3 mice had a mean of only 12.5% SPRET/Ei genome. They were further crossed with SWISS mice, resulting in N4 mice that contained 6.25% of SPRET/Ei genome. Only 1 out of 19 offspring survived a TNF challenge of 250 μg; again it was the only mouse that contained both loci on chromosomes 2 and 6 (Fig. 2).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

It has been shown that ES cells can be derived from many different mammalian species, including mouse, rat, rabbit, and humans [1619]. However, transmission of ES-derived genome to the germ cells and further to the offspring has proved impossible in species other than the house mouse M. musculus. Moreover, even within the M. musculus species, certain genetic backgrounds have been reported to be less permissive or even nonpermissive for germline-competent ES cell derivation [2022]. Here, we clearly demonstrate for the first time that ES cells can be derived from hybrid inter-species (C57BL/6 x SPRET/Ei)F1 blastocysts. The efficiency with which ES cells were derived from blastocysts obtained by natural mating (66%) was somewhat higher than from those obtained after superovulation (58%), which may be explained by the detrimental effect of the hormones used for superovulation on the development of preimplantation embryos [23]. However, because of the low number of blastocysts obtained by natural mating, no firm conclusions can be drawn.

The unusually high ratio of male (7) to female (9) ES cell lines could be a consequence of the conditioned medium used, since it was reported that efficiency of ES cell derivation using this medium is much higher than using a conventional medium. The fibroblast cell line used to condition the medium is a rabbit fibroblast cell line. The body temperature of a rabbit is about 38.5°C–39.5°C. Therefore, ES cells were derived and maintained at 38.5°C–39°C in the incubator. Previous ES cell–derivation experiments with inbred mouse strains have already shown that this temperature has no negative effect on the ES cell–derivation efficiency, since it allowed the establishment of ES cells from so-called non-permissive strains [10].

We further demonstrated that the hybrid ES cells are germline competent. The two female ES cell lines that were used were both able to transmit their genotype to the offspring of chimeric mice. In contrast, the male ES cell that was used gave no germline transmission, which was not unexpected due to the male sterility of the hybrid (C57BL/6 × SPRET/Ei) mice [24].

Derivation of hybrid ES cells from (129/Sv × M. casteneus) F1 blastocysts was already demonstrated [25]. However, both mouse strains belong to the same M. musculus species [2].

To our knowledge, this is the first report to show that transmission of the genome via ES cells is possible from mouse species other than M. musculus. There may be two possible explanations for this success. First, the ES cells were derived from F1 blastocysts. It is known that F1 mice can possess higher stress resistance than either of the parentals, so-called hybrid vigor [2], which may have facilitated the derivation of hybrid interspecies ES cells. It has already been demonstrated that by using F1 blastocysts, it was possible to derive ES cells containing 50% NOD (non-obese diabetic) genome, which could not be achieved when starting from pure NOD-derived blastocysts [26]. Second, ES cell derivation has been, until recently, only possible in certain inbred strains of M. musculus. Other inbred strains were reported to be nonpermissive for germline-competent ES cell derivation. However, we made use of the conditioned medium TX-ES, which was recently reported to be highly efficient in establishing germline-competent ES cell lines from several M. musculus strains, even those described to be nonpermissive for ES cell derivation [10].

To demonstrate the biological and functional relevance of the hybrid ES-derived SPRET/Ei genome, we studied the phenotype of the offspring of the chimeras by challenging them with a dose of TNF that is lethal for most inbred strains, but not for SPRET/Ei. The surviving mice were further back-crossed to SWISS until the N4 generation. As expected, only the mice that inherited both SPRET/Ei-derived TNF-protective loci on chromosomes 2 and 6 survived the challenge [9]. These N4 mice and further generations are excellent tools for the identification of the relevant protective genes—for example, by differential expression studies.

Furthermore, since we succeeded in deriving (C57BL/6 × SPRET/Ei)F1 hybrid ES cell lines that show a high level of chimerism and give a good rate of germline transmission, these lines may be used for the genetic targeting of the SPRET/Ei genome. This offers the possibility for using the F1 hybrid lines to study loci that confer the usually dominant traits of resistance to several types of stress, reported to be present in wild mice such as M. spretus [5]. Hence, F1 ES cells offer a powerful tool to target the SPRET/Ei allele and study the consequent phenotypes. Finally, the male F1 ES cells we generated can be used to study the deleterious gene interactions leading to hybrid sterility, which are observed in males derived from a M. musculus × M. spretus cross [24].

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

T.H. and J.S. are fellows with the Instituut voor de Aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen. Research was supported by the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen, the Interuniversitaire Attractiepolen, and Fortis Bank Verzekeringen. Hochepied and Schoonjans contributed equally to this work.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References