Polybrene/DMSO-assisted gene transfer. Generating stable transfectants with nanogram amounts of DNA.
Polybrene/DMSO-assisted gene transfer is a simple and versatile transfection strategy capable of producing high numbers of stable transfectants from adherent monolayer cultures with low (nanogram) quantities of exogenous DNA. The procedure involves two stages: adsorption and internalization. The former is mediated by polybrene (a polycation polymer) and favors the uniform coating of target cells with polybrene-DNA complexes. Following adsorption, the cells are permeabilized by a brief exposure to dimethyl sulfoxide (DMSO) to facilitate the uptake of DNA complexes. Diverse cell types can be exposed to a wide range of polybrene concentrations without adverse effects. By contrast, the key determinant of success is the DMSO permeabilization regime, which must be configured independently for each cell line. Protocols optimized for gene transfer in murine and human fibroblasts are presented along with a guide for the rapid optimization of the method. The advantages and limitations of the method are also discussed.
Health Canada, Life Sciences Division, Biotechnology, Sir F. G. Banting Research Centre, Ottawa, Ontario.
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Aubin
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M
Weinfeld
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Mirzayans
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Paterson
D016475:3T3 Cells; D000327:Adsorption; D000818:Animals; D001709:Biotechnology; D002478:Cells, Cultured; D004247:DNA / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D004121:Dimethyl Sulfoxide; D018014:Gene Transfer Techniques; D005821:Genetic Techniques; D006583:Hexadimethrine Bromide; D006801:Humans; D051379:Mice; D010539:Permeability; D014162:Transfection
D006583:Hexadimethrine Bromide; D004247:DNA; D004121:Dimethyl Sulfoxide