Poly-L-ornithine-mediated transfection of human keratinocytes.
Human keratinocytes are notoriously difficult to transfect. We have optimized a method for introducing plasmid DNA into keratinocytes that pairs the polycation poly-L-ornithine with a dimethylsulfoxide (DMSO) shock. The optimum poly-L-ornithine conditions for keratinocyte transfection entailed incubating the cells with 12 micrograms/ml poly-L-ornithine and 10 micrograms DNA for 6 h, followed by a 4-min 25% DMSO shock. Based on kinetic studies, 1 h is enough time to produce 10% positive cells in transient transfections, which increases up to an average of 20% after 6 h. Transfected cells survive passaging, and marker plasmids and selection can be used to yield stable transfectants at a rate twofold higher than in cells transfected with polybrene and DMSO. Transient transfection rates were significantly higher using poly-L-ornithine/DMSO than with the polybrene/DMSO or polybrene/glycerol methods previously reported. Overall, transfection mediated by poly-L-ornithine provides an efficient and inexpensive means of transiently or stably introducing DNA into keratinocytes.
Nead
M A
MA
Department of Microbiology and Immunology, University of Rochester, New York 14642, USA.
McCance
D J
DJ
D002470:Cell Survival; D002478:Cells, Cultured; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004357:Drug Synergism; D006583:Hexadimethrine Bromide / Q000494:pharmacology; D006801:Humans; D007231:Infant, Newborn; D015603:Keratinocytes / Q000166:cytology / Q000502:physiology; D007700:Kinetics; D008297:Male; D010455:Peptides / Q000494:pharmacology*; D014162:Transfection / Q000187:drug effects* / Q000379:methods
D010455:Peptides; C008973:polyornithine; D006583:Hexadimethrine Bromide; D004121:Dimethyl Sulfoxide