title: Tracking and quantitation of retroviral-mediated transfer using a completely humanized, red-shifted green fluorescent protein gene.
abstract: 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.
authors.affiliation: Gene Therapy Program, HGTRI, Des Moines, IA 50309, USA.
authors.forename: R R
authors.initials: RR
authors.lastname: Muldoon
authors.forename: J P
authors.initials: JP
authors.lastname: Levy
authors.forename: S R
authors.initials: SR
authors.lastname: Kain
authors.forename: P A
authors.initials: PA
authors.lastname: Kitts
authors.forename: C J
authors.initials: CJ
authors.lastname: Link
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D005434:Flow Cytometry; D015870:Gene Expression / Q000235:genetics*; D018014:Gene Transfer Techniques; D005819:Genetic Markers; D005822:Genetic Vectors / Q000302:isolation & purification*; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics*; D051379:Mice; D012190:Retroviridae / Q000235:genetics*; D037401:Scyphozoa / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D005819:Genetic Markers; D008164:Luminescent Proteins