Genetical Studies On The Expression Of Ovum Mutant Gene In Mice

Mice had been model animals to explore complex character in biological research currently. One of the most powerful advantages of mice is that mouse genome is highly homologous to human, and secondly there are many various types in mice, such as inbred, mutant, and genetically engineered mice lines, etc. Combined with easily managed and feeded, short growth cycle and other characteristics, mice are used in developmental biology, functional genomics and mechanism of human disease areas widely.The ovum mutant gene (Om) is located on chromosome 11 as a unique mutant gene of regulation of early embryonic development in mice. The DDK strains carrying Om gene exhibit special reproductive traits that the embryonic death in 3-5 days of pregnancy due to the disorders of blastocyst formation when DDK females cross with males of other inbred strains, whereas DDK males exhibit normal fertility in reciprocal crosses,and is known as DDK syndrome. It has been proved that the reproductive traits occur due to the incompatibility between ooplasmic factor of DDK and the sperm factor of other strains owing to the Om locus, which led to the hybrid zygote death in early embryonic development.According to this incompatibility theory, litters produced from backcrosses, such as F1 (Om/+)♀× Alien (+/+) ♂ and DDK (Om/Om)♀× F1(Oml+) ♂, should be half the size of those from a normal fertile cross. However, Zhao et al (2000) introduced Om gene into B6 genetic background to analyse embryo survival rate in backcrosses of heterozygous females and B6 males, who found that litter size decreased in a stepwise manner as their genetic background became more similar to that of B6 strains and embryonic mortality deviated from the semi-lethal rate, exhibited an increasing trend. Therefore, these results indicate that the presence of potential modifiers of Om in B6 stains that could increase embryonic mortality, also the modifiers only affect the fertility of heterozygous (Om/+) females and do not affect heterozygous (Om/+) males significantly.In addition, embryo mortality regulated by Om gene in early embryonic development was found within crosses between DDK females and other males of the same subspecies. Therefore, the research surrounding Om gene is also limited to intrasubspecies of M.m.domesticus in early studies. Zhao et al (2002) studied on the compatibility between ooplasmic factor and sperm gene in the intersubspecific crosses of DDK(M.m.domesticus) and MOM (M.m.molossinus), CASP (M.m.castaneus) strains, and extended the research of Om gene to intersubspecies of mice for the first time. It has reported that DDK females are Mly fertile when crossed to males of MOM and CASP strains, and showed that no incompatibilities exist between ooplasmic factor and sperm gene of DDK and MOM or CASP strains. Currently, only the relationship between ooplasmic factor and sperm factor of DDK strains and M.m.musculus subspecies remain unclear.Based on the previous studies, this research focused on two points:(1). To identify the modifier genes of Om present in B6 background, and further proved the number of potential modifiers and located on the chromosome of mice by QTL mapping methods. (2). Introduced PWK wild-derived inbred strain to investigate the interrelations between ooplasmic factor and sperm gene between DDK and M. m. musculus strain and the further completed the diagram of interrelations between ooplasmic factor and sperm gene both encoded at Om locus between DDK and other subspecies strains.1. Locating modifiers of Ovum mutant using crosses between DDK and B6 mice. In this study, F1 (B6♀×DDK♂) were backcrossed to B6 and female offspring (N2) were screened using two microsatellite markers linked to the Om locus, D11Mit36 and D11Mit66, to identify heterozygous females for the mapping population. Microsatellite markers were selected from the Mouse Genome Informatics database and the polymorphic markers were obtained through PCR screening with B6, DDK and F1 mice. Females in the [(Om/+) N2♀×XB6♂] crosses were checked daily for vaginal plugs and dissected at 12-15 day after pregnancy to record the live fetus (LF) phenotype. The linkage analyses were performed with R/qtl software and the logarithm of odds (LOD) scores were calculated with the permutation method. After loci significantly associated with the phenotype were located, the credible interval and the corresponding marginal likelihoods were identified by R-qtl. The results were as follows:1. Successfully constructed mapping populations and screened (Om/+) N2 samples 249, in which 200 samples are used for whole-genome scan, supplemented other 49 samples for further fine mapping.2. In whole-genome scan,58 polymorphic markers between DDK and B6 were screened out, and 12 polymorphic markers were screened for fine mapping in chromosome 9.3. A whole-genome scan was performed as an initial analysis using 200 N2 samples to ascertain the chromosome upon which the locus was located. The results