Abstract
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.
MeSH terms
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Cation Exchange Resins / metabolism
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Cation Exchange Resins / standards
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Cell Culture Techniques / methods*
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DNA / metabolism
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Gene Expression
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Gene Transfer Techniques*
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Genes, Reporter / genetics*
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Humans
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Indicators and Reagents / metabolism
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Indicators and Reagents / standards
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Integrin alpha6beta1
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Integrin beta1 / genetics
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Integrin beta1 / metabolism
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Integrins / genetics
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Integrins / metabolism
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Keratinocytes / cytology
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Keratinocytes / metabolism*
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Lipid Metabolism
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Lipids / standards
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Liposomes / metabolism
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Luciferases / genetics
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Luciferases / standards
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Phosphatidylethanolamines / metabolism
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Phosphatidylethanolamines / standards
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Stem Cells / cytology
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Time Factors
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Transfection / methods
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Transfection / standards*
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beta-Galactosidase / genetics
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beta-Galactosidase / standards
Substances
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Cation Exchange Resins
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Indicators and Reagents
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Integrin alpha6beta1
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Integrin beta1
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Integrins
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Lipids
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Lipofectamine
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Liposomes
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Phosphatidylethanolamines
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1,2-dielaidoylphosphatidylethanolamine
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DNA
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Luciferases
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beta-Galactosidase