Function of unique osteoblastic gene verified by human osteocalcin promoter in vivo

Aim: To construct specific shRNA expression vector driven by human osteocalcin promoter. Methods: The experiment was conducted in the Institute of Orthopaedics of Chinese PLA and Center for Plastic Surgery of Chinese PLA, Xijing Hospital, Fourth Military Medical University of Chinese PLA from November 2003 to September 2004. The human osteocalcin promoter was amplified by PCR using genomic DNA of HEK293 cells as templates. Then, the osteocalcin promoter, shRNA-coding sequence designed to knockdown luciferase gene and the polyadenylation signal were cloned into, the corresponding enzyme sites of pGL3-control vector in turns. After confirmed by double restriction enzyme digestion and DNA sequencing, the resulting plasmid pGL3-OCP-shLuc was used to co-transfect HEK293 and MG-63 cells with pGL3-control. Luciferase activities were assayed 48 hours after co-transfection, and the relative activities were calculated. Results: An 830 bp human osteocalcin promoter was amplified using specific primers. DNA sequencing results verified that the expression cassette of shRNA in pGL3-OCP-shLuc was consistent with that having been designed. After co-transfection of HEK293 and MG-63 with pGL3-OCP-shLuc and pGL3-control, the relative luciferase activities of HEK293 had no difference from those of the controls, but the relative luciferase activities of MG-63 were significantly decreased and the inhibition rate was about 69.8%. Conclusion: An shRNA expression vector driven by human osteocalcin promoter is successfully constructed, and the specific RNAi of bone tissue is verified, providing an basis for the introduction of RNAi into the field of gene therapy for osteosarcoma.