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 mu g/ml poly-L-ornithine and 10 mu g 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 fate 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.