p53基因表达沉默的稳定Vero细胞系的建立及特性分析  

Generation and Characteristic Analysis of a Stable Vero Cell Line with p53 Gene Expression Knocked-down Using Lentiviral Vector-mediated Short Hairpin RNA

作　　者：刘超[1] 邱亚峰[1] 沈阳[1] 刘庆伟[1] 马志永[1] 

机构地区：[1]中国农业科学院上海兽医研究所兽医公共卫生研究室,上海200241

出　　处：《生物技术通报》2010年第7期83-88,共6页Biotechnology Bulletin

基　　金：国家自然科学基金青年基金(30700593);中央级公益性科研院所基本科研业务费专项资金(2006JB06;2010JB20)

摘　　要：利用慢病毒介导的RNA干扰技术,建立稳定沉默p53基因的Vero细胞模型。设计针对p53基因的shRNA序列,构建p53shRNA慢病毒载体并感染Vero细胞;嘌呤霉素筛选阳性细胞,局部消化法挑选细胞克隆;利用RT-PCR和West-ernblot检测p53的表达水平;利用虫荧光素酶报告系统,分析p53的转录激活功能。RT-PCR和Westernblot结果显示,慢病毒介导的shRNA有效地沉默p53基因表达;与对照组细胞相比,干扰组细胞的p53的转录激活功能明显降低。慢病毒介导的shRNA高效、稳定地沉默了p53基因的表达,同时,有效地降低了p53的转录激活功能,为研究p53的生物学功能提供了有利的工具。The study was to establish a stable Vero cell line with p53 gene silencing using lentivirus-mediated RNA interference （RNAi）. Vero cells were infected with a recombinant lentivirus expressing a specific shRNA for p53 gene. The positive cell clones were selected using selective drug of Puromycin, and isolated using the method of partial digestion. The expression of p53 was detected by semi-quantitative RT-PCR and Western blot. The p53 transcription activity of the negative control and p53 RNAi Vero cells was ana- lyzed with a luciferase report system. The Vero cell line with p53 gene silencing was obtained. The analysis of semi-quantitative RT-PCR and Western blot showed that the expression of p53 was substantially down-regulated by RNAi. The p53 transcriptional activity in RNAi cells was significantly decreased compared with that in negative control cells. The results demonstrated that the lentiviral vector-mediated short hairpin RNA has effectively and specifically silenced p53 gene expression and consequently down-regulated the p53 transcriptional activity in the generated-p53 RNAi Vero cell line. This RNAi cell line was appeared to be a useful tool in studying the biological function of p53.

关 键 词：P53 慢病毒载体 RNA干扰 筛选 细胞系 

分 类 号：Q78[生物学—分子生物学]