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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front Med Chin    2009, Vol. 3 Issue (2) : 158-163     DOI: 10.1007/s11684-009-0042-5
RESEARCH ARTICLE |
Regulation of exogenous bFGF gene mediated by recombinant adeno-associated virus in vitro
Ke SONG, Nianjing RAO, Meiling CHEN, Yingguang CAO()
Center of Stomatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
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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     
Corresponding Authors: CAO Yingguang,Email:cyg0729@tjh.tjmu.edu.cn   
Issue Date: 05 June 2009
URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-009-0042-5     OR     http://academic.hep.com.cn/fmd/EN/Y2009/V3/I2/158
Fig.1  The expression of green fluorescence protein (GFP) in different doxycycline (Dox)-treated groups detected under fluorescence microscopy (×100). (a) - (d): Concentrations of Dox was 0, 0.5, 1 and 2 μg/mL respectively. The results showed that 1 μg/mL Dox could greatly reduce the expression of GFP.
Fig.2  Identification of the plasmid pAAV-S3-bFGF. The CDS of bFGF was cloned into the plasmid pAAV-S3-bFGF successfully. (a) The amplification of the complete CDS region of mouse bFGF gene from the plasmid pBluescript SKII-bFGF. The PCR products were separated by agarose gel electrophoresis. Lane 1: Marker; lane 2: negative control; lane 3: plasmid pBluescript SKII-bFGF. (b) The recombinant plasmid pAAV-S3-bFGF demonstrated by PCR analysis. Lane 1: Marker; lane 2: negative control; lane 3: plasmid pBluescript SKII-bFGF; lane 4: recombinant plasmid pAAV-S3-bFGF. (c) The recombinant plasmid pAAV-S3-bFGF demonstrated by restriction enzyme digestion analysis. Lanes 1 and 2: Marker; lane 3: plasmid pAAV-S3-bFGF which was not digested; lane 4: recombinant plasmid pAAV-S3-bFGF. bFGF: basic fibroblast growth factor; CDS: coding sequences; PCR: polymerase chain reaction.
Fig.3  Determination of the titer of recombinant virus containing vector genomes by dot-blot assay. The physical particle titer of rAAV2-tet-off-bFGF and rAAV2-EGFP was 2×10 and 1.8×10 vector genomes/mL, respectively. Dots 1-7: The hybridization blotting of multi-proportion dilution series of standard samples (3×10, 2×10, 1×10, 5×10, 2.5×10, 1.25×10, 6.25×10 vector genomes/mL); (a) rAAV2-tet-off-bFGF; (b) rAAV2-tet-off-bFGF 2-fold dilution sample; (c) rAAV2-tet-off-bFGF 4-fold dilution sample; (d) rAAV2- EGFP; (e) rAAV2- EGFP 2-fold dilution sample; (f) rAAV2-EGFP 4-fold dilution sample.
Fig.4  Detection of the mRNA expression of bFGF and osteogenic genes in MC3T3-E1 cells by a real time RT-PCR. The results showed that 1 μg/mL Dox could greatly reduce the expression of bFGF mRNA. (a) The real time PCR products were separated by agarose gel electrophoresis; (b)-(d): analysis of the mRNA expression of osteogenic genes. 1: MC3T3-E1 cells were transduced by rAAV2-EGFP as the negative control group; 2-5: MC3T3-E1 cells were transduced by rAAV2-tet-off-bFGF, and the concentrations of Dox were 0, 0.5, 1 and 2 μg/mL, respectively. : <0.05 as compared with group 1, : <0.05 as compared with group 2. bFGF: basic fibroblast growth factor; RT-PCR: reverse transcription-polymerase chain reaction; Dox: doxycycline; OC: osteocalcin.
Fig.5  Western blot analysis of bFGF protein in different groups. 1: MC3T3-E1 cells were transduced by rAAV2-EGFP as the negative control group; 2-5: MC3T3-E1 cells were transduced by rAAV2-tet-off-bFGF, and the concentrations of Dox were 0, 0.5, 1 and 2 μg/mL respectively. β-actin served as an internal control (housekeeping gene). The results showed that 1 μg/mL Dox could greatly reduce the expression of bFGF. : <0.01 as compared with group 1, : <0.01 as compared with group 2. bFGF: basic fibroblast growth factor; Dox: doxycycline.
Fig.6  ALP assay in MC3T3-E1 cells. 1: MC3T3-E1 cells were transduced by rAAV2-EGFP as the negative control group; 2-5: MC3T3-E1 cells were transduced by rAAV2-tet-off-bFGF, and the concentrations of Dox were 0, 0.5, 1 and 2 μg/mL respectively. : <0.01 as compared with group 1, : <0.01 as compared with group 2. bFGF: basic fibroblast growth factor; Dox: doxycycline; ALP: alkaline phosphatase.
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