Physical performance and soleus muscle fiber composition in wild-derived and laboratory inbred mouse strains.

We compared four inbred mouse strains in their physical performance, measured as a maximal treadmill running time, characteristics of soleus muscle, anatomic character, and growth. The strains used were Mus musculus domesticus [C57BL/6 (B6) and BALB/c], Mus musculus molossinus (MSM/Ms), and Mus spretus. Maximal running time was significantly different among these four mouse strains. Running time until exhaustion was highest in MSM/Ms and lowest in M. spretus. Maximal times for the laboratory mouse strains were nearly identical. Soleus muscle fiber type and cross-sectional area also differed significantly among the species. In particular, M. spretus was significantly different from the other inbred mouse strains. Growth in the wild-derived inbred mice appeared to be complete earlier than in the laboratory mice, and the body size of the wild strains was about half that of the laboratory strains. From these results, we propose that wild-derived inbred mouse strains are useful models for enhancing phenotypic variation in physical performance and adaptability.     

Altered Turnover of Hypoxanthine Phosphoribosyltransferase in Erythroid Cells of Mice Expressing Hprt a and Hprt b Alleles

We have previously shown that mice expressing Hprt a allele(s) have erythrocyte hypoxanthine phosphoribosyltransferase (HPRT) levels that are approximately 25-fold (Mus musculus castaneus) and 70-fold (Mus spretus) higher than in mice that express the Hprt b allele (Mus musculus domesticus; C57BI/6J; C3H/HeHa), and that these differences in erythrocyte HPRT levels are due to differences in the turnover rates of the HPRT A and B proteins as reticulocytes mature to erythrocytes. We show here that: the taxonomic subgroups of the genus Mus are essentially monomorphic for the occurrence of either the Hprt a or the Hprt b allele, with Hprt a being common in the aboriginal species (M. spretus, Mus hortulanus and Mus abbotti) and in several commensal species (Mus musculus musculus, M. m. castaneus, Mus musculus molossinus), while Hprt b is common in feral M. m. domesticus populations as well as in all inbred strains of mice tested; in all these diverse Mus subgroups there is a strict association of Hprt a with high and Hprt b with low levels of erythrocyte HPRT; and, the association between the occurrence of the Hprt a allele and elevated erythrocyte HPRT levels is retained following repeated backcrosses of wild-derived Hprt a allele(s) into the genetic background of inbred strains of mice with the Hprt b allele. Collectively, these observations indicate that the elevated and low levels of erythrocyte HPRT are specified by differences in the Hprt a and b structural genes.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA Sequence Analysis of Sry Alleles (Subgenus Mus) Implicates Misregulation as the Cause of C57bl/6j-Y(pos) Sex Reversal and Defines the Sry Functional Unit

The Sry (sex determining region, Y chromosome) open reading frame from mice representing four species of the genus Mus was sequenced in an effort to understand the conditional dysfunction of some M. domesticus Sry alleles when present on the C57BL/6J inbred strain genetic background and to delimit the functionally important protein regions. Twenty-two Sry alleles were sequenced, most from wild-derived Y chromosomes, including 11 M. domesticus alleles, seven M. musculus alleles and two alleles each from the related species M. spicilegus and M. spretus. We found that the HMG domain (high mobility group DNA binding domain) and the unique regions are well conserved, while the glutamine repeat cluster (GRC) region is quite variable. No correlation was found between the predicted protein isoforms and the ability of a Sry allele to allow differentiation of ovarian tissue when on the C57BL/6J genetic background, strongly suggesting that the cause of this sex reversal is not the Sry protein itself, but rather the regulation of SRY expression. Furthermore, our interspecies sequence analysis provides compelling evidence that the M. musculus and M. domesticus SRY functional domain is contained in the first 143 amino acids, which includes the HMG domain and adjacent unique region (UR-2).     

