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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.
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Keywords
tetracycline regulatory system
adeno-associated virus
basic fibroblast growth factor
gene regulation
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Corresponding Author(s):
CAO Yingguang,Email:cyg0729@tjh.tjmu.edu.cn
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Issue Date: 05 June 2009
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1 |
Drosse I, Volkmer E, Capanna R, De Biase P, Mutschler W, Schieker M. Tissue engineering for bone defect healing: an update on a multi-component approach. Injury , 2008, 39(Suppl 2): S9-20 doi: 10.1016/S0020-1383(08)70011-1
|
2 |
Cohen M M, Jr. The new bone biology: pathologic, molecular, and clinical correlates. Am J Med Genet A , 2006, 140(23): 2646-2706 doi: 10.1002/ajmg.a.31368
|
3 |
Wu X B, Dong X Y, Wu Z J, Cao H, Niu D B, Qu J G, Wang H, Hou Y. A novel method for purification of recombinant adeno-associated virus vectors on a large scale. Chin Sci Bull , 2001, 46(6): 485
|
4 |
Ponnazhagan S, Yoder M C, Srivastava A. Adeno-associated virus type 2-mediated transduction of murine hematopoietic cells with long-term repopulating ability and sustained expression of a human globin gene in vivo. J Virol , 1997, 71(4): 3098-3104
|
5 |
Wozney J M, Rosen V, Celeste A J, Mitsock L M, Whitters M J, Kriz R W, Hewick R M, Wang E A. Novel regulators of bone formation: molecular clones and activities. Science , 1988, 242(4885): 1528-1534 doi: 10.1126/science.3201241
|
6 |
Franceschi R T. Biological approaches to bone regeneration by gene therapy. J Dent Res , 2005, 84(12): 1093-1103 doi: 10.1177/154405910508401204
|
7 |
Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA , 1992, 89(12): 5547-5551 doi: 10.1073/pnas.89.12.5547
|
8 |
Freundlieb S, Schirra-Muller C, Bujard H. A tetracycline controlled activation/repression system with increased potential for gene transfer into mammalian cells. J Gene Med , 1999, 1(1): 4-12 doi: 10.1002/(SICI)1521-2254(199901/02)1:1<4::AID-JGM4>3.0.CO;2-Y
|
9 |
Song K, Rao N J, Chen M L, Cao Y G. Construction of adeno-associated virus system for human bone morphogenetic protein 7 gene. J Huazhong Univ Sci Technolog Med Sci , 2008, 28(1): 17-21
|
10 |
Conway J E, Rhys C M, Zolotukhin I, Zolotukhin S, Muzyczka N, Hayward G S, Byrne B J. High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and Cap. Gene Ther , 1999, 6(6): 986-993 doi: 10.1038/sj.gt.3300937
|
11 |
Knop D R, Harrell H. Bioreactor production of recombinant herpes simplex virus vectors. Biotechnol Prog , 2007, 23(3): 715-721 doi: 10.1021/bp060373p
|
12 |
Yang M, Ma Q J, Dang G T, Ma K T, Chen P, Zhou C Y. Adeno-associated virus-mediated bone morphogenetic protein-7 gene transfer induces C2C12 cell differentiation into osteoblast lineage cells. Acta Pharmacol Sin , 2005, 26(8): 963-968 doi: 10.1111/j.1745-7254.2005.00159.x
|
13 |
Hauswirth W W, Lewin A S, Zolotukhin S, Muzyczka N. Production and purification of recombinant adeno-associated virus. Methods Enzymol , 2000, 316: 743-761 doi: 10.1016/S0076-6879(00)16760-6
|
14 |
Han G C, Li Y, Wang J N, Wang R X, Chen G J, Song L, Xu, R N, Yu M, Wu X B, Qian J H, Shen B F. Active tolerance induction and prevention of autoimmune diabetes by immunogene therapy using recombinant adenoassociated virus expressing glutamic acid decarboxylase 65 peptide GAD (500-585). J Immunol , 2005, 174(8): 4516-4524
|
15 |
Yan H, Guo Y H, Zhang P, Zu L Y, Dong X Y, Chen L, Tian J W, Fan X L, Wang N P, Wu X B, Gao W. Superior neovascularization and muscle regeneration in ischemic skeletal muscles following VEGF gene transfer by rAAV1 pseudotyped vectors. Biochem Biophys Res Commun , 2005, 336(1): 287-298 doi: 10.1016/j.bbrc.2005.08.066
|
16 |
Ornitz D M, Itoh N. Fibroblast growth factors. Genome Biol , 2001, 2(3): REVIEWS3005
|
17 |
Naganawa T, Xiao L P, Coffin J D, Doetschman T, Sabbieti M G, Agas D, Hurley M M. Reduced expression and function of bone morphogenetic protein-2 in bones of Fgf2 null mice. J Cell Biochem , 2008, 103(6): 1975-1988 doi: 10.1002/jcb.21589
|
18 |
Agata H, Asahina I, Yamazaki Y, Uchida M, Shinohara Y, Honda M J, Kagami H, Ueda M. Effective bone engineering with periosteum-derived cells. J Dent Res , 2007, 86(1): 79-83 doi: 10.1177/154405910708600113
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