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

Rebecca R. Muldoon | Zendy; John Levy | Zendy; Steven R. Kain | Zendy; P. Kitts | Zendy; Charles J. Link | Zendy

Open Access

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

Author(s) -

Rebecca R. Muldoon,

John Levy,

Steven R. Kain,

P. Kitts,

Charles J. Link

Publication year - 1997

Publication title -

biotechniques

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 0.617

H-Index - 131

eISSN - 1940-9818

pISSN - 0736-6205

DOI - 10.2144/97221rr03

Subject(s) - green fluorescent protein , biology , gene , transfection , microbiology and biotechnology , genetic enhancement , gene expression , cell culture , plasmid , in vitro , chinese hamster ovary cell , viral vector , gene transfer , in vivo , recombinant dna , genetics

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.

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