shRNAs介导的Smad2基因在小鼠成纤维细胞NIH/3T3中表达的沉默  

THE SILENCE OF SHORT HAIRPIN RNAs INDUCED Smad2 IN NIH/3T3 FIBROBLAST CELLS

作　　者：郑嵘[1] 熊琪[1] 蒋思文[1] 左波[1] 李凤娥[1] 徐德全[1] 任竹青[1] 熊远著[1] 

机构地区：[1]华中农业大学农业部猪遗传育种重点开放实验室农业动物遗传育种与繁殖教育部重点实验室,武汉430070

出　　处：《解剖学报》2008年第5期661-665,共5页Acta Anatomica Sinica

基　　金：国家重点基础研究发展计划(973)资助项目(2006CB102102)

摘　　要：目的通过构建5个shRNA表达质粒,对转化生长因子β/Smads信号通路中受体调节性Smads蛋白中的Smad2的表达进行抑制。方法针对鼠Smad2基因的mRNA序列,设计合成了5条shRNA-Smad2 DNA序列,分别插入至shRNA表达载体中,获得了5个shRNA-Smad2表达质粒,将shRNA表达质粒转染NIH/3T3细胞,采用半定量RT-PCR和Western blotting方法,检测shRNA表达质粒对Smad2表达的抑制效果。结果编号为2.4的shRNA-Smad2表达质粒能够显著降低Smad2的表达,同时发现由不同的shRNA-Smad2表达质粒组成的RNAi池,产生的抑制效果比单个RNAi表达质粒的抑制效果明显提高。结论成功地构建了特异、高效抑制Smad2表达的shRNA表达质粒。Objective To construct five shRNA-expression plasmids and to investigate the expression of Smad2 in TGF- β Smads signal transduction treated with shRNA-expression plasmid. Methods Five shRNA-Smad2 DNA sequences from mRNA sequence of mouse Smad2 gene were designed and synthesized. DNA oligonucleotides encoding an appropriate shRNA were inserted to shRNA expression vector respectively. Five shRNA-Smad2 expression plasmids were obtained and then transfected into NIH/3T3 cells. The suppressed expression of Smad2 was assessed by RT-PCR and Westeru-blotting. Results The shRNA- expression plasmid numbered 2.4 could markedly reduce the expression of Smad2. The suppression effect of the RNAi-pool composed of four different plasmids was more obvious than that of any single. Conclusion The shRNA-expression plasmids were successfully constructed, which could specifically and effectively suppress the expression of Smad2. The method of using a mixture of RNAi plasmids to improve the RNAi efficiency was established.

关 键 词：RNA干扰 基因沉默 短发夹状RNA 转化生长因子β SMAD2 免疫印迹法 小鼠 

分 类 号：R813.3[医药卫生—放射医学]