Formation of stable cationic lipid/DNA complexes for gene transfer.
Stable cationic lipid/DNA complexes were formed by solubilizing cationic liposomes with 1% octylglucoside and complexing a DNA plasmid with the lipid in the presence of detergent. Removal of the detergent by dialysis yielded a lipid/DNA suspension that was able to transfect tissue culture cells up to 90 days after formation with no loss in activity. Similar levels of gene transfer were obtained by mixing the cationic lipid in a liposome form with DNA just prior to cell addition. However, expression was completely lost 24 hr after mixing. The transfection efficiency of the stable complex in 15% fetal calf serum was 30% of that obtained in the absence of serum, whereas the transient complex was completely inactivated with 2% fetal calf serum. A 90-day stability study comparing various storage conditions showed that the stable complex could be stored frozen or as a suspension at 4 degrees C with no loss in transfection efficiency. Centrifugation of the stable complex produced a pellet that contained approximately 90% of the DNA and 10% of the lipid. Transfection of cells with the resuspended pellet and the supernatant showed that the majority of the transfection activity was in the pellet and all the toxicity was in the supernatant. Formation of a stable cationic lipid/DNA complex has produced a transfection vehicle that can be stored indefinitely, can be concentrated with no loss in transfection efficiency, and the toxicity levels can be greatly reduced when the active complex is isolated from the uncomplexed lipid.
Hofland
H E
HE
Synthetic Vector Development, Somatix Therapy Corporation, Alameda, CA 94501, USA.
Shephard
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L
Sullivan
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SM
D016475:3T3 Cells; D000818:Animals; D001769:Blood; D002411:Cation Exchange Resins; D002417:Cattle; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics; D007202:Indicators and Reagents; D008055:Lipids; D008081:Liposomes; D051379:Mice; D010714:Phosphatidylethanolamines; D010957:Plasmids / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013096:Spermine / Q000031:analogs & derivatives; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000096:biosynthesis*
C098223:2,3-dioleyloxy-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethyl-1-propanaminium trifluoroacetate; D002411:Cation Exchange Resins; D003470:Culture Media; D007202:Indicators and Reagents; D008055:Lipids; C086724:Lipofectamine; D008081:Liposomes; D010714:Phosphatidylethanolamines; D011994:Recombinant Proteins; D013096:Spermine; C020888:1,2-dielaidoylphosphatidylethanolamine; D001616:beta-Galactosidase