Further mapping of quantitative trait loci for postnatal growth in an inter-sub-specific backcross of wild Mus musculus castaneus and C57BL/6J mice

We performed a quantitative trait locus (QTL) analysis of eight body weights recorded weekly from 3 weeks to 10 weeks after birth and two weight gains recorded between 3 weeks and 6 weeks, and between 6 weeks and 10 weeks in an inter-sub-specific backcross population of wild Mus musculus castaneus mice captured in the Philippines and the common inbred strain C57BL/6J ( M. musculus domesticus ), to elucidate the complex genetic architecture of body weight and growth. Interval mapping identified 17 significant QTLs with main effects on 11 chromosomes. In particular, the main effect of the most potent QTL on proximal chromosome 2 increased linearly with age, whereas other QTLs exerted effects on either the early or late growth period. Surprisingly, although wild mice displayed 60% of the body size of their C57BL/6J counterparts, the wild-derived allele enhanced growth at two QTLs. Interestingly, five of the 17 main-effect QTLs identified had significant epistatic interaction effects. Five new epistatic QTLs with no main effects were identified on different chromosomes or regions. For one pair of epistatic QTLs, mice that were heterozygous for the wild-derived allele at one QTL and homozygous for that allele at another QTL exhibited the most rapid growth in all four possible genotypic combinations. Out of the identified QTLs, several showed significant sex-specific effects.     

Gene flow of unique sequences between Mus musculus domesticus and Mus spretus

Allelic diversity has been examined from a variety of Mus musculus subspecies and Mus spretus strains by sequencing at a 453-bp unique sequence locus. One M. m. domesticus classic inbred strain, C57BL/KsJ, contained a sequence identical to that in the M. spretus wild-derived inbred strain SEG, and other wild M. spretus isolates. Such a result should have been precluded by the expected divergence between the species unless there has been interspecies gene flow. Examination of C57BL/KsJ for M. spretus-specific repetitive sequences shows that it is neither a mis-identified spretus strain nor a domesticus/spretus hybrid. Thus, in addition to the previously reported presence of small amounts of Mus spretus-specific repetitive DNA in M. m. domesticus, there is a detectable flow of unique sequence between the two species. There was also ancestral polymorphism observed among the spretus alleles. The difficulty of distinguishing ancestral polymorphism from horizontal transfer is discussed. Received: 14 May 1999 / Accepted: 5 November 1999     

A pronounced evolutionary shift of the pseudoautosomal region boundary in house mice

The pseudoautosomal region (PAR) is essential for the accurate pairing and segregation of the X and Y chromosomes during meiosis. Despite its functional significance, the PAR shows substantial evolutionary divergence in structure and sequence between mammalian species. An instructive example of PAR evolution is the house mouse Mus musculus domesticus (represented by the C57BL/6J strain), which has the smallest PAR among those that have been mapped. In C57BL/6J, the PAR boundary is located just ~700 kb from the distal end of the X chromosome, whereas the boundary is found at a more proximal position in Mus spretus, a species that diverged from house mice 2-4 million years ago. In this study we used a combination of genetic and physical mapping to document a pronounced shift in the PAR boundary in a second house mouse subspecies, Mus musculus castaneus (represented by the CAST/EiJ strain), ~430 kb proximal of the M. m. domesticus boundary. We demonstrate molecular evolutionary consequences of this shift, including a marked lineage-specific increase in sequence divergence within Mid1, a gene that resides entirely within the M. m. castaneus PAR but straddles the boundary in other subspecies. Our results extend observations of structural divergence in the PAR to closely related subspecies, pointing to major evolutionary changes in this functionally important genomic region over a short time period.     

Mitochondrial DNA Sequence Variation in the Eastern House Mouse, Mus Musculus: Comparison with Other House Mice and Report of a 75-Bp Tandem Repeat

The control region and flanking tRNAs were sequenced from 139 Mus musculus mitochondrial DNAs (mtDNAs) from mice collected at 44 localities extending from Germany to Japan. Among the 36 types of M. musculus mtDNA resolved, five have an added 75-bp direct repeat; the two copies within an individual differ by two to four base substitutions. Among 90 M. domesticus mtDNAs sequenced, 12 new types were found; 96 M. domesticus types have now been identified by sequencing this segment. Representative mtDNAs from M. castaneus, M. macedonicus, M. spicilegus and M. spretus were also sequenced. A parsimony tree for the M. musculus mtDNAs is about half as deep as the tree for the M. domesticus mtDNAs, which is consistent with the idea that M. musculus is genetically less diverse and younger than M. domesticus. The patterns of variation as a function of position are similar but not identical in M. musculus and M. domesticus mtDNAs. M. castaneus and M. musculus mtDNAs are allied, at a tree depth about three times as great as the start of intra-M. musculus divergence. The coalescence of the M. musculus and M. castaneus mtDNAs is about half as deep as their coalescence with the M. domesticus mtDNA lineages. The mtDNAs of the aboriginal M. macedonicus and M. spicilegus are each other's closest relatives, at a tree depth greater than the deepest intracommensal node. The mtDNA results support the view that the aboriginal M. spretus is the sister group of the other five species.     

