title: An efficient gene transduction system for studying gene function in primary human dermal fibroblasts and epidermal keratinocytes.
abstract: One of the critical challenges for cellular genetic studies in primary human skin cells is lack of a gene delivery system that provides efficient transduction and sustained expression of the transgenes. Due to the limited time of survival in culture, the processes of drug selection and clonal expansion for establishing gene stably expressing cell lines are not a realistic option for primary skin cells. We have examined various gene transduction techniques in primary dermal fibroblasts and epidermal keratinocytes of human skin. We report here that vectors based on the human immunodeficiency virus (HIV, lentivirus) offer more than 90% gene transduction efficiency and sustained expression of transgenes in both human skin cell types. In contrast, most of the commonly used techniques have at best 30% transduction efficiency in these cells. Using two previously reported migration control genes, protein kinase Cdelta and p38alpha-MAPK, as examples, we provide evidence that the unprecedented efficiency of the lentiviral system enables a clear detection of the genes' dominant negative effects, which are otherwise greatly compromised by ordinary transfection techniques. We believe that a wide application of this gene transduction system will greatly benefit studies of gene function in human skin cells.
authors.affiliation: Department of Medicine, Division of Dermatology and the USC/Norris Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, California 90033, USA. cherm@hsc.usc.edu
authors.forename: M
authors.initials: M
authors.lastname: Chen
authors.forename: W
authors.initials: W
authors.lastname: Li
authors.forename: J
authors.initials: J
authors.lastname: Fan
authors.forename: N
authors.initials: N
authors.lastname: Kasahara
authors.forename: D
authors.initials: D
authors.lastname: Woodley
mesh_terms: D005347:Fibroblasts / Q000502:physiology*; D006801:Humans; D015603:Keratinocytes / Q000502:physiology*; D016086:Lentivirus / Q000235:genetics; D011494:Protein Kinases / Q000235:genetics; D012879:Skin Physiological Phenomena / Q000235:genetics*; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D011494:Protein Kinases