Long-term expression of foreign genes in normal human epidermal keratinocytes after transfection with lipid/DNA complexes

Normal human epidermal keratinocytes were isolated and cultivated in scrum- free medium. The expression of the integrin subunits alpha (6) and beta (1) indicated that a high number of keratinocytes from the stem cell system wa s present. These cells were transfected with complexes made of different ca tionic lipids and marker genes. Effectene showed a 20-fold higher transfect ion efficiency, compared to Lipofectin and Lipofectamine, and a similar low toxicity. The transfection protocol was optimised. A DNA/lipid ratio of 0. 133 showed the highest transfection efficiency. Keratinocytes expressed the marker gene luciferase for 20 days. The maximum expression occurred after 3-4 days, where individual patches of fluorescent keratinocytes were detect ed. Transfected keratinocytes, cultivated at the air-liquid interface, expr essed the marker gene beta -galactosidase for at least 7 weeks.