Effect Of Sox2 Gene On The Epigenetic Modification Of Different Donor Cells In Sheep Somatic Cell Nuclear Transfer

The technology of somatic cell nuclear transfer (SCNT) in mammals has been developed rapidly and gradually improved, but the low efficiency of SCNT is always a bottleneck restricting its application and development in livestock farming. The types of donor nuclear and nuclear incomplete reprogramming are closely related to the cloning efficiency. Epigenetic modification of DNA methylation and histone acetylation are key factors in the process of nuclear reprogramming. Studies have confirmed that the Sox2 gene plays a key role in maintaining pluripotency of stem cell and embryo development. The neural stem cells with high expressionof the Sox2 gene used as donor nuclear have higher cloning efficiency in SCNT.In this study, we utilized Inner Mongolia sheep as the research object. Through electroporation, Sox2 gene was transferred into Bone Marrow Mesenchymal Stem Cells (BMSCs), Adipose Mesenchymal Stem Cells (ADSCs) and Epidermal Fibroblast Cells (SEFCs) of sheep which used as donor nuclear cells. We investigated the expression level of DNA Methyltransferase 1 (DNMT1) and Histone Deacetylase 2 (HDAC2) and analyzed the effect of Sox2 gene on the epigenetic modification of the three types of donor nuclear. At the same time, Sox2 gene were transferred into the mesenchymal stem cells as donor nuclear, and compared with reconstructed embryos using SEFCs as donor nuclear to analyze the effect of Sox2 gene on epigenetic modification and developmental ablity of reconstructed embryos. The main results were as follow:1) Isolated and obtained BMSCs, ADSCs and SEFCs cell lines, the cell growth curve was "S" type, and stem factor of Sox2, Oct4 and Nanog were expressed in BMSCs and ADSCs.2) Transferred pEGFPS-N1-Sox2 into BMSCs and ADSCs and SEFCs by electroporation, and screened positive cell lines. Sox2 gene was over expressed in three kinds of cells, and Sox2 gene can lower the expression of DNMT1 and HDAC2. DNMT1 respectively down 68%、25%、43%；and HDAC2 down about 15%.3) Successfully constructed 159 embryos using BMSCs transferred with Sox2 as the donors and 182 embryos using SEFCs as the donors. Embryos using BMSCs transferred with Sox2 had about 50% lower expression of DNMT1 and HDAC2 than embryos using SEFCs as the donors, which indicated that the Sox2 gene could improve the reprogramming of embryos.Results above lay necessary foundation to elucidate epigenetic modification mechanism of the influence of Sox2 gene on different nuclear donor cells, provide a new method for selection and genetic modification of different donor nuclear cells and improvement of the efficiency of SCNT, and establish a scientific basis for efficient and rapid improvement of sheep breeding by transgenic technology.