Transfection of mouse mesenchymal stem cells with recombinant adenovirus carrying mouse inducible costimulator gene

Objective Blockade of the inducible costimulator(ICOS) and its ligand signal pathway combined with mesenchymal stem cells(MSCs) may enhance the preventive ability of acute graft against host disease.This study was to construct a recombinant adenovirus vector carrying the mouse ICOS gene and transfect mouse MSCs so as to provide an experimental basis for transgene therapy.Methods The amplification products of ICOS by PCR with a pair of primers containing EcoRⅠ and SalⅠ restriction endonuclease sites were subcloned into the shuttle plasmid PDC318.The plasmid PDC318-mICOS was co-transfected with plasmid pPE3-F11B-EGFP containing the right arm of adenovirus 5 into 293 cells to produce a new recombinant adenovirus,followed by PCR identification.The adenovirus was packaged and propagated in 293 cells and purified by cesium chloride gradient centrifugation.The viral titer was determined by the method of 50% tissue culture infective dose(TCID50).Results The results of PCR assay indicated that the target gene was successfully inserted into the recombinant adenovirus vector.The viral titer was 6.54×109pfu/ml.Fluorescence microscopy showed many high-titer viruses in the transfected MSCs,and the expression of ICOS was 90.16%.Conclusion The recombinant adenovirus vector containing mouse ICOS was successfully constructed and high-titer viruses were obtained,and the vector could efficiently transfect mouse MSCs.