Applications of green fluorescent protein as a marker for studying gene transfer in human tumor cells

Objective To develop a technique for tracing tumor cells with enhanced green fluorescent protein (EGFP).Methods The retroviral vector LGSN,encoding EGFP,was constructed and the amphotropic virus was used to infect human tumor cell lines,including leukemia K562,mammary carcinoma MCF7,and bladder cancer 5637 cells.Forty-eight hours after transduction,stable EGFP-expressing tumor lines were selected in vitro with G418.The integration and expression of EGFP gene was examined using polymerase chain reaction,flow cytometry (FCM) and fluorescence microscopy.Results After transduction and selection,three resistant cell lines were established in which both EGFP gene and neomycin resistance gene NeoR was confirmed by DNA amplification.Compared to the uninfected cells,FCM analysis revealed EGFP expression in up to 90% (range 85 5%～90 0%)of cells containing LGSN vector.Similarly,EGFP signal was easily detected under fluorescence microscopy in all genetically tumor cells.Conclusion The retroviral vector carrying EGFP gene could mediate stable expression in tumor cells,indicating its roles in studying tumor growth,metastasis and angiogenesis.