利用体细胞核移植技术生产表达绿色荧光蛋白转基因猪胚胎的研究

试验对脂质体转染猪胎儿成纤维细胞的技术程序进行了筛选,以绿色荧光蛋白基因转染后的阳性细胞作为体细胞核移植的核供体,以体外成熟卵母细胞为核受体构建了绿色荧光蛋白转基因克隆猪胚胎,并对重构胚在体外发育情况以及绿色荧光蛋白表达情况进行了跟踪研究。结果显示,采用4.0μg.mL-1脂质体转染试剂,1.6μg.mL-1质粒DNA,转染6 h可以获得最佳转染效果,转染效率达4.48%;GFP转基因体细胞重构胚体外囊胚发育率为10%,GFP阳性胚胎率为48%。结果表明,脂质体转染试剂可以高效转染猪胎儿成纤维细胞,获得的阳性细胞具有支持猪全程发育的潜能。

Green fluorescent protein (GFP) transgenic pig embryos produced by somatic cell nuclear transfer

In this study,liposomal transfection system was screened and transgenic embryos were reconstr-ucted by nuclear transfer of GFP positive cells into enucleated in vitro matured oocytes,the development of reconstructed embryos both in vitro were observed,and GFP expression was checked as well.The results showed that porcine fetal fibroblast cultured with 4.0 μg·mL-1 lipotransfection regent and 1.6 μg·mL-1 plasmid DNA for 6 hours resulted in the highest transfection rate,which was 4.48%.In vitro blastocyst rate of GFP reconstructed embryos was 10%,GFP positive blastocyst rate was 48%.The results showed that plasmid regent could transfect PFF efficiently,and the positive cells have potentiality to support the reconstructed embryos develop to term.