Construction of adeno-associated virus system for human bone morphogenetic protein 7 gene.
To construct the recombinant adeno-associated virus (rAAV) vector with human bone morphogenetic protein 7 (BMP7) and observe the BMP7 mRNA expression in vitro, BMP7 CDS sequence was cloned into expression plasmid pAAV-MCS of AAV Helper Free System. The recombinant plasmid was identified with enzyme digestion and sequencing. The recombinant plasmid, pAAV-RC, pHelper were co-transfected into AAV-293 cells according to the calcium phosphate-based protocol. The viral stock was collected by 4 rounds of freeze/thaw. After purified and concentrated, the recombinant virus titer was determined by dot-blot assay. HEK293 cells were transfected with the recombinant virus at different MOI, and the expression of BMP7 mRNA was detected by RT-PCR. The results showed rAAV-BMP7 was constructed and packaged successfully. The physical particle titer was 2.5x10(11) vector genomes/mL. There was different expression level of BMP7 mRNA after transfecton. These data suggested that recombinant AAV mediated a stable expression of hBMP7 mRNA in 293 cells. The AAV production method may pave the way of an effective strategy for the jaw bone defection around dental implants.
Song
Ke
K
Center of Stomatological Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. songke_coco@163.com
Rao
Nianjing
N
Chen
Meiling
M
Cao
Yingguang
Y
D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins / Q000235:genetics* / Q000502:physiology; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D005786:Gene Expression Regulation; D005821:Genetic Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D016678:Genome; D006801:Humans; D010957:Plasmids / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics* / Q000502:physiology
C525819:BMP7 protein, human; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins; D017931:DNA Primers; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta