Construction Of Recombinant Osterix Adenovirus And The Primary Study About It's Effects On Osteoblast Cell Cycles

Objetive: To optimize the construction recombinant adenovirus of Osterix gene, and study the effects of the pAdEasy-Osx on the cell cycle of C2C12 cells.Methods:Osterix gene was amplified by RT-PCR and cloned into the transfer vector pAdTrack-CMV to construct a transfer plasmid pAdTrack-CMV-Osx. The plasmid was linearized with Pme I and cotransformed into E.coli BJ5183 cells with adenovirus genomic plasmid pAdEasy-1 by electroporation to achieve homologous recombination. After being digested with Pac I,the DNA of identified recombinant plasmid was transfected into QBI-293A cells with lipofectin to package and amplify adenovirus.After harvesting the virus particles, calculate the titer of the virus suspension. C2C12 cells were infected with the recombinant adenoviruses.The transcription and expression of the target gene was detected by RT-PCR and Western blotting, and its effect on cell cycle of C2C12 cells was examined by FCM(flow cytometer).Results:The pAdEasy-Osx was successfully constructed. After harvesting the virus particles, the titer of pAdEasy-Osx was about 2.25 ×10~8 v.p/ml. The transcription and expression of Osterix gene in C2C12 cells was proved by RT-PCR and Western blotting. The infection of pAdEasy-Osx resulted in significantly higher proliferative rate of C2C12 cells compared with uninfected ones, with cell number of G1 and G2 phase decreasing and that of S phase increasing.Conclusion: Recombinant Osterix adenovirus have been constructed successfully. Osterix gene can increase the growth of C2C12 cells.