Studies On Procedure And Identification Of Somatic Cell Nuclear Transfer In Porcine

Cloned pigs by somatic cell have great potential as models for human disease and as human xenotransplantation. This research focused on the nuclear transfer(NT) of porcine nuclear transfer and improve efficiency, this experiment studied the simplified condition and to try to found a high efficient platform for somatic cell nuclear transfer. Maturity of the porcine oocyte , preparation of donorcells, the construction of the cloned embryo, embryo transfer and identification of the cloned pig were studied in this paper, which optimize the experiment condition. We found a consummate system relatively, and produced the first cloned pig in Shanghai. There is result as follow:1. We used different basic maturation media to cultivate COCs. The maturing rate is higher when the COCs are maturated by NSCU-23(86.89% ) nd mTCM-199(89.94%). There is significant difference between TCM199(75.37%) and NCSU-23(mTCM199).It's said that NCSU-23 and mTCM-199 is more suitable in COCs maturation, which can get a good effect.2. Spindle-view system is used in enucleate of oocyte .The enucleate rate can get high to 90%. So this system is a safe and high efficient method of enucleate.3. Bama pig's cumulus cell, fetus and adult fibroblasts donors were chosen for donorcells. There were no statistical significances of reconstructed embryos developmental rates among cumulus cell, fetus and adult fibroblasts donors.4. No significant differences between B2 and NCSU-23 culture medium as for developmental rate of reconstructed embryos.5. A complete embryo transfer system was founded after several transplant operations. The receptor is better to use multiparity pig. The receptor is anesthetize by mixed anesthesia .About 200 cloned embryos and parthenogenetic embryo were put into the deep part of the egg calyx in spring or summer, which produced a cloned pig successfully. DNA analyses confirmed that the pig was genetically identical to the donor cells.It indicated that pigs can be cloned from the fetus fibroblasts donors by nuclear transfer.