RESUMO
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.
