Optimization Of Handmade Cloning And The Change Of Tetl In Early Embryogenesis In Pig

Somatic cell cloning has been widely used in production transgenic animals, species protection, livestock breeding, xenotransplantation, and disease models. However the application of this technology was limited because of the low efficiency of successs. It is generally believed that the low efficiency of cloning is due to the incorrect or incomplete nuclear reprogramming of the differentiated somatic cells after nuclear transfer. One of which is the abnormal DNA methylation and histone modifications. Therefore, it is crucial to improve the efficiency of somatic cell nuclear transfer.1. Optimization of handmade cloningIn order to improve the efficiency of handmade cloning, we optimized the process of HMC during the embryos reconstructed and tranfered. The results showed that the blastocyst rate can reach40%, then a total of235morula and blastocyst were transferred into uterus of three recipient sows that at natural estrus for4-5days. Two of them were pregnant but one aborted over60days. At114days, the pregnant sow gave birth to eight piglets, clone birth rate increased significantly relative to other laboratories.2. Immunofluorescence observation of Tetl on the oocytes and the parthenogenetic embryos at early developmental stageOocytes at0h (GV),12h,24h,36h and44h (oocytes at MⅡ stage) during the in vitro maturation were selected. Subsequently, the cumulus cells of these oocytes were removed as described above. For the matured MⅡ oocytes, some of them were parthenogenetic activated to collect the embryos at Oh,7.5h,12h,15h,24h and2-cell (36h). Both of the selected oocytes and the PA embryos were fixed with4%PFA and further were performed with immunofluorescence staining. Results after the confocal microscope observation showed that Tetl has a dynamic process both for the oocytes in vitro maturation and for the early development. It indicates that a dynamic regulation of Tetl on the maturation of oocyte and the reprogramming after activation of embryos. To fulfill understand of the function of Tetl on preimplantation developmental of pig embryos, further studies are needed.3. The effect of5-aza-CdR on methylation expression level of DNA methylation genes in oocytes and fibroblasts cells of pigOocytes were treated with different concentrations of5-aza-CdR for32-34h during their in vitro maturation and fetal fibroblasts established from Meishan pigs after24h recultivated were treated with different concentrations of5μM5-aza-CdR for another48h, and then the expression level of Tetl, Tet2, Tet3, Dnmtl, Dnmt2, Dnmt3a were analyzed by using Real-time PCR on the different treated groups. Results:After treated with different concentrations of5-aza-CdR not only Dnmts expression level changes significantly, but also Tet proteins expression levels change significantly in oocytes and fetal fibroblasts. It indicates that Dnmts and Tets maybe have an interaction.