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.

In contrast to infrequent and low-titer cross-neutralization of human immunodeficiency virus type 1 (HIV-1) isolates by HIV-2-and simian immunodeficiency virus (SIV)-positive sera, extensive cross-neutralization of * Corresponding author.

We have developed murine retroviral vectors (RVs) containing an optimized green fluorescent protein (GFP) gene to study retroviral gene transfer and expression in living cells. We used the codon "humanized", "red-shifted" GFP gene, hGFP-S65T, a gain of function variant of the wild-type GFP from the jellyfish Aequorea victoria. We cloned the hGFP-S65T gene into the RV plasmid pLNCX (pLNChG65T). A stable amphotropic RV-producer cell line (VPC), designated LNChG65T VPC, was generated that exhibited bright fluorescence in greater than 95% of the cells. Human A375 melanoma cells and IGROV ovarian carcinoma cells transduced from LNCh-G65T VPC demonstrated high levels of fluorescence. The expression of a single integrated hGFP-S65T gene in eukaryotic cells provides a powerful tool to study gene transfer, expression and functional studies in vitro and in vivo.