抑制人BI-1基因表达shRNA重组慢病毒表达载体的构建(英文)  

Construction of recombinant lentivirus vector expressing small hairpin RNA against human Bax inhibitor-1 gene expression

作　　者：吴丹[1] 王师尧[1] 王佩蓉[1] 金巍娜[1] 

机构地区：[1]北京大学医学部基础医学院医学遗传学系,北京市100191

出　　处：《中国组织工程研究与临床康复》2009年第50期9992-9996,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：Ph. D. Programs Foundation of the Ministry of Education of China,No.20070001801;National Fund for Fostering Talents of Basic Science,No.J0630853/J0108~~

摘　　要：背景:慢病毒介导的RNAi技术以其专一性、抑制效率高、作用持久等优点已广泛应用于基因功能研究中。该技术病毒包装、转染、以及shRNA序列设计都会对抑制效率产生影响。因此实验以人的Bax抑制子1(BI-1)为目的基因进行RNA干扰实验来探讨其影响因素。目的:为了利用慢病毒Lentivirus介导的RNAi技术寻找抑制人BI-1基因表达的siRNA。设计、时间及地点:单一样本观察,于2007-09/2008-12在北京大学医学部基础医学院医学遗传学系完成。材料:293T细胞、SH-SY5Y细胞为实验室保存;用Ambion公司(www.ambion.com)提供的网络在线工具设计了4个shRNA。方法:构建带有绿色荧光蛋白(EGFP)标签的、针对人BI-1基因不同区域设计的shRNA重组表达载体,然后与包装蛋白表达载体共转染293T细胞以包装成4种shRNA病毒粒子。通过流式细胞仪检测GFP的表达情况来摸索最佳包装条件。Real-timePCR以β-肌动蛋白mRNA被用作内参,将4种重组病毒和对照组病毒上清液侵染SH-SY5Y,检测内源性BI-1基因的敲低效率,筛选到最佳RNAi有效序列。主要观察指标:被不同包装病毒侵染的细胞内GFP的表达率。结果:pLentiLox3.7、rev、vsvg、rre等4质粒包装系统的最佳包装比例为2:1:1:1,包装48h收获的病毒粒子的感染效率最高,并且在包装后的24h之后换液会提高包装效率。靶定到人BI-1基因-2-17核苷酸(起始编码区域)的shRNARNA干扰效率最高。能够抑制40%正常基因表达。结论:实验摸索出Lentivirus介导的RNAi技术病毒包装的重要影响因素。为建立稳定的人BI-1基因表达敲低的神经细胞模型和研究BI-1异常表达参与的神经元凋亡疾病的病理研究打下基础。BACKGROUND： Lentivirus-mediated RNA interference technology has been widely used in the study of gene function because of its specificity, high inhibition efficiency and persistent effects. Virus packaging, transfection, as well as small hairpin RNA （shRNA）-coding sequence could affect the inhibition efficiency. Therefore, impact factors of RNA interference for human Bax inhibitor 1 （BI-1） was studied in this experiment. OBJECTIVE： To find the valid small interference RNA （siRNA） targeting human BI-1 gene by Lentivirus. DESIGN, TIME AND SETTING： Single sample observation was completed in Department of Medical Genetics, Basic Medical College, Peking University Health Science Center from September 2007 to December 2008. MATERIALS： 293T cell and SH-SY5Y cells were preserved in this laboratory; 4 shRNAs were designed through a use of online design tool by Ambion （www.ambion.com）. METHODS： Several recombinant plasmids which expressed shRNAs targeting different regions of the BI-1 gene and labeled enhanced green fluorescent protein as a fusion gene were constructed. Four types of shRNA-expressing vidons were obtained by cotransfection of shRNA-expressing plasmid and package protein-expressing plasmids into 293T cells. Green fluorescent protein expression was determined using flow cytometry to search the optimal package conditions. Real-time PCR and beta actin mRNA was used as an internal control, four types of recombinant viral supematant and control viral supernatant were added on the SH-SY5Y cells, knock-down efficacies of the endogenous BI-1 gene were determined, and the RNA interference effective sequences were optimized. MAIN OUTCOME MEASURES： Intracellular green fluorescent protein expression rate of the cells infected with different packaging virus. RESULTS： The optimal infective packaging virus could be obtained when the ratio of plasmids pLentiLox3.7, REV, VSVG and RRE was 2：1：1：1 and the supematants were collected 48 hours after transfection. Replacement of culture media 24

关 键 词：BI-1基因 RNA干扰 神经细胞模型 

分 类 号：R349.83[医药卫生—基础医学]