POLY-L-ORNITHINE-MEDIATED TRANSFECTION OF HUMAN KERATINOCYTES

Human keratinocytes are notoriously difficult to transfect, We have op timized a method for introducing plasmid DNA into keratinocytes that p airs the polycation poly-L-ornithine with a dimethylsulfoxide (DMSO) s hock, The optimum poly-l-ornithine conditions for keratinocyte transfe ction entailed incubating the cells with 12 mu g/ml poly-L-ornithine a nd 10 mu g DNA for 6 h, followed by a 4-min 25% DMSO shock. Based on k inetic studies, 1 h is enough time to produce 10% positive cells in tr ansient transfections, which increases up to an average of 20% after 6 h. Transfected cells survive passaging, and marker plasmids and selec tion can be used to yield stable transfectants at a fate twofold highe r than in cells transfected with polybrene and DMSO, Transient transfe ction rates were significantly higher using poly-L-ornithine/DMSO than with the polybrene/DMSO or polybrene/glycerol methods previously repo rted. Overall, transfection mediated by poly-L-ornithine provides an e fficient and inexpensive means of transiently or stably introducing DN A into keratinocytes.