Optimal parameters for the polybrene-induced DNA transfection of diploid human fibroblasts.
Recently it has been shown that Polybrene, in conjunction with dimethyl sulfoxide (DMSO) shock, can markedly increase frequency of DNA transfection of chicken embryo fibroblasts as compared with the frequency obtained with the standard calcium phosphate protocol. We have adapted this procedure for use with diploid human fibroblasts. Using plasmid DNA containing a dominant selectable marker gene (resistance to Geneticin), we have determined that treatment of the cells for 6 h in culture medium containing Polybrene at a concentration of 2 to 5 micrograms/ml, followed by a 4-min shock with 30% DMSO, resulted in the highest yield of transfectants, ca. 400/10(6) cells treated with as little as 100 ng of plasmid DNA. The selective agent could be added immediately after the DMSO shock. This allows transfection and selection to be carried out in the same dishes and ensures that each clone represents a unique event.
D002478:Cells, Cultured; D004269:DNA, Bacterial / Q000008:administration & dosage*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004351:Drug Resistance; D005347:Fibroblasts / Q000187:drug effects; D005839:Gentamicins / Q000494:pharmacology; D006583:Hexadimethrine Bromide / Q000494:pharmacology*; D006801:Humans; D008297:Male; D010413:Penis; D010957:Plasmids; D011073:Polyamines / Q000494:pharmacology*; D012641:Selection, Genetic; D014170:Transformation, Genetic
D004269:DNA, Bacterial; D005839:Gentamicins; D011073:Polyamines; D006583:Hexadimethrine Bromide; C010680:antibiotic G 418; D004121:Dimethyl Sulfoxide