Phenotypic Study Foxo3a Knockout Mice Created By TALEN-Mediated Gene Targeting And Transgenic Mice Of Buffalo Source ALK6 Point Mutations

It was reported that foxo3a played an important role in ovarian follicle activation of bubalus bubalis, pigs and mice, foxo3a-/- females could exhibit a kind of phenomenon of global follicular activation bringing about oocyte death ahead of time and early depletion of functional ovarian follicles, and secondary infertility. Natural mutation ALK6 Booroola sheep, small tail cold sheep, sheep, etc, in other words FecB gene phenotype, Single copy mutation (Genotype B+) ewe could increase by 1.11 lambs, productive double copy mutations (genotype BB) ewe could increased by 1.40-1.50 lambs. This research used 51bp-foxo3a knockout mice and source of the buffalo ALK6 point mutant mice as models, counted and analyzed the foals of different genotypes of mices, at the same time, observed the influence of related gene expression patterns.TALEN Genome-Editing-foxo3a mices:Established a PCR identification method of TALEN Genome-Editing-foxo3a mices (51 bp missing). By the means of agarose gel electrophoresis which used PCR products as substrates, foxo3a-/- mice had a 602 bp single stripe, foxo3a+/+ mice had a 653 bp single stripe, foxo3a-/+ mice had two strips(602 bp and 653 bp). Use T7E1 enzyme cut off the gene editing incision further, it could observe that foxo3a-/+ mice produced 3 stripe, foxo3a-/- mice and wild type mices produced different single size stripe. Because there were 270th,285th and 270th PSTI enzyme loci in the 653 bp genome sequence of wild type mices. Therefore the PCR products of the wild type mice would produce about 110 bp and 260 bp two bands; The DNA sequence of foxo3a-/+ could be digested about 543 bp and 110 bp and 260 bp three stripes; The one of foxo3a-/- mices only could be digested 543 bp and 110 bp two stripes.Biological information analysis shows that the enzyme 51 bp happened to contain the foxo3a 32nd threonine site, which was an important phosphorylation site. Choose foxo3a-/+ and foxo3a-/+ combination to mate, PCR was carried out on the offspring rats electrophoresis, PCR and enzyme digestion and sequencing identification showed that offspring to the total number of foal only 43, including 24 foxo3a-/+ mices, single knock positive rate is 55.81%,9 foxo3a-/- mices, foxo3a-/- positive rate was 20.93%, total positive rate was 76.74%, positive rate in accordance with Mendel fixed rate. Choose identified foxo3a-/- mothers to count the litter size and counted four litters, average foal was 7±1.02, which was significantly lower than that of wild mices (9.71±0.71) (P<0.05). foxo3a-/+ litter size had counted 13 nest, an average number was 7.38±0.54, also significantly lower than that of wild type mice (P<0.05). foxo3a-/+ mice and wild-type mice mating statistical foal number eight, the average foal number was 7.38±0.54 only. Using SPSS analysis results showed that compared with wild type mice, the litter number of foxo3a-/- mice decreased significantly (P= 0.013, P< 0.05), foxo3a-/+ mice also decreased significantly (P=0.015, P <0.05). There was not significant difference on the litter size of foxo3a-/-mice and foxo3a-/+ mice (P=0.674).Buffalo ALK6 point mutations FecB gene transgenic mice:To investigate ALK6 point mutations FecB gene wae able to improve the water buffalo fertility, this experiment used buffalo source ALK6 point mutations transgenic mice as models, observed the litter size and gene expression levels.The results showed that mice and buffalo ALK6 gene encoding the same number of amino acids were 502, there were no signal peptide, and molecular weight respectively were 56.9444 Kda and 56.9234 Kda, PI was 7.48 and 7.78 respectively, subcellular localization in the plasma membrane, had a phosphorylation sites in 374,24th,109th and 109th were glycosylation modification site. The average foal number of Buffalo source ALK6 point mutations transgenic mice was 11.6±0.91, which was on average more 1.89 than the wild type mice (foals number was 9.71±0.71), but they were no significant statistical difference (P= 0.297, P>0.05). QRT-PCR results showed that genetically engineered mice ALK6 mRNA expression significantly decreased in the ovaries and testes of buffalo source ALK6 point mutations (P<0.05), the sum of buffalo ALK6 and mouse ALK6 mRNA was less than the wild type mice. Conclusion:The litter size of Buffalo source ALK6 mutant transgenic mice had increased, but the difference was not significant, this have a connection with ALK6 gene expression in ovarian tissues. Conclusion: this study laid the foundation of the research to clarify foxo3a and FecB genes involved in the molecular mechanism of mammal reproduction efficiency.