Construction and identification of recombinant plasmids containing short hairpin RNA targeting c-Met gene

OBJECTIVE To construct recombinant plasmids containing short hairpin RNA that targets c-Met gene,to assay the expression of c-Met mRNA and proteinum in Hep-2 cells after transfected with recombinant plasmids,and to select the most inhibitive c-Met-shRNA sequence.METHODS Three pairs fragment of shRNAs that targeted c-Met gene were designed.The recombinant plasmids pSilencer2.0/c-Met-shRNA containing short hairpin RNA targeting c-Met gene was constructed and transfected via cationic liposome Lipofectamine2000 into Hep-2 cells.The transfection efficacy was tested by RT-PCR and Western-Blot method,The difference in c-Met gene and proteinum expression levels between cells transfected group and the control group were compared.RESULTS The successful construction of recombinant plasmids was confirmed by DNA sequencing of the inserted segments.By RT-PCR and Western-Blot method, the strong effect of transfection of pSilencer2.0/c-Met-shRNA down-regulated c-Met expression in Hep-2 cells and recombinant plasmid 2 was confirmed.CONCLUSION The recombinant plasmids pSilencer2.0/c-Met-shRNA was constructed successfully and made it possible in the further studies to understand the effect of c-Met to the biological behaviour of Hep-2 cells.