应用RNA干扰技术体外抑制兔成纤维细胞HGPRT的表达及其核移植研究(英文)  

RNAi-mediated Stable Silencing of HGPRT Expression in Rabbit Fibroblasts and SCNT Embryo Production

作　　者：郭毅[1] 张传山[2] 李善刚[1] 李锋[1] 谷瑞环[1] 邢凤英[1] 李垚[1] 姚刚[2] 陈学进[1] 

机构地区：[1]上海交通大学医学院实验动物科学部,上海200025 [2]新疆农业大学动物医学学院,乌鲁木齐830052

出　　处：《生物化学与生物物理进展》2009年第7期872-879,共8页Progress In Biochemistry and Biophysics

基　　金：supported by grants from Important Project of Basic Research for Science and Technology in Shanghai Government(08JC1413900 );Agricultural Development by Science and Technology Program in Shanghai Government (2006)5-3; Shanghai LeadingAcademic Discipline Project(S30201)~~

摘　　要：次黄嘌呤-鸟嘌呤磷酸核糖基转移酶(hypoxanthine guaninephos phoribosyltransferase,HGPRT)的功能缺失与痛风、肾结石和雷纳综合症(Lesch-Nyhan Syndrome)等疾病相关.制作HGPRT基因表达降低的模式动物,将有利于人们对这种疾病的发病机理和治疗做进一步的研究.构建了针对HGPRT基因表达的shRNA干扰载体,并将质粒转染兔成纤维细胞,获得携带该干扰片段的转基因细胞系,经PCR鉴定转基因成纤维细胞克隆阳性率为83.3%.RT-PCR及Western blot检测结果表明转基因干扰成纤维细胞系HGPRT mRNA和蛋白质表达量明显降低.最后,以转基因成纤维细胞进行核移植,囊胚率为27.8%,与正常来源的成纤维细胞囊胚率相比较差异不显著.说明,通过RNAi可稳定干扰兔成纤维细胞HGPRT基因的表达,为进一步通过核移植技术建立HGPRT RNAi转基因兔模型创造条件.The hypoxanthine-guanine phosphoribosyl transferase （HGPRT） gene mutation is responsible for gouty arthritis, kidney stone, and Lesch-Nyhan Syndrome （LNS）. It has been reported that the expression of HGPRT is decreased or even absent in these diseases. Rabbits are an ideal model for studying the pathology of these diseases. Therefore, the development of an HGPRT-knockdown rabbit model will be highly beneficial in such studies. Stable HGPRT-knockdown transgenic fibroblast lines were generated by transfecting rabbit fibroblasts with RNA interference （RNAi） plasmids. Polymerase chain reaction （PCR） analyses indicated that the average positive rate was 83.3%. The mRNA and protein levels of HGPRT in the transgenic fibroblast lines were significantly lower than that in the control. Transgenic rabbit blastocysts were derived after performing nuclear transfer. The results show that RNAi can be used to stably knock down expression of the HGPRT in rabbit fibroblasts and further improvements in related technologies will facilitate the use of this method for the generation of HGPR T-knockdown rabbits.

关 键 词：RNA干扰 兔成纤维体细胞 次黄嘌呤-鸟嘌呤磷酸核糖基转移酶(HGPRT) 核移植 

分 类 号：Q7[生物学—分子生物学]