title: Retrovirus-mediated gene transfer to cystic fibrosis airway epithelial cells: effect of selectable marker sequences on long-term expression.
abstract: Retrovirus-mediated gene transfer offers the potential for stable long-term expression of transduced genes in host cells subsequent to integration of vector DNA into the host genome. Using a murine amphotropic retrovirus vector containing an interleukin-2 receptor (IL-2R) gene as a reporter and a neomycin phosphotransferase (neor) gene as a dominant selectable marker, we measured the efficiency of retrovirus-mediated gene transfer and the stability of transduced gene expression in a cystic fibrosis tracheal epithelial cell line (CFT1). The use of the IL-2R cell surface marker as a reporter of infection permitted both quantitation of vector gene expression and flow cytometric sorting of cells transduced with the vector. In initial studies, the optimal conditions for retrovirus-mediated gene transfer were determined. The presence of a polycation was required for optimal transduction efficiency. The efficiency of infection of CFT1 cells was increased by repetitive exposure to virus such that it was possible to transduce approximately 80% of the cells following three successive daily exposures. The long-term stability of expression of the non-selected IL-2R gene was also evaluated. A slow decline in the percentage of cells expressing IL-2R was seen with cells that were maintained under constant selection pressure for expression of the neor gene, which was expressed from an internal promoter. Similar results were obtained when cultures were selected initially for neor gene expression and maintained without selection thereafter. In contrast, stable expression was observed in CFT1 cells for at least one year following multiple infections in the absence of G418 selection. In conclusion, (i) transduction of foreign genes into human airway epithelial cells using an amphotropic retrovirus vector can be highly efficient in the presence of appropriate polycations and multiple exposures; and (ii) stable expression of a non-selected gene in these epithelial cells is better maintained without selection.
authors.affiliation: UNC Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill 27599.
authors.forename: J C
authors.initials: JC
authors.lastname: Olsen
authors.forename: L G
authors.initials: LG
authors.lastname: Johnson
authors.forename: M L
authors.initials: ML
authors.lastname: Wong-Sun
authors.forename: K L
authors.initials: KL
authors.lastname: Moore
authors.forename: R
authors.initials: R
authors.lastname: Swanstrom
authors.forename: R C
authors.initials: RC
authors.lastname: Boucher
mesh_terms: D002460:Cell Line; D003550:Cystic Fibrosis / Q000235:genetics*; D004847:Epithelial Cells; D005786:Gene Expression Regulation; D005819:Genetic Markers / Q000235:genetics*; D006801:Humans; D019868:Kanamycin Kinase; D008979:Moloney murine leukemia virus / Q000235:genetics*; D010770:Phosphotransferases / Q000235:genetics; D011533:Proviruses / Q000235:genetics; D012313:RNA / Q000032:analysis; D015375:Receptors, Interleukin-2 / Q000235:genetics; D013997:Time Factors; D014132:Trachea / Q000166:cytology / Q000378:metabolism*; D014162:Transfection
chemical_list: D005819:Genetic Markers; D015375:Receptors, Interleukin-2; D012313:RNA; D010770:Phosphotransferases; D019868:Kanamycin Kinase