Construction of human osteoinductive factor gene lentivirus vector and stable over-expression in human aortic smooth muscle cells

Objective: To construct human osteoinductive factor(OIF) recombinant lentivirus vector and over-express OIF stably in human aortic smooth muscle cells(HASMCs) . Methods: OIF-3FLAG was amplified by PCR and cloned into vector(pMSCV PIG) . The lentivirus vector pMSCV PIG-OIF-3FLAG was constructed and indentified by sequencing. pMSCV PIG-OIF-3FLAG or pMSCV PIG with helper vectors of VSVG and GAG-POL were co-transfected into 293T cells. Infectious lentivirus was harvested at 48 h post-transfection. The lentivirus was added in HASMCs. After 48 h, puromycin was added for 4 d. When the cells reached 90%, HASMCs over-expressed pMSCV PIG-OIF-3FLAG stably. Human OIF expression was verified by quantitative real-time PCR and western blot analysis. Results: The lentivirus vector pMSCV PIG-OIF-3FLAG was successfully constructed and confirmed by enzyme digestion and sequencing. HASMCs were infected by lentivirus supernatant. Over-expression of OIF in HASMCs was revealed by real-time PCR and western blot. Conclusions: Human OIF gene recombined lentivirus vector and a cell model which over-expessing OIF stably in HASMCs were successfully established.