Induction Of Goat IPS Cells With Exogenous Factors

Four factors, oct4, sox2, klf4 and c-myc were transduced into murine embryonic fibroblasts by Yamanaka and his colleagues in 2006, after some time, these murine embryonic fibroblasts were induced into a embryonic stem cell-like state, and these derived cells were named as "induced pluripotent stem cells".Based on its potential application in clinical therapy, induced pluripotent stem cells were widely studied by many researchers and groups on its safty, efficency of induction, its application in animal and human disease, as well as the generation of iPS cells with somatic cells from different kinds of animals.On an attempt to generate goat induced pluripotent stem cells in this study, goat ear fibroblasts were isolated from adult goat, and human oct4, sox2, klf4 and c-myc were transduced into goat ear fibroblasts with lentivirus, then these cells were maintained on a culture meduim standardized for embryonic stem cells with different feeders and were treated with different chemicals. The main findings were as follows:1. Packeging system pMD2.G, psPAX2 and lentiviral vectors containing human oct4, sox2, klf4, c-myc were obtained after abstraction and identification with double digesting.2. Murine embryonic fibroblasts isolated from Kunming mice and goat ear fibroblasts isolated from adult goats were treated with mitomycin C and would be used as feeders, as well as pig fetal fibroblasts and goat fetal fibroblasts.3. Lentivirus supernatants were collected after lentiviral vectors and packeging system being cotransfected into 293T cells with liposome 2000, RNA was isolated from lentivirus supernatants and cDNA was synthesized, then lentivirus were titered by quantitative PCR.4. lentivirus were transduced into goat ear fibroblasts which were then maintained on a culture meduim standardized for embryonic stem cells with different feeders and were treated with different chemicals(vitamin C, VPA, vitamin C+VPA), and pig fetal fibroblasts, goat fetal fibroblasts were also treated as above. Results showed that we only got induced pluripotent stem cells with goat ear fibroblasts feeder and there were no differences between treatments with different chemicals. 5. Alkaline Phosphatase staining and immunocytochemical staining were performed on induced pluripotent stem cells and results showed that these cells exhibited Alkaline Phosphatase activity and expressed embryonic stem cell makers, including SSEA-1 and oct4.6. The RNA of induced pluripotent stem cells was isolated and RT-PCR results showed that these induced pluripotent stem cells expressed all four exogenous factors and embryonic stem cell marker genes including oct4 and nanog, but not eras, dexl or gdf3.