Construction of FAK shRNA vector driven by the tumor-specific survivin promoter

Objective To construct a eukaryotic FAK shRNA expression vector driven by the tumor-specific survivin gene promoter and to evaluate its effect on HepG2 FAK mRNA expression.Methods The Survivin gene promoter was amplified by PCR,and then cloned to pGEMT vector and recombinated as pGEMT/pSurv,which was identified by double-enzyme digestion and sequencing.FAK shRNA sequences were designed and chemically synthesized,and then conjugated into the pGenesil-1 and recombined as pGenesil-FAK shRNA vectors,which were identified by double-enzyme digestion and sequencing.The U6 promoter in pGenesil-FAK shRNA vectors was then replaced by survivin gene promoter and recombined as pGenesil-pSurv-FAK shRNA vectors.The pGenesil-pSurv-FAK shRNA vectors were tranfected into HepG2 cell,and the transfection efficiency was observed by fluorescence microscope.After 48-hour transfection,the FAK mRNA expression level in HepG2 was evaluated by RT-RCR.Results The eukaryotic FAK shRNA expression vector pGenesil-pSurv-FAK shRNA driven by the tumor-specific survivin gene promoter was constructed successfully.The expression of FAK mRNA in HepG2 cell was down-regulated after transfection.Conclusion FAK shRNA expression vector driven by the tumor-specific Survivin gene promoter can downregulate the expression of FAK mRNA in HepG2,which may lay the foundation for further study of targeting FAK as an alternative treatment of HCC.