Construction of adenoviral vector encoding human VEGF_(121) cDNA and its expression in vitro

Objective To construct the adenoviral vector bringing hVEGF 121 cDNA for evaluation of the possibility of VEGF gene therapy in ischemic bone disease. Methods Human vascular endothelial growth factor (hVEGF 121 ) cDNA obtained from the plasmid pCDI/ VEGF 121 was cloned into plasmid pshuttle and further cloned to Adeno X Viral DNA. The recombinant adenoviral plasmid was identified and then transferred to the adenoviral packaging cell HEK293 by lipofectamine mediated gene transfer method to pack the virus. After titilating the virus, the mouse bone marrow stromal cells (MSC) were transfected by the adenovirus and the expression of VEGF gene was detected. Results The recombinant Adeno VEGF 121 was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequencing analysis. After MSCs were tranfected by the virus, RT PCR showed that hVEGF 121 mRNA was transcripted from the hVEGF 121 gene. Western blot and immune histochemistry showed VEGF 121 protein was expressed in transgene MSCs. Conclusion The recombinant adenoviral vector bringing hVEGF 121 cDNA was successfully constructed and the transgene MSC expressed hVEGF gene in vitro, it provided the further foundation of VEGF gene therapy for bone ischemic diseases.