重组慢病毒介导的nmd途径因子upf1和smg1可诱导干扰细胞株的构建  

Construction of Cell Strain Containing Inducible Interference of NMD Factor UPF1 and SMG1 Mediated by Lentiviral System

作　　者：柴宝峰[1] 常文娟[1] 申泉[1] 王刚[1] 

机构地区：[1]化学生物学与分子工程教育部重点实验室,山西大学生物技术研究所,山西太原030006

出　　处：《山西大学学报（自然科学版）》2012年第2期376-382,共7页Journal of Shanxi University(Natural Science Edition)

基　　金：国家自然科学基金(31172078;30770294);山西省自然科学基金(2009011040-1);山西省留学归国基金项目

摘　　要：无义介导的mRNA降解途径是一个比较完善的异常mRNA的降解机制,结合在外显子拼接复合体上的多种蛋白决定NMD途径对异常转录物的识别和降解的启动,其中UPF1和SMG1发挥主要功能.UPF1是一个RNA解旋酶和RNA依赖的ATP酶;而SMG1具有磷脂酰肌醇激酶活性,负责UPF1的磷酸化.本研究构建了含有UPF1和SMG-1基因发夹结构的诱导开关基因表达干扰质粒.利用慢病毒介导转化哺乳动物细胞HEK293T细胞得到重组病毒,经鉴定后感染细胞AD_293,目的基因在细胞中得以高效表达.通过继代培养和单克隆化,得到强力霉素诱导干扰UPF1和SMG-1表达的稳定细胞株.Nonsense-mediated decay(NMD) is well known by the lucid definition of being a RNA surveillance mechanism that ensures the speedy degradation of mRNAs containing premature translation termination codons.The definition of aberrant transcription and start of NMD are determined by the protein complex binding on EJC,where UPF1 and SMG1 play key role for NMD path.UPF1 is a RNA helicase and a RNA-dependent ATPase.SMG1 is responsible for UPF1 phosphorylation as a phosphatidylinositol 3-kinase.In this study,we construct the pHIV-7-derived lentiviral vector pTIG(pHIV7-TetR-IRES-GFP) encoding a U6tetO-promoted short hairpin RNA(shRNA) cassette containing UPF1 and SMG1.The resultant plasmids were transfected into HEK293T cell with the help of packaging plasmids to result in recombinant virus harboring hairpin RNA cassette.The resultant virus were then used to infect AD_293 cell to observe the expression of hairpin RNA gene.We have obtained the cell strains expressing hairpin RNA of UPF1 and SMG1,and these cell strains could be used for next research on the mechanism of NMD and gene screen.

关 键 词：nmd途径 upf1 smg1 PTC RNA干扰 SHRNA 

分 类 号：Q813[生物学—生物工程]