Construction of a recombinant adenovirus co-expressing bone morphogenic proteins 9 and 6 and its effect on osteogenesis in C3H10 cells.
- Author:
Xiaohong DIE
1
;
Qing LUO
;
Cong CHEN
;
Guangjin LUO
;
Quan KANG
Author Information
- Publication Type:Journal Article
- MeSH: Adenoviridae; genetics; Bone Morphogenetic Protein 6; genetics; Genetic Vectors; Growth Differentiation Factors; genetics; HEK293 Cells; Humans; Osteoblasts; cytology; Osteogenesis; Plasmids; Recombinant Fusion Proteins; genetics; Transfection
- From: Journal of Southern Medical University 2013;33(9):1273-1279
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct a recombinant adenovirus co-expressing bone morphogenic protein (BMP) 9 and BMP6 and observe its effect on the osteogenesis in C3H10 cells.
METHODThe full-length sequences of BMP9 and BMP6 were amplified from AdEasy vector by PCR and cloned into the shuttle plasmid pASG2 vector to construct the co-expression shuttle plasmid pASG2-BMP9, 6 followed by homologous recombination with plasmid pAdeasy-1 in BJ5183. After confirmation by restriction endonuclease digestion, the recombinant vector was transfected into HEK293 cells, and high-titer recombinant adenovirus (Ad-BMP9, 6) was collected after amplification. Ad-BMP9, 6 was then transduced into C3H10 cells in vitro, and the mRNA expression of BMP9 and BMP6 was detected by RT-PCR. The osteogenic capability of the transfected cells was observed by alkaline phosphatase staining and calcium-alizarin red staining.
RESULTSAdBMP9,6 was constructed successfully and effectively infected in C3H10 cells, in which high expressions of BMP6 and BMP9 were detected. C3H10 cells infected with Ad-BMP9,6 showed stronger alkaline phosphatase and calcium-alizarin red staining than the cells transfected by either BMP9 or BMP6 alone.
CONCLUSIONThe recombinant adenovirus co-expressing BMP9 and BMP6 we constructed shows a more potent effect than the adenoviruses expressing either BMP9 or BMP6 alone in inducing the osteogenic differentiation of C3H10 cells into osteoblasts.