Fine haplotype structure of a chromosome 17 region in the laboratory and wild mouse

Extensive linkage disequilibrium among classical laboratory strains represents an obstacle in the high-resolution haplotype mapping of mouse quantitative trait loci (QTL). To determine the potential of wild-derived mouse strains for fine QTL mapping, we constructed a haplotype map of a 250-kb region of the t-complex on chromosome 17 containing the Hybrid sterility 1 (Hst1) gene. We resequenced 33 loci from up to 80 chromosomes of five mouse (sub)species. Trans-species single-nucleotide polymorphisms (SNPs) were rare between Mus m. musculus (Mmmu) and Mus m. domesticus (Mmd). The haplotypes in Mmmu and Mmd differed and therefore strains from these subspecies should not be combined for haplotype-associated mapping. The haplotypes of t-chromosomes differed from all non-t Mmmu and Mmd haplotypes. Half of the SNPs and SN indels but only one of seven longer rearrangements found in classical laboratory strains were useful for haplotype mapping in the wild-derived M. m. domesticus. The largest Mmd haplotype block contained three genes of a highly conserved synteny. The lengths of the haplotype blocks deduced from 36 domesticus chromosomes were in tens of kilobases, suggesting that the wild-derived Mmd strains are suitable for fine interval-specific mapping.     

Genetics and evolution of hybrid male sterility in house mice

Comparative genetic mapping provides insights into the evolution of the reproductive barriers that separate closely related species. This approach has been used to document the accumulation of reproductive incompatibilities over time, but has only been applied to a few taxa. House mice offer a powerful system to reconstruct the evolution of reproductive isolation between multiple subspecies pairs. However, studies of the primary reproductive barrier in house mice-hybrid male sterility-have been restricted to a single subspecies pair: Mus musculus musculus and Mus musculus domesticus. To provide a more complete characterization of reproductive isolation in house mice, we conducted an F(2) intercross between wild-derived inbred strains from Mus musculus castaneus and M. m. domesticus. We identified autosomal and X-linked QTL associated with a range of hybrid male sterility phenotypes, including testis weight, sperm density, and sperm morphology. The pseudoautosomal region (PAR) was strongly associated with hybrid sterility phenotypes when heterozygous. We compared QTL found in this cross with QTL identified in a previous F(2) intercross between M. m. musculus and M. m. domesticus and found three shared autosomal QTL. Most QTL were not shared, demonstrating that the genetic basis of hybrid male sterility largely differs between these closely related subspecies pairs. These results lay the groundwork for identifying genes responsible for the early stages of speciation in house mice.     

cis-acting determinants of basal and lipid-regulated apolipoprotein A-IV expression in mice

The levels of apolipoprotein A-IV (apoA-IV) mRNA are regulated by dietary lipid in the liver of both the mouse and rat. Thirteen different inbred mouse strains were fed a high lipid diet, and the effect on apoA-IV liver mRNA levels was examined. It was found that each strain responded in one of two ways. Mice of four strains had higher liver apoA-IV mRNA levels as compared with syngeneic mice fed a normal chow diet. Mice of the other nine strains had decreased liver apoA-IV mRNA levels as compared with syngeneic mice fed a normal chow diet. Using F1 hybrids between mice from BALB/c, C3H, and C57BL/6 and between 129 and C57BL/6, as well as recombinant inbred strains derived from a cross between BALB/c and C57BL/6, we have shown that both the normal level of liver apoA-IV mRNA in the chow-fed mice and the lipid-dependent regulation of apoA-IV mRNA levels are controlled by cis-acting genetic elements. The apoA-IV mRNA levels in mice fed a normal diet varied dramatically among strains, with the largest difference (90-fold) being between the 129/J inbred strain and the C57BL/6J strain. In addition, we have examined the expression of apoA-IV during mouse development. ApoA-IV mRNA is expressed early in mouse liver (16 days postcoitum), whereas others have shown previously that rat liver apoA-IV mRNA is undetectable until 14 days after birth. ApoA-IV mRNA levels in the intestine and apoA-I mRNA levels in the liver and intestine, by contrast, mirror the pattern seen in the rat.     

Update of mouse microsatellite database of Japan (MMDBJ).

We updated a database of microsatellite marker polymorphisms found in inbred strains of the mouse, most of which were derived from the wild stocks of four Mus musculus subspecies, M. m. domesticus, M. m. musculus, M. m.castaneus and M. m. molossinus. The major aim of constructing this database was to establish the genetic status of these inbred strains as resources for linkage analysis and positional cloning. The inbred strains incorporated in our database are A/J, C57BL/6J, CBA/J, DBA/2J, SM/J, SWR/J, 129Sv/J, MSM/Ms, JF1/Ms, CAST/Ei, NC/Nga, BLG2/Ms, NJL/Ms, PGN2/Ms, SK/CamEi and SWN/Ms, which have not or have only been poorly incorporated in the Whitehead Institute/MIT (WI/MIT) microsatellite database. The number of polymorphic microsatellite loci incorporated in our database is over 1,000 in all strains, and the URL site for our database is located at http:// www.shigen.nig.ac.jp /mouse/mmdbj/mouse.html.     

Mosaic genealogy of the Mus musculus genome revealed by 21 nuclear genes from its three subspecies

Patterns of genetic variation provide insight into the evolutionary history of a species. Mouse (Mus musculus) is a good model for this purpose. Here we present the analysis of genealogies of the 21 nuclear loci and one mitochondrial DNA region in M. musculus based on our nucleotide sequences of nine inbred strains from three M. musculus subspecies (musculus, domesticus, and castaneus) and one M. spicilegus strain as an outgroup. The mitochondrial DNA gene genealogy of those strains confirmed the introgression pattern of one musculus strain. When all the nuclear DNA data were concatenated to produce a phylogenetic tree of nine strains, musculus and domesticus strains formed monophyletic clusters with each other, while the two castaneus strains were paraphyletic. When each DNA region was treated independently, the phylogenetic networks revealed an unnegligibly high level of subspecies admixture and the mosaic nature of their genome. Estimation of ancestral and derived population sizes and migration rates suggests the effects of ancestral polymorphism and gene flow on the pattern of genetic variation of the current subspecies. Gene genealogies of Fut4 and Dfy loci also suggested existence of the gene flow between M. musculus and M. spicilegus or other distant species.     

The chromosome 11 region from strain 129 provides protection from sex reversal in XYPOS mice

C57BL/6J (B6) mice containing the Mus domesticus poschiavinus Y chromosome, YPOS, develop ovarian tissue, whereas testicular tissue develops in DBA/2J or 129S1/SvImJ (129) mice containing the YPOS chromosome. To identify genes involved in sex determination, we used a congenic strain approach to determine which chromosomal regions from 129Sl/SvImJ provide protection against sex reversal in XYPOS mice of the C57BL/6J.129-YPOS strain. Genome scans using microsatellite and SNP markers identified a chromosome 11 region of 129 origin in C57BL/6J.129-YPOS mice. To determine if this region influenced testis development in XYPOS mice, two strains of C57BL/6J-YPOS mice were produced and used in genetic experiments. XYPOS adults homozygous for the 129 region had a lower incidence of sex reversal than XYPOS adults homozygous for the B6 region. In addition, many homozygous 129 XYPOS fetuses developed normal-appearing testes, an occurrence never observed in XYPOS mice of the C57BL/6J-YPOS strain. Finally, the amount of testicular tissue observed in ovotestes of heterozygous 129/B6 XYPOS fetuses was greater than the amount observed in ovotestes of homozygous B6 XYPOS fetuses. We conclude that a chromosome 11 locus derived from 129Sl/SvImJ essentially protects against sex reversal in XYPOS mice. A number of genes located in this chromosome 11 region are discussed as potential candidates.     

Reduced nucleotide variability at an androgen-binding protein locus (Abpa) in house mice: evidence for positive natural selection.

Previous work has shown that the gene for the alpha subunit of androgen-binding protein, Abpa, may be involved in premating isolation between different subspecies of the house mouse, Mus musculus. We investigated patterns of DNA sequence variation at Abpa within and between species of mice to test several predictions of a model of neutral molecular evolution. Intraspecific variation among 10 Mus musculus domesticus alleles was compared with divergence between M. m. domesticus and M. caroli for Abpa and two X-linked genes, Glra2 and Amg. No variation was observed at Abpa within M. m. domesticus. The ratio of polymorphism to divergence was significantly lower at Abpa than at Glra2 and Amg, despite the fact that all three genes experience similar rates of recombination. Interspecific comparisons among M. m. domesticus, Mus musculus musculus, Mus musculus castaneus, Mus spretus, Mus spicilegus, and Mus caroli revealed that the ratio of nonsynonymous substitutions to synonymous substitutions on a per-site basis (Ka/Ks) was generally greater than one. The combined observations of no variation at Abpa within M. m: domesticus and uniformly high Ka/Ks values between species suggest that positive directional selection has acted recently at this locus.     

