title: DNA-mediated gene transfection into primary cultures of adult mouse keratinocytes.
abstract: An efficient and reproducible technique for the transfection of primary cultures of adult mouse keratinocytes has been developed. The procedure involves culturing the primary adult mouse epidermal cells at 32 degrees C in an enriched media until they reach 70 to 95% confluency, followed by transfection with exogenous DNA in a low potassium environment. Using chloramphenicol acetyl transferase (CAT) transient gene expression assays and various strong viral promoter/CAT constructs, the transfection procedure was optimized for media formulation, plasmid DNA concentration, carrier DNA concentration, incubation temperature, incubation period, and cell density. Optimized parameters include the use of 6 micrograms plasmid DNA and 10 micrograms pUC19 carrier DNA per 60-mm tissue culture dish. Since primary keratinocytes undergo a well-characterized pattern of differentiation in vitro in response to extracellular calcium concentrations, this transfection procedure should provide a useful model in which to study both tissue- and differentiation-specific gene expression.
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, Omaha, Nebraska 68198.
authors.forename: N A
authors.initials: NA
authors.lastname: Betz
authors.forename: K J
authors.initials: KJ
authors.lastname: Wolterman
authors.forename: J J
authors.initials: JJ
authors.lastname: Reiners
authors.forename: J C
authors.initials: JC
authors.lastname: Pelling
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003470:Culture Media; D005260:Female; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013696:Temperature; D014162:Transfection
chemical_list: D003470:Culture Media; D015500:Chloramphenicol O-Acetyltransferase