RNA干扰诱导DNA修复基因hMGMT表达沉寂的研究  

Study of RNA Interference Induced Expression Silencing of DNA Repair Gene hMGMT

作　　者：赖延东[1] 吕嘉春[1] 李秀英[2] 宾晓农[1] 吴建军[1] 

机构地区：[1]广州医学院化学致癌研究所,广东广州510182 [2]暨南大学药学院基因组药物研究所,广东广州510632

出　　处：《癌变．畸变．突变》2006年第1期9-11,共3页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：国家自然科学基金项目(No.30371196;30200235);广东省重点科技攻关资助项目(No.2002B30104);广东省医学科研资助项目(B2001075)

摘　　要：背景与目的构建能特异诱导人DNA修复基因hMGMT表达沉寂的RNA干扰载体。材料与方法通过两步法聚合酶链反应(Polymerasing chain reaction,PCR)扩增hMGMT特异RNA干扰表达盒,连入质粒载体构建RNA干扰质粒pU6-MGMTi,与pGEFP-C1共转染人支气管上皮16HBE(Human bronchial epithelial)细胞,G418筛选后,采用RT-PCR和Western Blot分别检测mRNA和蛋白水平的表达抑制情况。结果成功构建hMGMT特异RNA干扰质粒pU6-MGMTi,转染16HBE细胞的mRNA和蛋白表达受到显著的抑制。结论应用RNA干扰表达盒以及共转染技术,构建MGMT基因表达沉寂的细胞株,为进一步阐明MGMT基因的功能创造条件。BACKGROUND ＆ AIM： To construct the RNA interference vector which can specially induce the expression silencing of human DNA repair gene hMGMT. MATERIAL AND METHODS： The hMGMT specific siRNA expression cassette was made by two steps PCR, and then linked with pUC19 to get pU6-MGMTi, co-transfected with pEGFP-C1 into 16HBE and screened by G418. The expression level of mRNA and protein was detected by RT-PCR and Western Blot, respectively. RESULTS： hMGMT-specific RNA interference vector pU6-MGMTi was constructed successfully. Transfected 16HBE cells MGMT mRNA and protein expression level were dramatically suppressed. CONCLUSION： Silencing of MGMT expression cell line was built by RNA interference expression cassette and co-transfection technology, which offered condition for studying the gene function of MGMT.

关 键 词：hMGMT基因 RNA干扰 16HBE 

分 类 号：Q342.3[生物学—遗传学] R730.1[医药卫生—肿瘤]