The regulatory effect of basicfibroblast growth factor(bFGF)mediated by recombinant adeno-associated virus(AAV)in vitro was investigated.Recombinant plasmid pAAV-S3-bFGF,and pSVneo were co-transfected into BHK-21 cells...The regulatory effect of basicfibroblast growth factor(bFGF)mediated by recombinant adeno-associated virus(AAV)in vitro was investigated.Recombinant plasmid pAAV-S3-bFGF,and pSVneo were co-transfected into BHK-21 cells,then the recombinant AAV genome was replicated and packaged with the helper virus HSV1-rc/ΔUL2.The titer of the recombinant rAAV2-tet-off-bFGF was determined by dot-blot assay.MC3T3-E1 cells were infected with rAAV2-tet-off-bFGF.Regulatory effects of Doxycycline(Dox)on bFGF and osteogenic factors were assayed quantitatively by real-time reverse transcription-polymerase chain reaction(RT-PCR)and Western blot.The physical particle titer of rAAV2-tet-off-bFGF successfully constructed was 1.8�1012 vector genomes/mL,and the virus could infect MC3T3-E1 cells effectively.In MC3T3-E1 cells treated with Dox,the expression levels of exogenous bFGF and osteogenic factors declined to varying degrees.It was concluded that rAAV2-tet-off-bFGF could infect MC3T3 cells efficiently,and this recombinant system could be regulated success-fully by Dox in vitro.展开更多
基金supported by the National Natural Science Foundation of China(Grant No.30572065/C03031103).
文摘The regulatory effect of basicfibroblast growth factor(bFGF)mediated by recombinant adeno-associated virus(AAV)in vitro was investigated.Recombinant plasmid pAAV-S3-bFGF,and pSVneo were co-transfected into BHK-21 cells,then the recombinant AAV genome was replicated and packaged with the helper virus HSV1-rc/ΔUL2.The titer of the recombinant rAAV2-tet-off-bFGF was determined by dot-blot assay.MC3T3-E1 cells were infected with rAAV2-tet-off-bFGF.Regulatory effects of Doxycycline(Dox)on bFGF and osteogenic factors were assayed quantitatively by real-time reverse transcription-polymerase chain reaction(RT-PCR)and Western blot.The physical particle titer of rAAV2-tet-off-bFGF successfully constructed was 1.8�1012 vector genomes/mL,and the virus could infect MC3T3-E1 cells effectively.In MC3T3-E1 cells treated with Dox,the expression levels of exogenous bFGF and osteogenic factors declined to varying degrees.It was concluded that rAAV2-tet-off-bFGF could infect MC3T3 cells efficiently,and this recombinant system could be regulated success-fully by Dox in vitro.
