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 serum-free medium. The expression of the integrin subunits alpha6 and beta1 indicated that a high number of keratinocytes from the stem cell system was present. These cells were transfected with complexes made of different cationic lipids and marker genes. Effectene showed a 20-fold higher transfection 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 detected. Transfected keratinocytes, cultivated at the air-liquid interface, expressed the marker gene beta-galactosidase for at least 7 weeks.
Zellmer
S
S
Institute of Biochemistry, Medical Faculty, University Leipzig, Germany. zellmers@medizin.uni-leipzig.de
Gaunitz
F
F
Salvetter
J
J
Surovoy
A
A
Reissig
D
D
Gebhardt
R
R
D002411:Cation Exchange Resins / Q000378:metabolism / Q000592:standards; D018929:Cell Culture Techniques / Q000379:methods*; D004247:DNA / Q000378:metabolism; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter / Q000235:genetics*; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism / Q000592:standards; D039121:Integrin alpha6beta1; D019012:Integrin beta1 / Q000235:genetics / Q000378:metabolism; D016023:Integrins / Q000235:genetics / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D050356:Lipid Metabolism; D008055:Lipids / Q000592:standards; D008081:Liposomes / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000592:standards; D010714:Phosphatidylethanolamines / Q000378:metabolism / Q000592:standards; D013234:Stem Cells / Q000166:cytology; D013997:Time Factors; D014162:Transfection / Q000379:methods / Q000592:standards*; D001616:beta-Galactosidase / Q000235:genetics / Q000592:standards
D002411:Cation Exchange Resins; D007202:Indicators and Reagents; D039121:Integrin alpha6beta1; D019012:Integrin beta1; D016023:Integrins; D008055:Lipids; C086724:Lipofectamine; D008081:Liposomes; D010714:Phosphatidylethanolamines; C020888:1,2-dielaidoylphosphatidylethanolamine; D004247:DNA; D008156:Luciferases; D001616:beta-Galactosidase