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 gene transfer systems. We have developed an efficient retroviral-based method that allows rapid identification of gene transfer in living mammalian cells. Cells were generated containing a gene for an improved (humanized, red-shifted) version of the Aequorea victoria green fluorescent 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 melanoma 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. These vectors represent a substantial improvement over currently available gene transfer marking systems. Bright, long-term expression of the hRGFP gene in living eukaryotic cells will advance the study of gene transfer, gene expression, and gene product function in vitro and in vivo particularly for human gene therapy applications.
Levy
J P
JP
Laboratory of Molecular Oncology, Central Iowa Health Systems, Des Moines 50309, USA.
Muldoon
R R
RR
Zolotukhin
S
S
Link
C J
CJ
D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D002469:Cell Separation; D017931:DNA Primers / Q000235:genetics; D005434:Flow Cytometry; D005453:Fluorescence; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics*; D051379:Mice; D012190:Retroviridae / Q000235:genetics; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins