Inclusion of cholesterol in DOTAP transfection complexes increases the delivery of DNA to cells in vitro in the presence of serum.
The contrast between the relative efficiency of transfection by cationic lipid reagents in vitro and that in vivo is well recognised. One suggested reason for this is the presence of competing polyanionic surfaces in blood and other biological fluids, and even in vitro transfections have to be performed in low-serum medium. In this study we have shown that by preparing cationic lipid reagents based on DOTAP with cholesterol as a second constituent of the bilayer we can achieve significant levels of in vitro transfection in serum concentrations of up to 80% (DOTAP alone did not transfect at all in these conditions). In an effort to explain the behaviour of DOTAP/cholesterol mixes under these conditions, we examined the effect of serum on the transfection complex. We could detect protein bound to each type of cationic lipid complex, but there was no difference in the amount nor in the type of protein bound. DNA within either type of complex which were incubated with increasing amounts of serum remained resistant to digestion with DNase I, and there was no reduction in the condensation of the DNA as measured by ethidium bromide fluorescence. Finally, we measured the attachment and uptake into cells by the different complexes in the presence of serum and showed that more DOTAP-cholesterol than DOTAP complexes attach to and are taken up by cells in the presence of serum. We suggest that improved cell binding and uptake may be the main mechanism by which cholesterol acts to maintain transfection in the presence of serum.
Crook
K
K
MRC Human Genetics Unit, Edinburgh, UK.
Stevenson
B J
BJ
Dubouchet
M
M
Porteous
D J
DJ
D000818:Animals; D001769:Blood; D002784:Cholesterol; D005229:Fatty Acids, Monounsaturated; D015316:Genetic Therapy; D008081:Liposomes; D000644:Quaternary Ammonium Compounds; D014162:Transfection / Q000379:methods*
D005229:Fatty Acids, Monounsaturated; D008081:Liposomes; D000644:Quaternary Ammonium Compounds; D002784:Cholesterol; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane