Function Research Of Testis-specific Novel Genes T279 And TSC21 During Spermatogenesis Of Testis In Mice

Part ?:Establishment of testis-specific new gene T279 and TSC21 gene knockout mice modelsObjectiveThe testis specific genes T279 and TSC21 knockout mice were established by gene targeting technique to study the function of these two testis-specific new genes in mouse testes.MethodsOrder 129 BAC clones which contain T279 and TSA21 genes,amplify retrieving homologous arms and Loxp-neo-Loxp fragment by PCR method,PL253 plasmid were used for ligation and homologous recombination between different fragment,construct targeting vector and transform it into ES cells by electroporate method,Southern blot were used to identify homologous recombination occurred ES cell clones.The identified ES cell clones were injected into the blastocysts by microinjection,and blastocysts were implanted into the uterus of pseudo-pregnant mice to develop chimeric mice,after backcross and intercross,we can screen out T279 and TSC21 knockout mice from the birth of offspring mice.ResultsThe correct structure of the targeting vector was confirmed by the results of restriction endonuclease enzyme digestion and DNA sequencing analysis.Southern blot result showed that homologous recombination-positive ES cell clones exist.T279 and TSC21 gene knockout targeting vector were successfully constructed and homologous recombination-positive ES cell clones is obtained.The identified ES cells were injected into blastocysts and implanted into the uterus of pseudo-pregnant mice.The chimeric mice were successfully obtained.T279 and TSC21 gene knockout mice have been obtained.ConclusionTestis specific gene T279 and TSC21 gene knockout mice were successfully constructed.Part ?:Function research of testis-specific novel genes T279 and TSC21 during spermatogenesis of testis in miceObjectiveReproduction and amplification of gene knockout mice models.phenotypic analysis was used to preliminary explore the function of T279 and TSC21 during the process of mouse spermatogenesis.Hope that this study can explore new methods for the diagnosis and treatment of male infertility.MethodsRT-PCR and Real-Time PCR were used to analyze the expression profile of gene T279 and gene TSC21 in mice.Mating experiments,spermatogenesis evaluation,epididymal sperm analysis,testicular tissue section HE staining methods were used to preliminary explore the impacts on spermatogenesis and fertility,which caused by complete deletion of this newly discovered testis-specific gene T279 and TSC21.ResultsThe complete deletion of T279 gene is likely to lead to an increase in sperm abnormality in male mice,but the fertility of T279 knockout male mice has not been significantly affected.Partial deletion of TSC21 gene did not cause significant effect on the spermatogenesis and fertility of male mice.There was no TSC21 knockout homozygous offspring.ConclusionTesticular specific genes T279 and TCS21 are likely to play an important role in the development of testicular spermatozoa in male mice.The complete knockout deletion of T279 gene is likely to lead to abnormal spermatogenesis in male mice,leading to an increase in sperm deformity in male mice.The complete deletion of the TSC21 gene is likely to lead to lethal abnormalities in progeny during embryonic development.