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

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.