Higher differentiation among subspecies of the house mouse (Mus musculus) in genomic regions with low recombination

In the early stages of reproductive isolation, genomic regions of reduced recombination are expected to show greater levels of differentiation, either because gene flow between species is reduced in these regions or because the effects of selection at linked sites within species are enhanced in these regions. Here, we study the patterns of DNA sequence variation at 27 autosomal loci among populations of Mus musculus musculus, M. m. domesticus, and M. m. castaneus, three subspecies of house mice with collinear genomes. We found that some loci exhibit considerable shared variation among subspecies, while others exhibit fixed differences. We used an isolation-with-gene-flow model to estimate divergence times and effective population sizes (N(e) ) and to disentangle ancestral variation from gene flow. Estimates of divergence time indicate that all three subspecies diverged from one another within a very short period of time approximately 350,000 years ago. Overall, N(e) for each subspecies was associated with the degree of genetic differentiation: M. m. musculus had the smallest N(e) and the greatest proportion of monophyletic gene genealogies, while M. m. castaneus had the largest N(e) and the smallest proportion of monophyletic gene genealogies. M. m. domesticus and M. m. musculus were more differentiated from each other than either were from M. m. castaneus, consistent with greater reproductive isolation between M. m. domesticus and M. m. musculus. F(ST) was significantly greater at loci experiencing low recombination rates compared to loci experiencing high recombination rates in comparisons between M. m. castaneus and M. m. musculus or M. m. domesticus. These results provide evidence that genomic regions with less recombination show greater differentiation, even in the absence of chromosomal rearrangements.     

Variation in the Major Urinary Protein Multigene Family in Wild-Derived Mice

The levels of expression and genomic organization of genes coding for the major urinary proteins (MUPs) were examined in several stocks of wild-derived mice. Levels of MUP mRNA in the liver varied considerably with M. musculus Brno and M. castaneus males having several-fold more MUP RNA than inbred C57BL/6 males, whereas M. hortulanus, M. caroli and M. cervicolor displayed levels much lower than C57BL/6. Analysis of RNA with MUP cDNAs specific to two different subfamilies of MUP genes revealed that M. caroli and M. cervicolor primarily expressed a MUP mRNA that was less abundant in C57BL/6, suggesting differential expression of subfamilies of genes within the MUP multigene complex. Although inbred males usually have five-fold more MUP mRNA than inbred females, male to female ratios for wild-derived stocks ranged from one to several hundred. Southern blots of genomic DNA hybridized to MUP subfamily probes revealed differences in restriction fragment sizes as well as possible variation in the number of MUP genes in some species. Analysis of urinary proteins from hybrids between C57BL/6 and M. spretus suggested that low MUP expression in M. spretus females was due to cis-acting genetic elements.     

Regulatory and Structural Genes for Lysozymes of Mice

The molecular and genetic basis of large differences in the concentration of P lysozyme in the small intestine has been investigated by crossing inbred strains of two species of house mouse (genus Mus). The concentration of P in domesticus is about 130-fold higher than in castaneus. An autosomal genetic element determining the concentration of P has been identified and named the P lysozyme regulator, Lzp-r. The level of P in interspecific hybrids (domesticus X castaneus) as well as in certain classes of backcross progeny is intermediate relative to parental levels, which shows that the two alleles of Lzp-r are inherited additively. There are two forms of P lysozyme in the intestine of the interspecific hybrid--one having the heat stability of domesticus P, the other being more stable and presumably the product of the castaneus P locus. These two forms occur in equal amounts, and it appears that Lzp-r acts in trans. The linkage of Lzp-r to three structural genes (Lzp-s, Lzm-sl, and Lzm-s2), one specifying P lysozyme and two specifying M lysozymes, was shown by electrophoretic analysis of backcrosses involving domesticus and castaneus and also domesticus and spretus. The role of regulatory mutations in evolution is discussed in light of these results.     

