标题: Breaking the Species Barrier: Derivation of Germline-Competent Embryonic Stem Ce [打印本页] 作者: 江边孤钓 时间: 2009-3-5 10:37 标题: Breaking the Species Barrier: Derivation of Germline-Competent Embryonic Stem Ce
a Molecular Genetics of the Mouse, Department for Molecular Biomedical Research, Ghent University and Flanders Interuniversity Institute for Biotechnology, Ghent, Belgium;* u2 a; P _& E# k
; b4 l1 y' h+ eb Thromb-X N.V., Center for Molecular and Vascular Biology, Catholic University of Leuven, Leuven, Belgium , X! t% A+ Y6 N0 d 5 b6 o( G& H% | u9 j* |' D# PKey Words. Embryonic stem cell ? Mus spretus ? Germline transmission ? TNF 4 S! o1 ?1 D8 M, S5 @& r- O, J' Q9 h
Correspondence: Claude Libert, Ph.D., Department of Molecular and Biomedical Research, University of Ghent/VIB, Technologiepark 927, B-9052 Zwijnaarde, Belgium. Telephone: 00-32-9-331-3700; Fax: 00-32-9-331-3609; e-mail: Claude.Libert@dmbr.UGent.be* z* h2 S# H+ i7 [
" U* E0 ^1 I+ e, H: y- `. OABSTRACT : D# q& X8 z v# E/ i+ ?: F* w * o) V8 D, \" F, lThe 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 . 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 . 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 . 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. , H6 f3 U- n: x o3 @" u- K+ Z+ X* o* Y0 m" {- O
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 . 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 , skin cancer , and thymic lymphomas . 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 .& X' D( G0 k6 k- [
* V, E" ^" z+ K# FSince 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 . 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 x SPRET/Ei)F1 hybrid blastocysts, allowing the SPRET/Ei genome to be genetically manipulated.! I: w7 T! P+ l% n2 j
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MATERIALS AND METHODS& e( P' @/ f0 [2 O! l# S/ A5 }! w
# h+ T8 E% r* JES Cell Derivation of (C57BL/6 x SPRET/Ei)F1 Blastocysts |1 c; j0 T9 s* s# I9 f" w6 B7 \3 [4 L& W8 a
Because of the dominant nature of SPRET/Ei phenotypes, we decided to derive ES cells from (C57BL/6 x SPRET/Ei)F1 blastocysts. We used the highly efficient conditioned medium TX-ES , 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. 8 }0 A* s0 f; G* A% H ( \% `6 v, x/ a: M F# bTable 1. Establishment of ES cells from (C57BL/6x SPRET/Ei)F1 blastocysts3 X @5 t& p* y( {
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Germline Transmission of (C57BL/6 x SPRET/Ei)F1 ES Cells : h' A/ }$ v6 g- m3 o7 K6 ]6 B4 P; T9 E! S
Due to male hybrid sterility of F1 animals , the female ES cells were our primary interest. The ES cell lines were genotyped using a Y-specific probe . Of the 16 F1 ES cell lines, nine were male and seven were female (data not shown)." n, W |- P1 b# B3 k1 g
$ \: ?; u* p' D+ h" \8 BTo 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).+ D' N8 F* Q1 `4 Y+ u
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Table 2. Germline transmission of (C57BL/6 x SPRET/Ei)F1 embryonic stem cells/ q9 G' J' k. W! B9 @. C3 }
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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.; ~4 V; q' Z# i$ n' S
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Table 3. SPRET/Ei-specific markers in chimeric offspring derived from embryonic stem cell line B6/SPRET#18 5 F# f1 Q- K Q+ y- G- H1 u8 z 9 w. l" c) E# IFigure 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.8 U5 r/ J5 K* i% g
* m# m L) a% vMost 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 . 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).8 F! U9 W6 \9 o. N1 ?. O/ P1 ^
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DISCUSSION# m- R" ]0 ^' r% |; A+ [" @; O
3 R5 I. i# l3 A) A6 Y& ~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. 3 x, m+ X! ~* U! O6 u3 F# p; e7 N$ H( ]2 p7 |
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