转GFP基因核移植牛胚胎的研究

Study on Transgenic GFP Gene Bovine Embryos Production by Nuclear Transfer

试验采用脂质体转染法与电穿孔法,以携带绿色荧光蛋白(GFP)-新霉抗性(neo-)双标记基因的pMSCV质粒转染胎牛耳成纤维细胞为供体与体外成熟的牛卵母细胞为受体构建克隆胚。研究了体外成熟培养液中添加EGF(表皮生长因子)对转基因胚的影响,不同转染方法构建供体细胞对重构胚发育的影响和在不同体外培养系统中的发育效果。结果显示,体外成熟培养液中添加EGF 30 ng/mL组的卵母细胞成熟率最高,但对后期转基因重构胚的囊胚发育率的影响,以添加EGF 20ng/mL组的最高;以胎牛耳成纤维细胞为供体细胞,不同转染方法转染供体细胞构建重构胚,其囊胚发育率差异不显著(P>0.05);mSOFaa+颗粒细胞单层细胞共培养体系中的转基因囊胚发育率最好,该体系更适合体细胞核移植法生产转基因牛胚胎。

pMSCV containing GFP and neogene was transfected into the cattle fetal auris fibroblast cells by lipofection or electrofusion was the donor. 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 maturation and transgenic embryos in vitro culture, donor cells gene-transfeetion method, conducted to investigate the effect of three in vitro culture systems on the development of bovine embryos and to observe the development of bovine transgenie embryos in vitro in the optimal culture system will be studied. 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, blastocyst development rate. The cleavage rate and blastocyst development rate adding 25 ng/mL EGF to medium group were significantly higher than those in control group; and its effect on cell proliferation after grafting. The different gene transfection 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.