| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1997: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1996: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
None of currently available gene transfer system has possessed the property of site-specific integration of interested genes to the preferred site on the host genome. Only recombinant adeno-associated virus (rAAV) is potentially possible to have this property, because the genome of wild-type AAV integrates specifically to a defined site, called AAVS1, on the chromosome 19. Rep protein (s) ; encoded viral gene, mediates site-specific integration via complex formation between AAV ITR (inverted terminal repeat) and AAVS1. The currently available rAAV without rep gene has accordingly lacked the property of site-specific integration. Rep proteins are known to be cytostatic and an inhibitor of sp1 promoters under stable expression. Actually, it is difficult to express Rep protein only in the moment of integration event. In this study, we examined direct delivery of Rep Protein to 293 cells with AAV vector plasmid by lipofection, instead of cotransfection of Rep-expression plasmids. Rep 78 Pr
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otein (psub 201 ; 318-2227 nt) was expressed as a fusion protein (MalE-REP) of maltose binding protein in E.coli. and purified through Eamylose resin. After digestion of Mal-REP by Factor X,REP fraction was prepared by passing through amylose resin for removal of Maltose binding protein. 293 cells were lipofected with Rep protein (MalE-REP or REP) and AAV vector plasmid (XF) containing the tk promoter driven neoR gene flanked by ITRs, and culture in the presence of G418. Specific integration of AAV vector plasmid was detected by mested-PCR amplification from 3'-vector ITR to AAVS1, southern analysis by AAVS1 oligo, and direct sequencing of PCR bands. The 3'-ITR of AAV vector plasmid is integrated at the sequence followed to rep binding site into the AAVS1 locus (nt 991,1014, or 1161). Both of ITR and AAVS1 integration sites were very similar to those by wild-type AAV (AAVS1 1078,1138 nt) or cotransfection of Rep-expression plasmids (AAVS1 1001-1146 nt). These results suggested that specific integration essentially and sufficiently requires only Rep 78 and vector ITR.This strategy is easy to control amount of Rep protein in the targeted cells and may be useful for site specific integration of the interested genes into the chromosome 19, although the further studies remains for the optimal condition of lipofection. Less
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