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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.

Tracking and Quantitation of Retroviral-Mediated Transfer Using a Completely Humanized, Red-Shifted Green Fluorescent Protein Gene