title: Effect of recombinant adeno-associated BMP-4/7 fusion gene on the biology of BMSCs.
abstract: The aim of the study was to investigate the effect of recombinant adeno-associated virus (AAV) with morphogenetic protein 4/7 fusion gene on the ossification of rabbit bone marrow stromal cells (BMSCs). The genes BMP-4 and BMP-7 were obtained through one-step reverse transcriptase polymerase chain reaction from human placental cells. The BMP-4/7 fusion gene was generated through recombination. The rabbit BMSCs were transfected with the recombinant AAV vectors carrying AAV-BMP-4/7 with various multiplicity of infection (MOI) values. Expression of fusion gene BMP-4/7 protein was determined by the ELISA method. The ossification of cells was evaluated by observing alkaline phosphatase (ALP) and osteocalcin (OC) activity after transfection for 7 and 14 days. We successfully constructed the recombinant AAV with the BMP-4/7 fusion gene. A 29-20 kDa protein was shown by SDS-PAGE electrophoresis following transfection with AAV-BMP-4/7. The fusion protein BMP-4/7 in BMSCs transferred by AAV showed a positive correlation with various MOI values. There was significantly higher ALP and OC activity in the AAV-BMP-4/7 transfection groups than in the AAV-EGFP groups (t(ALP)=896.88, P<0.001, t(OC)=543.24, P<0.01). The fusion gene BMP-4/7 is capable of enhancing the expression of BMPs and possesses significant ossification activity through AAV.
authors.affiliation: Department of Orthopedics, the First Hospital of Harbin Medical                     University, Harbin 150001, P.R. China.
authors.forename: Shao-Hui
authors.initials: SH
authors.lastname: Yuan
authors.forename: Zhenggang
authors.initials: Z
authors.lastname: Bi
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D055415:Bone Morphogenetic Protein 4 / Q000235:genetics* / Q000378:metabolism; D055419:Bone Morphogenetic Protein 7 / Q000235:genetics* / Q000378:metabolism*; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism* / Q000473:pathology; D015675:Osteocalcin / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D055415:Bone Morphogenetic Protein 4; D055419:Bone Morphogenetic Protein 7; D011993:Recombinant Fusion Proteins; D015675:Osteocalcin; D000469:Alkaline Phosphatase