Abstract: 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. Method 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 trasnfected by either BMP9
or BMP6 alone. Conclusion 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.