癌变·畸变·突变2012，Vol.24Issue(4)：290-294,5.DOI:10.3969/j.issn.1004-616x.2012.04.012
CYP2E1基因RNA干扰载体的构建与表达
Construction and identification of lentivirus vectors interfering CYP2E1 gene expression in L02 cells
摘要
Abstract
OBJECTIVE: To construct and identify the lentivirus vectors that interfere CYP2E1 gene expression in human hepatocytes (L02 cells), so that these vectors could be applied for further studies of CYP2E1 in metabolism of environmental pollutants and drugs. METHODS: Three pairs of shRNA were designed and synthesized, then ligated to the lentivirus PLK0.1 vector. The lentivirus vectors with shRNA targeting human CYP2E1 Mrna were transfected into 293FT cells by Lipofectamine, then the lentivirus supernatant was obtained and used for infecting L02 cells. After one week of selection with puromycin, the CYP2E1 -silent cells were obtained, then the efficiency of gene knockdown were determined by Real-time PCR and western blot. RESULTS: PCR and western blot data showed that shRNA were successfully inserted into PLKO.lvector, restructured PLKO.lvector was transfected into 293FT cells and high-titer lentivirus was formed- The lentivirus was transducted into L02 cells, CYP2E1-silent cells were obtained after selection. PCR data showed the highest inhibitory efficiency was approximately 86.9% for CYP2E1 expression, and western blot data indicated that no obvious band came out in L02 cells with shRNA3 interfering target. CONCLUSION: CYP2E1 -silent cells were successfully constructed by using lentivirus-mediated RNA interference technology.关键词
CYP2E1/RNA干扰/慢病毒/载体Key words
CYP2E1/RNA interference/lentivirus/vector分类
生物科学引用本文复制引用
毛吉炎,徐新云,何晓阳,毛侃琅,黄海燕,刘庆成..CYP2E1基因RNA干扰载体的构建与表达[J].癌变·畸变·突变,2012,24(4):290-294,5.基金项目
深圳市科技计划项目(20ll01015) （20ll01015）
