构建骨骼肌特异表达人FS基因载体及其稳定转染绵羊胎儿成纤维细胞

Construction of a Skeletal Muscle Specific Expression Vector of Follistatin and Its Stable Transfection into Sheep Fetal Fibroblasts Cells

旨在构建骨骼肌特异表达人卵泡抑制素(follistatin,FS)基因载体并得到其稳定转染的蒙古绵羊胎儿成纤维细胞系,为后期通过体细胞核移植方法制作转FS基因克隆绵羊奠定基础。首先通过RT-PCR方法克隆得到人FS基因cDNA序列,然后与猪骨骼肌特异表达启动子α-actin以及红色荧光蛋白表达元件连接,构建成FS基因骨骼肌特异表达载体pCFCDS。脂质体介导外源表达载体转染绵羊胎儿成纤维细胞,经G418筛选后得到稳定转染的绵羊转基因细胞克隆。PCR鉴定外源基因在细胞基因组中的整合,分析转基因细胞系的核型和生长状况。结果显示,成功构建得到绵羊骨骼肌特异表达人FS基因的真核表达载体,并得到其稳定转染的转基因绵羊胎儿成纤维细胞系,为后期通过体细胞核移植方法制作转FS基因克隆绵羊奠定基础。

The objective of the experiment is to construct a skeletal muscle specific expression vector of FS and then obtain the transgenic cell line which would pave the way to obtain the transgenic cloned sheep.The cDNA of FS was cloned by RT-PCR.Then pCFCDS,a skeletal muscle specific expression vector of FS,was constructed by connecting with α-actin promoter,as well as a red florescence protein expression unit.The sheep fetal fibroblast cells were transfected with the expression vector by LipofectamineTM 2000.Cell clones stably expressing red florescence were obtained after screening by G418.The recombinant of exogenous DNA was identified by polymerase chain reaction,and the karyotype as well as growth curve of transgenic cells were analysed.The result showed that a skeletal muscle specific expression vector of FS was constructed successfully.The exogenous genes have been integrated into sheep fetal fibroblast cells genome stably.These results have paved the way to obtain the FS transgenic sheep by nuclear transfer in the future.