RNA干扰EGFR基因家族真核表达载体的构建  

Construction of eukaryotic expression plasmid experssing shRNA targeting EGFR gene family

作　　者：黄环[1] 吴永忠[2] 李少林[3] 郭启帅[1] 林远洪[1] 彭志平[3] 

机构地区：[1]重庆医科大学附属第一医院肿瘤科,重庆400016 [2]重庆市肿瘤研究所,重庆400030 [3]重庆医科大学基础医学院放射医学教研室,重庆400016

出　　处：《重庆医科大学学报》2008年第4期399-402,共4页Journal of Chongqing Medical University

基　　金：重庆市自然科学基金资助课题(2005-BB-5134)

摘　　要：目的:构建针对表皮生长因子受体(Epidermal growth factor recepter,EGFR)家族的小分子发夹状RN(A Small hairpinRNA,shRNA)干扰质粒表达载体,为本课题下一步研究提供有力工具。方法:选择放疗不敏感的卵巢癌细胞SKOV3中高表达的EGFR家族同源性基因为靶基因,根据shRNA的设计原则,设计实验组及对照组shRNA。体外合成2段编码短发夹RNA序列的DNA单链,退火,克隆到相应载体pGensil-1的polⅢ启动子下游。结果:经酶切和DNA测序鉴定成功构建了EGFR家族同源性基因的RNA干扰表达载体,分别命名为pGensil-1-E及pGensil-1-C。为下一步体外、体内实验奠定了基础。Objective：To construct the eukaryotic expression plasmid expressing shRNA targeting EGFR gene family as a tool for following experiments in vitro and in vivo. Methods ：EFGR family highly expressed in low radiosensitive ovarian cancer cells SKOV3,chosen highly expressed homologous genes in EFGR family as targeted gene. According to guidelines for shRNA design,designed experimental shRNA and negative control shRNA. In vitro synthesized 2 of the coding sequence of short hairpin RNA DNA single-strands, annealing, and cloned into the corresponding vector pGensil-1 of pol Ⅲ promoter downstream,named pGensil-1-E and pGensil-1-C. Confirmed by restrict endonuclease digestion and DNA sequencing. Conclusions：Eukaryotic expression plasmid experssing shRNA targeting homologous genes in EFGR family was constructed successfully.

关 键 词：EGFR家族 RNA干扰 真核细胞表达载体 

分 类 号：Q784[生物学—分子生物学]