逆转录病毒载体介导shRNA对人胚胎肾细胞中p53基因的沉默作用

目的：检测逆转录病毒载体介导的小发夹环RNA（small hairpin RNA，shRNA）对人胚胎肾细胞（HEK293）中p53基因的干扰作用。方法：将人H₁启动子由XholⅠ和EcoRⅠ酶切位点插入CMV启动子上游，人CD₄基因替代匀霉素抗性基因，作为检测标志。针对人野生型p53基因设计的shRNA被连接在H₁启动子的下游，构建含有shRNA的逆转录病毒载体LTRH₁-p53，即RNAi表达载体；利用流式细胞术检测逆转录病毒感染HEK293细胞72 h后细胞内P53蛋白水平的变化。结果：成功构建LTRH₁-p53载体，该载体感染HEK293细胞后72 h，与感染空病毒载体LTRH₁组相比，P53蛋白表达降至空病毒载体LTRH₁组的69％（P<0.01）。结论：LTRH₁-p53表达载体成功使p53基因沉默，shRNA表达载体的使用为基因功能研究提供有力工具。

Small hairpin RNA and retroviral vector-mediated silencing of p53 in HEK293 cells

Objective To establish a retroviral vector of small hairpin RNA(shRNA) expression in which the sense and antisense sequences targeting wild type human p53 were linked together with a 9-nucleotide loop to detect the interfering role of p53 gene in mammalian cells.Methods The human H_1 promoter was inserted into the upstream of the cytomegalovirus(CMV) promoter by using Xhol I and EcoR I sites.The resistanting hygromycin gene was replaced with the human CD_4 gene.These oligonucleotides were annealed and ligated the downstream of the H_1 promoter.All oligonucleotides were synthesized with Qiagen.HEK293 was infected with the shRNA vector and the expression of p53 was detected 72 h after infection by flow cytometry.Results The retroviral vector was successfully constructed and the expression of P53 protein was definitely suppressed in the HEK293 72 h after infection.(Conclusion The) applying of shRNA expression vector is possible to provide a prompt and promising method for evaluating the gene function of mammalian cells.