title: SV40 Immortalization of feline fibroblasts as targets for MHC-restricted cytotoxic T-cell assays.
abstract: CTL assays in outbred cats have been difficult to perform because of a lack of a good source of syngeneic target cell. Primary fibroblasts from cats are widely used as target cells for MHC-restricted cytotoxic T-cell (CTL) assays, but their limited life-spans of 8-10 culture passages can be problematic for longitudinal studies. To circumvent the life-span limitations of primary fibroblast cultures, we developed a procedure for immortalizing feline primary fibroblast cells by transfection with a molecular clone of simian virus 40 (SV40). Fibroblast cultures from skin biopsies of 28 cats were immortalized using this procedure and have been passaged for longer than 6 months without showing any phenotypic difference from the original primary cells. Non-SV40 transfected feline fibroblasts from a selection of animals in the same group survived for only 6-8 weeks before reaching senescence. The immortalized fibroblasts expressed SV40 T-antigen and Class I MHC protein, and were successfully used as target cells in 51Cr release CTL assays in feline immunodeficiency virus (FIV)-infected cats and in vitro stimulated allogeneic T-cell cultures.
authors.affiliation: Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, 1925 Coffey Rd., Columbus, OH 43210,USA.
authors.forename: S
authors.initials: S
authors.lastname: Köksoy
authors.forename: A J
authors.initials: AJ
authors.lastname: Phipps
authors.forename: K A
authors.initials: KA
authors.lastname: Hayes
authors.forename: L E
authors.initials: LE
authors.lastname: Mathes
mesh_terms: D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D002415:Cats / Q000276:immunology*; D002472:Cell Transformation, Viral / Q000235:genetics / Q000276:immunology*; D016922:Cellular Senescence / Q000502:physiology; D002860:Chromium Radioisotopes; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods / Q000662:veterinary*; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D005347:Fibroblasts / Q000166:cytology* / Q000276:immunology; D005455:Fluorescent Antibody Technique / Q000662:veterinary; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis / Q000235:genetics; D000949:Histocompatibility Antigens Class II / Q000096:biosynthesis / Q000235:genetics; D016089:Immunodeficiency Virus, Feline / Q000302:isolation & purification; D007519:Isoantigens / Q000276:immunology; D013539:Simian virus 40 / Q000276:immunology*; D013047:Specific Pathogen-Free Organisms; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014162:Transfection / Q000662:veterinary
chemical_list: D000952:Antigens, Polyomavirus Transforming; D002860:Chromium Radioisotopes; D018984:Epitopes, T-Lymphocyte; D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; D007519:Isoantigens