针对多效蛋白基因的小干扰RNA的设计及其稳定表达载体的构建  

Design of Pleiotrophin-Specific siRNA and Development of a Vector-Based siRNA Expression System

作　　者：胡迎春[1] 刘敏丽[2] 霍艳英[1] 项晓琼[1] 李刚[1] 

机构地区：[1]军事医学科学院放射与辐射医学研究所,北京100850 [2]重庆医科大学病理学教研室,重庆400016

出　　处：《生物技术通讯》2006年第4期509-511,共3页Letters in Biotechnology

基　　金：国家自然科学基金项目(30471946);北京市自然科学基金项目(7052056)

摘　　要：目的:设计多效蛋白基因特异性的小干扰RNA(siRNA),并构建一系列能在哺乳动物细胞内稳定表达这些siRNA的表达质粒,以便为在体外研究多效蛋白基因的功能打下基础。方法:设计并合成具有多效蛋白基因特异性的一组寡核苷酸片段,并克隆到pSilencer3.1-H1hygro载体;用脂质体LipofectAMINE2000转染和潮霉素筛选等方法建立能稳定表达相应siRNA的一组Pten-/-细胞克隆;利用Northern印迹检测这些细胞内多效蛋白基因的表达情况。结果:设计并构建了3个针对多效蛋白基因的siRNA表达质粒,并证明其中的1种质粒能在Pten-/-细胞内稳定表达相应的siRNA,并显著地抑制了该细胞多效蛋白基因的表达。结论:设计并构建出的针对多效蛋白基因的siRNA表达载体所表达的siRNA具有较强的RNA干涉功能,为多效蛋白基因的功能研究奠定了实验基础。Objective： To design pleiotrophin-specific siRNA and construct a group of plasmids expressing various pleiotrophin-specific siRNA in mammalian cells. Methods： A group of double strand oligonucleotide fragments were synthesized and cloned into pSilencer 3.1-H1 hygro vector. LipofectAMINE 2000 and hygromycin B screening were used to establish Pten-/- cell lines with stable expression of pleiotrophin-specific siRNA. The mRNA levels of pleiotrophin in the Pten-/- cells were analyzed using Northern blot. Results： Three plasmids expressing pleiotrophin-specific siRNA have been designed and construced, and one of them was proven to be able to effectively knockdown pleiotrophin gene in Pten-/- cells. Conclusion： The vectors constructed are able to express the pleiotrophin-specific siRNA and they are potent in me- diating pleiotrophin-specific RNA interference in Pten-/- cells.

关 键 词：小干扰RNA 多效蛋白基因 基因表达 

分 类 号：Q75[生物学—分子生物学] Q78