敲除成纤维细胞生长因子5基因对阿尔巴斯绒山羊胎儿皮肤成纤维细胞系的影响

摘要

In this study, in order to provide a nuclear for somatic cell nuclear transfer and production of transgenic animals, cationic liposome-mediated transfection of homologous recombination FGF5 gene knockout vector to somatic cell. After positive and negative selection obtained 51 cell clones, which remaining 12 clones after propagation, and ultimately get 4 positive cell clones by the molecular indentification of DNA and RNA levels, namely established the FGF5-knockouted Inner Mongolia Arbas cashmere goat fetal skin fibroblast cell line. Compared with normal somatic cell, the four FGF5 -knockouted positive cell clones had occurred very significant changes in cell morphology, traits and growth characteristics: bigger cell volume; fuzzy boundaries, reduced stereoscopic;growth rate dramatic decline;trypsin digestion time required was significantly increased. This study showed that these changes might be due to FGF5 gene knockout, cell transfection, drug selection and other roles.%本研究为体细胞核移植生产转基因动物提供核供体,以阳离子脂质体介导转染同源重组成纤维细胞生长因子5(fibroblast growth factor 5,FGF5)基因打靶载体至体细胞中,经正负双筛选获得了51个细胞克隆,扩繁后剩余12个克隆,最终经DNA及RNA水平的分子生物学鉴定得到4个阳性细胞克隆,即建立了敲除FGF5基因的内蒙古阿尔巴斯绒山羊胎儿皮肤成纤维细胞系.与普通细胞相比,所得的阳性细胞克隆在细胞形态、性状及生长特性等方面均发生了极其显著的变化:细胞体积增大；边界模糊,立体感降低；生长速率明显下降；胰蛋白酶消化所需时间明显增加.研究结果表明上述变化可能是受到了敲除FGF5基因、细胞转染和药物筛选作用等的影响所致.