Genetic differences in the histochemically defined structure of oligosaccharides in mice

A wide range of tissues from three interfertile species of mice and an interspecific hybrid was examined with lectins conjugated to peroxidase to localize specifically glycoconjugates containing terminal alpha-N-acetylgalactosamine, alpha-galactose, and alpha-fucose, and the terminal disaccharide galactose-(beta 1----3)-N-acetylgalactosamine. This battery of lectins disclosed marked heterogeneity of glycoconjugates in different histological sites in a given animal and even between cells in a presumably homogeneous cell population within an organ. No variation with any lectin was observed between individuals of two closely related inbred strains of Mus domesticus at any specific histological or cytological site. In contrast, littermates of an outbred strain of Mus castaneus differed in binding of certain lectins at various sites, attesting to a genetic basis for individual variation. Hybrids between castaneus and domesticus mice also showed individual variation. Moreover, extensive differences between the mouse species were demonstrable with every lectin in glycoconjugates of stored secretions, Golgi cisternae, and apical or basolateral plasmalemma in many cell types. Totaling the differences in tabulated staining intensities for each possible species pair gave a measure of the overall extent of difference at 53 histological sites. According to this measure, the three species are about equally divergent from one another. Some differences between species appeared to depend on histological rather than histochemical variation, as, for example, a greater abundance of granular duct cells in the sublingual and submandibular glands in Mus hortulanus. Other differences were apparently derived from pathological change, as exemplified by casts and lymphoid infiltrates in kidney and structurally atypical submandibular gland lobules in Mus castaneus, and possibly by infiltrating cells in intestinal lamina propria and epithelium in Mus castaneus and hortulanus.     

Lactate dehydrogenase isozymes in xenotropic virus producing mice

Lactate dehydrogenase (LDH; E.C. 1.1.1.27) isozymes were compared in three inbred strains of mice, and two strains of wild mice, as well as the F1 hybrids and other genetic crosses involving two of the inbred strains. The strains examined were NZB/B1NJ, 129/J and C57BL/6J, Mus musculus molossinus and M. musculus castaneus. Genetic crosses were made between the xenotropic virus-producing NZB and the non-virus producing 129/J mice. Tissue specificity of LDH in these strains was studied using homogenates of kidney, liver, spleen and thymus. Polymorphism of the enzyme was studied by agarose gel electrophoresis. Enzyme polymorphism in the tissues of NZB and 129/J has not been previously reported. The liver and spleen tissues of 129/J showed the absence of LDH-1 and LDH-2 isozymes. Thymic homogenates of NZB showed a lack of expression of LDH-1, LDH-2 and LDH-3 isozymes. The F1, F2 and the backcross progeny from genetic crosses involving NZB, and 129/J mice showed an isozyme pattern more similar to the non-virus-producing 129/J strain than the virus-producing NZB. Evidence of genetic regulation at the LDH-B subunit appears to be the reason for the differential expression of the isozymes in NZB and 129/J strains. The other inbred strain of mice, C57BL/6J, also showed a greater similarity to the 129/J strain than NZB. The two strains of wild mice were similar in their expression of LDH-isozymes between each other and to the 129/J strain, with respect to the liver and spleen tissues.     

Efficient production of intersubspecific hybrid mice and embryonic stem cells by intracytoplasmic sperm injection

Recently, mice and embryonic stem (ES) cells with allelic polymorphisms have been used extensively in the field of genetics and developmental biology. In this study, we examined whether intersubspecific hybrid mice and ES cells with these genotypes can be efficiently produced by intracytoplasmic sperm injection (ICSI). Frozen-thawed spermatozoa from wild-derived strains, JF1 (Mus musculus molossinus), MSM (M. m. molossinus), HMI (M. m. castaneus), and SWN (M. m. spp.), were directly injected into mature oocytes from laboratory mice ([C57BL/6 x DBA2]F1; M. m. domesticus). The in vitro and in vivo developmental capacity of F1 embryos was not significantly different among the groups (P > 0.05), and term offspring were efficiently obtained in all groups (27%-34% of transferred embryos). However, the mean body and placental weights of the offspring differed significantly with genotype (P < 5 x 10(-10)), with the HMI hybrid greatest in both body and placental weights. In an application study using these F1 offspring, we analyzed their mitochondrial DNA using intersubspecific polymorphisms and found the consistent disappearance of sperm mitochondrial DNA in the F1 progeny. In a second series of experiments, we generated F1 blastocysts by injecting MSM spermatozoa into C57BL/6 oocytes and used them to generate hybrid ES cell lines. The ES cell lines were established at a high efficiency (9 lines from 20 blastocysts) and their allelic polymorphisms were confirmed. Thus, ICSI using cryopreserved spermatozoa allows the efficient and immediate production of a number of F1 hybrid mice and ES cell lines, which can be used for polymorphic analysis of mouse genetics.