Regulation of exogenous bFGF gene mediated by recombinant adeno-associated virus in vitro

Regulation of exogenous bFGF gene mediated by recombinant adeno-associated virus in vitro

Abstract

The regulatory effect of basic fibroblast 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 successfully by Dox in vitro.

Keywords

tetracycline regulatory system / adeno-associated virus / basic fibroblast growth factor / gene regulation

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Acknowledgements

The study was supported by the National Natural Science Foundation of China (Grant No. 30572065/C03031103).

版权

2014 Higher Education Press and Springer-Verlag Berlin Heidelberg