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iPS cells in livestock breeding new varieties and cell reprogramming mechanism is of great significance. In this study, lentivirus infections method fused with green fluorescent protein pSox2, hOct4, pMyc and pKlf4 four kinds defining factor-induced bovine fetal skin fibroblasts iPS cells, and the formation of iPS cell clones were identified. The results were as follows: bovine fetal fibroblasts after lentiviral infection begins after the first 15 d morphological changes by long spindle becomes irregular spherical edge. After receiving the feeder layer growth of cell adhesion, cell morphological changes not only increase in the number, and gathered together like dense clonal growth was flat, clear boundary. High magnification observation bovine iPS clones large nuclei, nuclear matter with higher rates observed under fluorescent bovine iPS clones, some cells are able to express green fluorescent protein. Further subcultured bovine iPS cell clones is growing and spread to a dozen generations maintained a steady growth. Cryopreservation - recovery of cells continue to maintain stable performance subculture. When the cells after receiving the feeder layer iPS cell clones with stable proliferation and passaged expressing green fluorescence intensity gradually weakened, and clones expressing green fluorescent protein gradually reduced. Colchicine bovine iPS cell clones karyotyping showed that iPS cells induced bovine chromosome number of 60, with a normal karyotype cattle coincide; alkaline phosphatase staining was positive. Identified by RT-PCR clone iPS cell pluripotency genes Oct4, Sox2, Nanog, c-Myc, and Klf4 expression, the results Oct4, Nanog, c-Myc, and Klf4 were positive, Sox2 is negative, and as a control bovine fetal fibroblast cells were not above five gene expression. Immunofluorescence staining of bovine iPS cell clones pluripotency genes related proteins were detected, Oct4 and Nanog protein was positive, but the rest of protein expression showed a negative. Bovine iPS cell clones in vitro suspension culture of 10 d, can be observed the formation of embryoid bodies, and tend to clump EBs integration. Bovine iPS cells into nude multi dorsal subcutaneous multiple injections, six weeks after the injection site successfully grown in nude multiple sizes teratoma teratoma removed irregular spherical shape, wrapped in a layer of white film, soft, strong invasiveness, teratoma surface distribution of many vessels and subcutaneous tissue in nude mice connected. The tissue sections and HE staining can be observed in the growth of teratoma in nude mice by the presence of inner, middle and outer three germ layers from the organization. Conclusion: defining factor pSox2, hOct4, pc-Myc and pKlf4 successfully bovine fetal bovine fibroblasts induced into iPS cells.
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