[Construction and identification of recombinant adeno-associated virus expressing siRNA]

Adeno-associated virus (AAV) mediated RNA interference can be used to inhibit the expression of homologous genes in different mammalian cells. In this study, a transfer plasmid (pAAV-EGFP-H1) containing the H1 promoter and EGFP-expressing cassette was constructed based on the backbone of pAAV-MCS. Using calcium phosphate precipitation method, pAAV-EGFP-H1 was co-transfected into AAV-293 cells with helper plasmids and infective recombinant AAV was generated. EGFP gene was selected as the interfering target. EGFP gene was removed from pAAV-EGFP-H1 and a new transfer plasmid pAAV-H1 was constructed. Recombinant AAV-H1-shEGFP then infected 293 cells which was pre-transfected with plasmid pEGFP-N1. After 72 hours, the interference effect on EGFP expression was investigated by fluorescence microscope, fluorescence quantitative PCR and fluorescence activated cell sorting (FACS). All results showed that rAAV-H1-shEGFP could effectively reduce more than 60 percent of EGFP expression in 293 cells. The study demonstrates that a recombinant AAV transfer plasmid for RNAi is constructed, and the generated recombinant AAV can be used for further investigation on RNAi research.