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Papers In Press, published online ahead of print November 1, 2004
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Vascular Biology, Baker Heart Research Institute, Melbourne, Vic 8008
Corresponding Author: Dmitri.Sviridov{at}Baker.edu.au
This study was aimed at developing a method for high-efficiency transient transfection of macrophages. Seven methods were evaluated for transient transfection of murine macrophage cells RAW 264.7. The highest transfection efficiency was achieved with DEAE-Dextran, however the proportion of cells expressing the reporter gene did not exceed 20%. It was subsequently found that the CMV plasmid promoter in these cells becomes methylated. When cells were treated with the methylation inhibitor 5-azacytidine, methylation of the plasmid promoter was abolished and a dose-dependent stimulation of reporter gene expression was observed with expression achieved in more than 80% of cells. Treatment of cells with 5-azacytidine also caused increased efficiency of transfection of macrophages with plasmids driven by RSV, SV40 and EF-1alpha promoters and transient transfection of human hepatoma cells HepG2. Inhibition of methylation also increased the amount and activity of sterol 27-hydroxylase (CYP27A1) detected in RAW 264.7 cells transfected with a CYP27A1 expression plasmid. Treatment of cells with 5-azacytidine alone did not affect either cholesterol efflux from non-transfected cells or expression of ABCA1 and CYP27A1. However, transfection with CYP27A1 lead to a 2-4-fold increase of cholesterol efflux. We conclude that treatment with 5-azacytidine can be used for high-efficiency transient transfection of macrophages.
Revised on October 14, 2004
Accepted on October 28, 2004
Demethylation using the epigenetic modifier, 5-azacytidine, increases the efficiency of transient transfection of macrophages
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