High-frequency transfection of cultured human epidermal basal cells that differentiate to form a multilayered tissue

Primary cultures of human keratinocytes form a multilayered tissue. By incubating the tissue cultures in Ca2(+)-free medium the differentiated cell layers can be stripped off leaving a basal cell monolayer. We have developed a method for high-frequency transfection of these epidermal basal cells with genes inserted into Epstein-Barr virus-based expression vectors. Using the Escherichia coli lac z gene as a marker gene, the transient and long-term expression and the fate of the transfected cells were studied. During regeneration of the multilayered tissue most of the transfected basal cells enlarge and undergo differentiation, but a minor population remains as basal cells. Incubation with the tumor promotor 12-O-tetradecanoylphorbol-13-acetate results in an increase in the proportion of transfected keratinocytes that are small, suggesting a relative expansion of the immature cell pool.