indicated that LOD scores of one major modifier locus higher than threshold which was located on chromosome 9, near the microsatellite marker D9Mit273 (49 cM). Two additional loci detected on chromosomes 8 and 15 have maximum LOD values about 1.0 less than the threshold significance loci.4. For the fine mapping, an additional 12 polymorphic markers were selected for screening 249 N2 samples. The 1000 permutation tests indicated that LOD scores of 2.0 (p=0.01) are significant for LF and the maximum LOD score,3.37, was observed near the microsatellite marker D9Mit73. A credible interval for this major locus was approximately 11.4 cM, including the region from 34.43 to 45.8 cM. According to the QTL effect analysis, the LF of individuals with homozygous genotype at the D9Mit73 locus (2.32 ± 0.31) was significantly lower than for individuals who were heterozygous (3.86 ± 0.24). The LOD linkage peaks together with the correlation between the lethal phenotype and the major locus, indicating that the locus in the B6 strain was a modifier of Om.According to the above results, the next step includes increased microsatellite markers to refine credible interval of the modifier locus and determine other modifier loci in B6 strains. On the other hand, candidate genes screened within the specific region of chromosome 9 and verified the function by bioinformatics methods. With this work, we hope to contribute to the investigation of the mechanism of modifiers and ovum mutant gene, and provide a foundation for studing regulating mechanisms of related genes in early embryonic development of mice.(2) Investigating the compatibility between sperm factor and ooplasmic factor in the intersubspecific crosses of PWK and DDK mice. In this study, the reciprocal crosses between DDK and PWK strains were constructed to record conception rate, litter size and other phenotypic data, and B6 strains were introduced as a control. The mating reproduction rate between DDK and PWK strains were determined by the comparative analysis of reproduction phenotypic data in each group. Subsequently, through observation and analysis of reproduction phenotypic data in combination of F1 backcrosses, further determined the compatibility between DDK and PWK strains. In previous research, due to incompatibility between sperm factor and ooplasmic factor by Om, the transmission ratio of Om locus in N2 mice deviated significantly from 50%:50%. In order to fully determine the compatibility between ooplasmic factor and sperm gene in intersubspecific crosses of DDK and PWK strains, microsatellite markers which link to Om locus were used to analyse the genotype of N2 samples. According to the transmission ratio of Om locus in N2 samples to get the final judgment of compatibility between DDK and PWK strains. The results were as follows:1. In this study, the 28 groups of reproductive data were collected from combination groups between DDK females and PWK males, and 95 groups from F1 males backcrosses. The DDK females are fully fertile when crossed to males of PWK strains by comparative analysis of the reproductive performance of the various combinations.2. Investigated the transmission ratio of Om locus of F1 males. The total 600 N2 of backcross generation samples were detected by microsatellite markers, D11Mit36 and D11Mit66, which closely linked to the Om locus. The results showed that homozygous and heterozygous proportion of Om locus in N2 samples were 50%:50%, which indicated the compatibility between ooplasmic factor of DDK with sperm factor of PWK strains.3. The 34 groups of reproductive data were collected from combination between PWK females and DDK males, and 80 groups from F1 females backcrosses. The PWK females are fully fertile when crossed to males of DDK strains by comparative analysis of the reproductive performance of the the various combinations.4. Investigated the transmission ratio of Om locus of F1 females. The total 593 N2 of backcross generation samples were detected by the common microsatellite markers. The results showed the proportion of (+/Om) and (Om/Om) in progeny samples of backcrosses were 50%:50%, which determined the compatibility between ooplasmic factor of PWK with sperm factor of DDK strains.5. Combining all the reproductive performance of reciprocal crosses between DDK and PWK strains and the transmission ratio of Om locus, the results indicated that no incompatibilities exist between ooplasmic factor and sperm gene in intersubspecific crosses of DDK and PWK strains.Based on the findings above, this study identified compatibilities exist between PWK strains and DDK, which improve the schematically summarized the interrelations among DDK strains with the same subspecies strains (B6 strain, etc., Mus musculus domesticus), as well as with different subspecies strains (MOM strains, M.m.molossinus; CASP strains, M.m.castaneus; and PWK strains, M.m.musculus), between ooplasmic factor and sperm factor which encoded by Om locus, and further expand the research of Om gene and DDK mice strains.