Construction of VEGF eukaryotic expression vector and identification of transfected mesenchymal stem cells

Objective To construct rat VEGF164 eukaryotic expression vector pcDNA3.1(-)/VEGF164,and to examine the exogenous protein expression in transfected bone marrow mesenchymal stem cells for gene therapy in lung injury.Methods VEGF164 eukaryotic expression vector was constructed and identified with recombinant DNA technology.Vector pcDNA3.1(-)/VEGF164,pcDNA3.1(-) and Lipofectamine 2000TM were transfected into rat marrow mesechymal stem cells of plasmid group,empty plasmid group and liposome group,respectively,with liposome-mediated technique.In vitro VEGF expression was assessed by immunohistochemistry and Western blot.Results The sequencing of pcDNA3.1(-)/VEGF164 was completely correct.Scattered yellow granules was only observed in pcDNA3.1(-)/VEGF164-transfected MSCs by immunohistchemistry.Furthermore,Western blot showed significantly higher level of VEGF in cytolytic product in plasmid group than those in the other two groups.Conclusion Eukaryotic expressing vector of pcDNA3.1(-)/VEGF164 has been successfully constructed and transfected into rat bone marrow mesenchymal stem cells with effective expression of target gene and protein,providing a pontential gene therapy for lung injury.