[Construction of a recombinant adenovirus co-expressing bone morphogenic proteins 9 and 6 and its effect on osteogenesis in C3H10 cells].
OBJECTIVE
To construct a recombinant adenovirus co-expressing bone morphogenic protein (BMP) 9 and BMP6 and observe its effect on the osteogenesis in C3H10 cells.


METHODS
The 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.


RESULTS
AdBMP9,6 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.


CONCLUSIONS
The 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.
Ministry of Education Key Laboratory of Child Development and Disorders/Key Laboratory of Pediatrics in Chongqing/Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Children's Hospital, Chongqing Medical University, Chongqing 400014, China.E-mail: dxhwork@foxmail.com.
Xiaohong
X
Die
Qing
Q
Luo
Cong
C
Chen
Guangjin
G
Luo
Quan
Q
Kang
D000256:Adenoviridae / Q000235:genetics*; D055418:Bone Morphogenetic Protein 6 / Q000235:genetics*; D005822:Genetic Vectors; D055427:Growth Differentiation Factor 2; D055412:Growth Differentiation Factors / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D010006:Osteoblasts / Q000166:cytology; D010012:Osteogenesis; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection
C525816:BMP6 protein, human; D055418:Bone Morphogenetic Protein 6; C525887:GDF2 protein, human; D055427:Growth Differentiation Factor 2; D055412:Growth Differentiation Factors; D011993:Recombinant Fusion Proteins