J. Lipid Res. Neurobiology of Lipids (ISSN1683-5506)
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Originally published In Press as doi:10.1194/jlr.D400014-JLR200 on November 1, 2004

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Journal of Lipid Research, Vol. 46, 356-365, February 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Methods

Demethylation using the epigenetic modifier, 5-azacytidine, increases the efficiency of transient transfection of macrophages

Geneviève Escher, Anh Hoang, Suzan Georges, Urbain Tchoua, Assam El-Osta, Zygmunt Krozowski and Dmitri Sviridov1

Baker Heart Research Institute, Melbourne, Victoria, 8008, Australia

1 To whom correspondence should be addressed. e-mail: 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 RAW 264.7 cells. The highest transfection efficiency was achieved with DEAE-dextran, although the proportion of cells expressing the reporter gene did not exceed 20%. It was subsequently found that the cytomegalovirus 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-1{alpha} promoters and transient transfection of human HepG2 cells. 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 nontransfected cells or expression of ABCA1 and CYP27A1. However, transfection with CYP27A1 led to a 2- to 4-fold increase of cholesterol efflux.

We conclude that treatment with 5-azacytidine can be used for high-efficiency transient transfection of macrophages.

Abbreviations: apoA-I, apolipoprotein A-I; CMV, cytomegalovirus; CYP27A1, sterol 27-hydroxylase; GFP, green fluorescent protein; LXR, liver X receptor

Supplementary key words cholesterol efflux • CYP27A1 • atherosclerosis • lipoproteins


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