脂质体法构建广西巴马小型猪转基因体细胞及转基因克隆胚的生产

脂质体法是一种传统的向哺乳动物细胞导入外源基因的方法,然而其转染效率受多种因素影响。本研究将从脂质体和质粒DNA量、转染暴露时间以及混合孵育时间等四方面进行探索,以寻找最佳的转染体系。试验结果表明最佳转染体系为：脂质体1.25μL、DNA 0.7μg、脂质体-DNA混合孵育0 min以及转染暴露6 h。随后我们用优化过的转染体系对新生广西巴马小型猪肾脏成纤维细胞进行转绿色荧光蛋白（green fluorescent protein,GFP）操作,获得了（26.45±2.11）%的转染率,并以此为核供体经体细胞核移植技术成功构建表达GFP的广西巴马小型猪克隆胚胎,同时证实转基因克隆胚的体外发育能力比得上非转基因克隆胚。这为我们构建用于人类疾病研究的基因修饰广西巴马小型猪奠定了基础。

Construction of Guangxi Bama Minipig Transgenic Somatic Cells via Liposome Transfection Method and Production of Transgenic Cloned Embryos

Liposome transfection is a traditional method to delivery exogenous gene into mammalian cells. However, its efficiency is influenced by many factors. This study would optimize the quantities of liposome reagent and plasmid DNA, and the times of exposing and mixing. Experimental results show that the optimal transfection system is： The liposome 1.25 μL, DNA 0.7 μg, liposome-DNA mixing for 0 min and exposed for 6 h in incubation. Then we use the optimized transfection system to construction green fluorescent protein （GFP） transgenic flbroblasts from newborn Guangxi Bama minipig kidney. The transfection efficiency is （26.45±2.11）%. In addition, we use these transgenic fibroblasts via somatic cell nuclear transfer （SCNT） method to construct transgenie cloned embryos, and find the in vitro developmental capacity of transgenic cloned embryos are comparable to those derived from non-transgenic fibroblasts. This laid a solid foundation for our future studies on genetically modified Guangxi Bama minipigs for human disease research.