Construction and expression detection of eukaryotic expression vector of humanized green fluorescent protein gene

Objective:To construct the eukaryotic expression vector pcDNA3GFP for determination of expression of green fluorescent protein(GFP).Methods:GFP cDNA was obtained from plasmid pCIGFP with single Not I digestion and was inserted into the same restriction site of pcDNA3GFP. The upright insertion was determined with BamH I digestion.Results:With transfection of pcDNA3GFP encapsulated by Lipofect AMINE PLUS TM into the cultured MDCK cells,G418 was used for selection of positive clones expressing green fluorescence. GFP positive clones of MDCK cells formed 20 days after transfection with fluorescent microscopy.Conclusion:It was demonstrated that GFP was successfully expressed in MDCK cells,implying that GFP was a good reporter and selection marker molecule in mammalian cells.