SET基因shRNA慢病毒载体的构建及其在肝细胞中的表达鉴定  

Construction and identification of lentivirus vectors interfering with gene expression in liver cells

作　　者：刘建军[1] 蒋英芝[1] 叶金波[1] 李杰[1] 杨细飞[1] 周丽[1] 黄海燕[1] 黄新凤[1] 

机构地区：[1]深圳市疾病预防控制中心深圳市现代毒理学重点实验室,广东深圳518055

出　　处：《癌变．畸变．突变》2013年第1期44-47,52,共5页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：国家自然科学基金资助项目(81273126;30972454);深圳市重点实验室提升发展项目(CXB201005260068A)

摘　　要：目的:构建SET基因shRNA慢病毒表达载体,为研究SET在三氯乙烯毒性机制中的作用提供技术支持。方法:通过NCBI检索SET序列,设计合成5对shRNA片段,退火后连接到慢病毒载体pLVX-shRNA1 vector。挑取筛选的单菌落,经PCR和测序鉴定后提取质粒。将质粒共转染到293T细胞,收集病毒上清,转导入L-02肝细胞中,利用嘌呤霉素筛选得到SET缺陷型细胞,最后利用荧光定量PCR和Western blot鉴定干扰效果。结果:PCR和测序结果证明双链shRNA正确插入慢病毒载体pLVX-shRNA1 vector,共转染293T细胞得到高滴度病毒,转导L-02细胞筛选出SET基因缺陷型细胞。结论:利用慢病毒介导的RNAi技术成功构建了SET基因缺陷型细胞。OBJECTIVE： To construct and identify the lentivirus vectors that interfere with SET gene expression in liver cells, which would provide technical support for studying the role of SET in the mechanism of TCE toxicity. METHODS ： Based upon SET sequences retrieved through NCBI and literature reviews, 5 pairs of shRNA were designed and synthetized, annealed and ligated to the lentivirus pLVX-shRNA1 vector. Before plasmids were extracted, single colonies were picked and identified by PCR and sequencing. The lentivirus vector with shRNA targeting human SET mRNA were transfected into 293T cells, then the lentivirus supernatant was obtained and used for infecting L-02 cells. After selection with puromycin, the SET-deficient cells were obtained. The efficiency of gene knockdown was determined by real-time PCR and Western blot. RESULTS： shRNA was inserted into pLVX-shRNA1 vector, the reconstructured vector was transfected into 293T cells and high-titer lentivirus was formed. The lentivirus was transduced into L-02 cells, and SET-deficient cells were obtained after selection. CONCLUSION： SET-deficient cells were successfully constructed by using lentivirus-mediated RNA interference technology. [KEY WORDS1 SET; RNAinterference; lentivirus; vector

关 键 词：SET RNA干扰 慢病毒 载体 

分 类 号：Q782[生物学—分子生物学] Q786