RETROVIRAL TRANSFER AND EXPRESSION OF A HUMANIZED, RED-SHIFTED GREEN FLUORESCENT PROTEIN GENE INTO HUMAN TUMOR-CELLS

Over two-thirds of the current gene therapy protocols use retroviral g ene transfer systems. We have developed an efficient retroviral-based method that allows rapid identification of gene transfer in living mam malian cells. Cells were generated containing a gene for an improved ( humanized, red-shifted) version of the Aequorea victoria green fluores cent protein (hRGFP) from a retroviral vector. The hRGFP gene was used to produce an amphotropic vector producer cell line that demonstrated vibrant green fluorescence after excitation with blue light. A375 mel anoma cells transduced with the retroviral vector demonstrated stable green fluorescence. Both PA317 murine fibroblasts and A375 human cell lines containing the vector were easily detected by FACS analysis. The se vectors represent a substantial improvement over currently availabl e gene transfer marking systems. Bright, long-term expression of the h RGFP gene in living eukaryotic cells will advance the study of gene tr ansfer, gene expression, and gene product function in vitro and in viv o particularly for human gene therapy applications.