Construction and identification of lentivirus vectors interfering CYP2E1 gene expression in L02 cells

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 PLKO.l 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.l vector,restructured PLKO.l vector 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.