title: Recombinant  adeno-associated virus BMP-4/7 fusion gene confers ossification activity in  rabbit bone marrow stromal cells.
abstract: The  biological effects of transfection of an adeno-associated virus (AAV) vector  with bone morphogenetic proteins 4 and 7 (BMP-4/7) fusion gene (AAV-BMP-4/7)  were determined in rabbit bone marrow stromal cells (BMSCs). BMP-4 and BMP-7  genes were obtained through one-step reverse transcriptase polymerase chain  reaction from human placental cells. The BMP-4/7 fusion gene was then generated  through recombination. Rabbit BMSCs were transfected with the recombinant AAV  vectors carrying AAV-BMP-4/7 with multiplicity of infection values. Cell growth  curves were drawn to evaluate the biological effects of AAV-BMP-4/7 on cell  activity. The transfection efficiency was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium  bromide assay. The ossification of cells was evaluated by observing alkaline  phosphatase (ALP) and osteocalcin (OC) activity after transfection for 7 and 14  days. The cells were then transfected with AAV-BMP-4/7 and AAV-enhanced green  fluorescent protein. We successfully constructed the recombinant  adeno-associated virus with the BMP-4/7 fusion gene. The transfection  efficiency of AAV-BMP-4/7 was approximately 72% without significant biological  effects on cell activity. Cell ossification was significant after transfection  with AAV-BMP-4/7. The 1 x 10(5) vg/cell  multiplicity of infection value of transfection efficiency was more than 5 x 10(4) vg/cell  (59.38%). Significantly higher ALP and OC activity occurred in the AAV-BMP-4/7  transfection groups than in the AAV-enhanced green fluorescent protein groups (t(ALP) =  896.88, P < 0.001; t(OC) =  543.24, P < 0.01). The AAV-BMP-4/7 fusion gene can highly efficient  transfect rabbit BMSCs cultured in vitro and it has significant  ossification activity.
authors.affiliation: Department of Orthopedics, First Hospital of Harbin Medical University, Harbin, China.
authors.forename: S H
authors.initials: SH
authors.lastname: Yuan
authors.forename: C B
authors.initials: CB
authors.lastname: Gao
authors.forename: C U
authors.initials: CU
authors.lastname: Yin
authors.forename: Z G
authors.initials: ZG
authors.lastname: Yin
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D055415:Bone Morphogenetic Protein 4 / Q000235:genetics*; D055419:Bone Morphogenetic Protein 7 / Q000235:genetics*; D049109:Cell Proliferation; D048430:Cell Shape; D000229:Dependovirus / Q000378:metabolism*; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D015675:Osteocalcin / Q000378:metabolism; D010012:Osteogenesis; D010957:Plasmids / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics*; D014162:Transfection
chemical_list: D055415:Bone Morphogenetic Protein 4; D055419:Bone Morphogenetic Protein 7; D011993:Recombinant Fusion Proteins; D015675:Osteocalcin; D000469:Alkaline Phosphatase