利用RNAi抑制牛骨髓间充质干细胞中朊蛋白基因(PRNP)的表达  

Inhibition of gene expression of PRNP in bovine bone marrow mesenchymal stem cell by RNA interference technology

作　　者：宋少康[1,2] 董雅娟[1,2,3] 柏学进[1,2,3] 王文明[1,2] 刘玮[1,2] 牛召姗[1,2] 赵仕全[1,2] 杨莉[1,2] 

机构地区：[1]青岛农业大学动物胚胎工程中心,山东青岛266109 [2]山东省黑牛繁育工程技术研究中心,山东青岛266109 [3]山东布莱凯特黑牛科技股份有限公司,山东淄博256306

出　　处：《中国兽医学报》2012年第11期1683-1688,共6页Chinese Journal of Veterinary Science

基　　金：国家"抗病转基因牛新品种培育"资助项目(2008ZX08007-004)

摘　　要：利用RNAi技术,根据牛源PRNP基因cDNA设计3段siRNA序列和1个阴性对照序列,分别将其连接到RNA干扰载体pRNAT-U6.1/Neo上构建成shRNA载体,并将shRNA载体转染牛骨髓间充质干细胞(BMSC);通过Real-time PCR和Western Blotting筛选抑制效果最佳的载体;并用800mg/L G418对转染最佳载体的细胞进行药物筛选。结果显示:成功构建了3个靶向shRNA载体和1个阴性对照shRNA载体;转染后48h在荧光镜下检测各组均可观察到绿色荧光的表达;Real-time PCR和Western Blotting结果显示,3个靶向shRNA载体在不同时间段均在一定程度上下调了PRNP mRNA的表达,抑制了朊蛋白PrPC的生成,得到了1个最佳干扰载体sh3;通过药物筛选出了稳定转染的细胞单克隆。本研究获得了1个有效抑制朊蛋白基因表达的shRNA载体,并筛选出稳定转染的细胞单克隆,上述结果可为抗疯牛病体细胞核移植提供供体细胞。In order to provide the donor cell for the production of the anti-mad cow disease cloned transgenic cattle,the bovine bone marrow mesenchymal stem cells（BMSC） were used to inhibit the prion protein（PRNP） gene expression. We used the technology of RNA interference （RNAi） to cut down the PRNP gene expression,three candidate shRNAs（shl ,sh2,sh3） targeting the cod- ing sequence of bovine prnp gene and a negative control were designed,then they were ligated into the vector of pRNAT-U6.1/Neo respectively,and were transfected into BMSC. Real-time PCR and Western bloting screened the best inhibitory effect of the carrier; and we selected cells which transfected the best carrier in cell culture medium with 800 mg/L G418. We successfully constructed three targeting shRNA vector and a negative control shRNA vector. The green fluorescencecan had been observed by flu- orescence microscope after 48 h transfection. By the means of reabtime PCR and Western blotting, we could examine that at different periods three targeted shRNA can reduce the PRNP mRNA expression and inhibit the generation of prion protein（PrP^c）. We can draw that sh3 is one of the best interference carrier. The positive single stable transfection cell clones were also got by drug screening. In conclusion, the best shR- NA vector for effective inhibiting the prion protein gene expression and the stably transfected monoelonal cells had been obtained. This study provides donor cells for anti-mad cow somatic cell nuclear transfer.

关 键 词：疯牛病 PRNP 朊蛋白PrPc shRNA BMSC 

分 类 号：S852.65[农业科学—基础兽医学]