Establishment And As Donor Cell For Somatic Cell Nuclear Transfer Of Sheep Mesenchymai Stem Cells

The world’s first mammalian somatic cell cloned sheep "Dolly " had been cultivated successfully in1997. From then on, somatic cell nuclear transfer technology has been developing day by day. The selection of donor cells, which can directly impact on the efficiency of clone,is one of the vital factor to the success of somatic cell clone.Up to now,there are different kinds of somatic cells were used as donor cells for cloning successfully, such as various types of fibroblasts, mammary epithelial cells, reproductive system cells, lymphocytes, etc. However, it is low efficiency, high cost in cloning process. Mesenchymal stem cells (Mesenchymal stem cells, MSCs) are adult stem cells derived from the mesoderm,which is present in connective tissue and organ mass Organ interstitial extensively, has the potential of highly proliferative, self-renewal and differentiation. Researchers had obtained clone cattle, pigs and dogs successfully which use mesenchymal stem cells as nuclear donor。But there are no related reports of sheep.In this study, we established sheep fat and bone marrow mesenchymal stem cells, identified its biological characteristics and stem cell characteristics, then we induced mesenchymal stem cells differentiate into neurons, cardiomyocytes and pancreatic islet-like cells in tissue. After staining and RT-PCR identification, we confirmed that the differentiated cell performanced cell differentiation-specific morphology and expressed specific genes. Based on this, we used bone marrow mesenchymal stem cells rather than commonly fibroblast nuclear as donor cells to construct clone embryos by nuclear transfer, then Compared the early development ability with cloned embryos of fibroblasts. After embryo transfer, birthed the first case of adult stem cells cloned sheep. The results are as follows:1-. Established bone marrow mesenchymal stem cells (Bone marrow-derived mesenchymal stem cells, BMSCs) and adipose-derived mesenchymal stem cells (adipose-derived stromal cells ADSCs) of fetal, juvenile and adult sheep successfully. Two types of mesenchymal stem cells showed fusiform and easy to form a colony in vitro. The doubling time of BMSCs from three developmental stages sheep was30.82±0.05h,30.74±0.03h and31.2±0.06h respectively, which of fetal and adult ADSCs were34.02±0.06h and34.10±0.04h, cell growth curve showed "S" type; After identification by RT-PCR, showed sheep MSCs expressed stem cell-specific pluripotency gene Oct4, Sox2and Nanog. But, the BMSCs were not expressed CD34, CD45and HLA.2、We subcultured sheep MSCs long-term in vitro, find the MSCs can maintain its genetic stability well. Conducted karyotype analysis of P10, P20and P30of BMSCs and ADSCs, the normal karyotype rates were:P10:90.74%(49/54) and86.05%(37/43); P20:86.11%(31/36) and84.00%(42/50); P30:81.63%(40/49) and86.67%(39/45).3、We multi-induced sheep MSCs, found which can differentiate into neuron-like cells, cardiac cells, osteoblasts, fat cells and pancreatic islet cells cross the mesoderm. After staining differentiated neural cells with aniline blue, indicated the presence of Nissl bodies, RT-PCR detected the expression of neuron-specific gene GFAP and NSE; differentiated cardiomyocytes by PAS staining showed there were lots of glycogen in cells, and expressed cell-specific gene GATA-4and NKX-2.5; Alizarin red staining testified that there were crimson bone nodules from osteogenic cells, also expressed osteoblast-specific gene Osteocalcin and CFBA; Oil Red O stained lipid droplets in fat cells into red, and expressed fat cell-specific gene ion and Leptin of PPAR successfully; dithizone staining manifest that islet-like cells were stained into scarlet,which expressed islet-specific genes Insulin and PAX4.4、we used MSCs as the nuclear donor and enucleated oocytes as receptor to build reconstruct embryos, fusion rate of reconstructed embryos of BMSCs from Fetus, infant and adult were:80.62%,83.43%,78.46%;while which of blastocyst were:0.74%,1.71%,0%. fusion rate of reconstructed embryos of ADSCs from fetal and adult were81.48%,79.23%; blastocyst rates were:1.12%,0%.5、After transferring reconstructed embryos into20receptor sheep, three of which were pregnant; five cloned sheep derived from stem cells were generated, and three survived. The RCP value between donor cells and cloned sheep were higher than99.999%through microsatellite DNA paternity test, proved that cloned sheep were mesenchymal stem cells cloned sheep.To sum up, we had established bone marrow and adipose-derived mesenchymal stem cells of sheep successfully, then made use of bone marrow mesenchymal stem cells as nuclear donor cells, we cultivated adult stem cells cloned sheep successfully. Our research laid the foundation of clinical application and livestock transgenic clone of mesenchymal stem cells.