TALEN介导猪瘟病毒结构蛋白基因E0敲入山羊乳腺上皮细胞β-乳球蛋白基因座

CSFV E0 gene knocked-in β-lactoglobulin locus in goat mammary epithelial cells by TALEN

验证整合于山羊β-乳球蛋白基因座的猪瘟病毒E0(CSFV E0)基因是否能够利用内源性启动子调控该基因表达,为制备转CSFV E0基因山羊做好准备。利用脂质体转染的方法将实验室已构好的针对山羊β-乳球蛋白基因第一外显子的TALENs表达载体和包含CSFV E0基因的打靶载体共转染山羊乳腺上皮细胞,用药物(G418)进行筛选获得单克隆,并对获得的克隆进行5′端和3′端鉴定,鉴定都为阳性的克隆进行激素诱导24h后,收集培养液和细胞。通过RT-PCR和Western blot技术检测证明,转基因阳性细胞能够表达并分泌出CSFV E0蛋白。本试验为以转CSFV E0基因细胞为供核体进行核移植制作转基因动物奠定了基础。

To verify whether the classical swine fever virus E0 （CSFV E0） gene integrated into the goat β-lactoglobulin locus can regulate the expression of the gene using an endogenous promoter and prepare for the preparation of the swine fever E0 （CSFV E0） gene goat. TALENs expression vector for the first exon of the goat beta lactoglobulin gene and containing structural protein E0 gene of CSFV targeting vector which had been constructed by laboratory was co transfected into goat mammary epithelial cells by using liposome mediated transfection method, then by drug （G418） screening to obtain monoclonal which needed to identify of 5＇ and 3＇ end. The clones iden tified as positive clones were induced by hormone,and culture medium and cells were collected af ter induction. By RT PCR and Western blot technology, it is proved that the transgenic posilive ceils can express and secrete CSFV E0 protein. This study lays the foundation for the CSFV E0 gene transfected cells as donor cells in nuclear transfer to obtain the transgenic animals.