牛朊蛋白基因prnp敲除载体的构建及真核细胞转染  

Construction of prnp gene knockout vector and its transfection in eukaryotic cell

作　　者：张海林[1] 程胖[1] 兰杰[1] 宋永利[1] 张涌[1] 

机构地区：[1]西北农林科技大学动物医学院农业部动物生殖生理与胚胎工程重点实验室,杨凌712100

出　　处：《生物工程学报》2010年第3期297-304,共8页Chinese Journal of Biotechnology

基　　金：抗病转基因牛新品种培育(No.2008ZX08007-004)资助~~

摘　　要：利用正负筛选策略(Positive-negative selection,PNS)对中靶细胞进行富集是提高体细胞基因打靶效率常用的策略之一。将动物的朊蛋白基因prnp敲除,使其不能表达朊蛋白(传染性海绵状脑病的致病蛋白),从而使其具有抵抗Prion病感染的能力。本研究采用正负筛选策略,构建了牛prnp基因的双等位基因敲除载体,经内切酶Sac Ⅱ线性化后,再通过电穿孔转染牛胎儿成纤维细胞,分别用600μg/mL G418、200nmol/mL Ganciclovir(GCV)进行正负药物筛选,最终获得了176个药物抗性细胞克隆,进一步采用PCR、测序、间接免疫荧光试验及Western blotting试验对细胞克隆进行鉴定,结果表明,其中的9个细胞克隆为中靶细胞,证明牛prnp基因被成功敲除。本研究为牛prnp的敲除提供了可行性依据,并为体细胞核移植生产敲除朊蛋白基因的转基因动物提供供体细胞。It is one of the frequently utilized strategies for positive-negative selection to elevate the gene targeting efficiency in somatic cells by enriching targeted colonies. Knocking out prnp in animals by gene targeting can prevent it from expressing Prion protein （Pathogenic protein of transmissible spongiform encephalopathy）, which enables it to resist infection of Prion. We constructed a bovine prnp biallelic targeting vector via the positive-negative selection strategy, and transfected the linearized vector into the bovine fetal fibroblasts through electroporation. Then, we selected cells in cell culture medium with G418 under a concentration of 600 μg/mL followed by Ganciclovir （GCV） under a concentration of 200 nmol/mL. In the end, we successfully obtained 176 cell clones. All these clones were identified by means of sequencing, immunofluorescence and western blotting, respectively, confirming that there existed 9 positive cell clones. The results showed that the bovine prnp gene was successfully knocked out. Conclusively, we provide an effective way to knockout bovine prnp gene, which could serve as the basis for producing prion protein gene knockout transgenic cloned cattle.

关 键 词：基因打靶 正负筛选策略 朊蛋白 牛胎儿成纤维细胞 PRNP 间接免疫荧光 Western BLOTTING 

分 类 号：Q78[生物学—分子生物学]