Construction of pSUPER-H1 RNAi system inhibiting the expression of VEGF

Objective To construct the expressing vector of siRNA in order to inhibit vascular endothelial growth factor expression.Methods We constructed the plasmid containing PolⅢ H1 promoter(pSUPER-H1).Three pairs of oligonucleotides comprised of 64 bases were chemically synthetized and annealed.pSUPER-H1 vectors were linearized with BglⅡ and HindⅢ.The annealed oligonucleotides were inserted into downstream of H1 promoter to construct the recombinant shRNA plasmid(pSUPER-H1-VEGF).Recombinant pSUPER-H1-VEGF vectors were then transformed into E.coli.of competence.The positive clones were selected and recombinant pSUPER-H1-VEGF plasmids were extracted.The plasmids were digested with EcoRⅠ and HindⅢ and identified by agarose gel electrophoresis.Results Agarose gel electrophoresis demonstrated that 64bp oligonucleotides were inserted into the expected sites.Sequence analysis showed that the inserted sequences were exactly correct.Conclusion Successful construction of recombitant pSUPER-H1-VEGF plasmids provides a tool to express siRNA in vivo mediated by vectors.