Adenovirus-mediated Expression of Human Endothelial Nitric Oxide Synthase and Enhanced Green Fluorescent Protein in Rat Mesenchymal Stem Cells

Objective To investigate the expression of human endothelial nitric oxide synthase(heNOS) and enhanced green fluorescent protein(EGFP) in rat mesenchymal stem cells cultured in vitro.Methods Mesenchymal stem cells(MSCs) were isolated from bone marrow of new-born SD rats and expanded in vitro.MSCs were identified with their abilities to differentiate into adipogenic and osteogenic lineages and their immunophenotype of CD44,CD45 and CD90.The vector pShuttle-heNOS-EGFP was linearized with Pme I and transformed into ultracompetent BJ 5183 bacteria containing pAdEasy-1.The positive clone of homologous recombination(pAdEasy-heNOS-EGFP) was identified by PCR,endonuclease digestion,and DNA sequencing.The positive recombinant adenoviral plasmid was digested with Pac I and transfected AD 293 cells with LipofectamineTM 2000 to package recombinant adenovirus particles.Thereafter,the recombinant adenovirus infected MSCs.The expression of heNOS and EGFP in MSCs were detected by immunofluorescent staining and fluorescent microscopy,respectively.The non-infected MSCs served as control.Results Homologous recombination occurred between pShuttle-heNOS-EGFP and pAdEasy-1 within BJ 5183 to generate pAdEasy-heNOS-EGFP.The recombinant adenovirus Ad-heNOS-EGFP was confirmed to be successfully packaged within AD 293 cells with PCR,and efficiently infected MSCs.The green and red fluorescence were seen in MSCs infected with Ad-heNOS-EGFP under fluorescent microscope,which demonstrated the expression of EGFP and heNOS in MSCs.Conclusion Exogenous heNOS cDNA could be efficiently transduced into MSCs mediated by adenoviral vector and overexpressed within MSCs,which would provide a new strategy to cell-based therapy of erectile dysfunction.Moreover,EGFP expression is a useful tool for directly monitoring the infection of target cells and the expression of gene of interest.