[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.
Yin
Jun
J
College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China. sy-yinjun@163.com
Li
Hou-da
HD
D000818:Animals; D002460:Cell Line; D004274:DNA, Recombinant / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics*; D066246:ErbB Receptors / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection
D004274:DNA, Recombinant; D034741:RNA, Small Interfering; D066246:ErbB Receptors