Highly efficient transfection of mammalian cells by electric field pulses. Application to large volumes of cell culture by using a flow system.
We have transfected mammalian cells with plasmid DNA by application of electric pulses. Chinese hamster ovary cells were chosen as a model in order to study and to optimize the transfection protocol. A plasmid carrying the gene coding for beta-galactosidase activity was used to determine transient expression of the electrotransferred activity at the cell level. Optimum transient expression for cells in suspension was obtained by application of 10 square wave pulses of 5-ms duration and 0.6-kV/cm intensity. Under the best conditions, transfection frequencies as high as 50-60% could be obtained and appeared to be highly dependent on the age of the cell culture. The method was applicable to plated cells growing in a petri dish or on microcarriers. The possibility of extension of the technique to large volumes of cells is presented. A flow system, composed of a peristaltic pump connected to the electropulser chamber, allowed large volumes of cells to be treated with flow rates in the order of several milliliters/minute. Transfection frequencies for the large volumes were 25% for cells in suspension and 35% for cells on microcarriers. These results open new perspectives in large-scale transfection technology of cells however they are grown.
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Centre de Recherches de Biochimie et de Génétique Cellulaires du CNRS, Toulouse, France.
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D000818:Animals; D016466:CHO Cells; D002463:Cell Membrane Permeability; D002470:Cell Survival; D002478:Cells, Cultured; D006224:Cricetinae; D004558:Electric Stimulation / Q000379:methods; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase