鲁西黄牛成纤维细胞MSTN基因敲除研究

肌肉生长抑制素(Myostatin,MSTN)又称GDF-8,属于转化生长因子超家族,是在骨骼肌中广泛表达的一类糖蛋白。试验根据已知牛MSTN基因的序列设计引物,通过PCR技术克隆获得同源长短臂,利用pPNT质粒为骨架构建含有loxP-Neo-loxP结构的打靶载体,然后利用脂质体转染鲁西黄牛成纤维细胞,G418和GANC双向筛选,收集打靶细胞,PCR法进行鉴定,初步确定筛选到的细胞为MSTN基因敲除细胞,为后续试验获得MSTN基因敲除牛奠定了基础。

Study on myostatin knockout of Luxihuangniu fibroblast

Myostatin(MSTN),a secreted growth and differentiation factor(GDF-8),a member of the transforming growth factor βsuper family,is one kind of skeletal muscle growth negative regulative factor.Firstly,two pairs of primers were designed according to sequence of myostatin of bovine which had been known.The long and short homologous arms were amplified from the luxihuangniu genome by long fragment PCR technique.Then,MSTN knock-out vector contained loxP-Neo-loxP structure was constructed.Thirdly,MSTN knock-out vector was transfected into Luxihuangniu fibroblasts by lipofectamine,and the cells were selected by culture in the medium containing G418 and GANC.At last,these cells were identified by PCR examination.The results showed that MSTN gene knock-out vector occurred in the target fixed income,which laid a good foundation for the follow-up experiments to obtain the MSTN knock-out Luxihuangniu.