Study On Transgenic GFP Gene In Bovine Embryos Production By Somatic Cell Nuclear Transfer

This study was to optimize a system for production of transgenic bovine embryos by nuclear transfer of somatic cells.First purpose is to confirm the effect that somatic cell nuclear transfer was used at the field.Second purpose is to check out blastula ratio of somatic cell nuclear transfer.And third purpose is to improve the environments of transgenic bovine embryos and provide theoretic basis of transgenic bovine embryos quality and quantity for IVC.A P^MSCV with green fluorescent protein-mycophenolate resistance marker gene was transfected into fetal fibroblast cells that was the donor.The maturation of bovine oocyte in vitro was the receptor.The embryo cloning was constructed by the donor and receptor.The experiment was to examine researchful effect which bovine embryos were produced with Parthenogenesis in vitro,was to examine researchful effect of HYA,different time and concentration of oocyte interaction in vitro,P^MSCV containing GFP and neo-gene was transfected into the cattle fetal auris fibroblast cells which was the donor by lipofection or electrofusion.The maturation of bovine oocyte in vitro was receptor.The embryo cloning was constructed by the donor and receptor.Under different conditions,such as adding epidermal growth factor(EGF) to the culture medium improved the nuclear maturatio and transgenic embryos in vitro culture,donor cells gene-transfection method,Conducted to investigate the effect of four vitro culture systems on the development of bovine embryos and to observe the development of bovine transgenic embryos in vitro in the optimal culture system will be studied.Under different conditions,such as electric field intensity and DC pulse frequency, the passage of the number of cells of transgenic embryos and integration will be studied.The result shows that the rate of the integration was the highest under 1.6 kV/cm of the electric field strength and DC pulse repetition frequency of 1.The donor cells of five passages were beneficial to transfer embryo.To examine the effects of EGF on the nuclear and cytoplasm maturation of cattle oocytes in vitro culture,the best concentration of EGF was 30 ng/ml then the oocytes were activated by 6-dimethylaminopurine combined with ethanol, examining the cleavage rate and blastocyst development rate.The cleavage rate and blastocyst development rate adding 20ng/ml EGF to medium group were significantly higher than those in control group;and its effect on cell proliferation after grafting.The different genetransfection method was beneficial to transfer embryo.The rate of the integration was the blastocyst development rate of cattle oocytes significantly(P＞0.05);The highest mSOFaa +granulosa cell monolayer co-culture system.