Journal of Lipid Research
Volume 46, Issue 2, February 2005, Pages 356-365
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Methods
Demethylation using the epigenetic modifier, 5-azacytidine, increases the efficiency of transient transfection of macrophages

https://doi.org/10.1194/jlr.D400014-JLR200Get rights and content
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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-1α 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.

cholesterol efflux
CYP27A1
atherosclerosis
lipoproteins

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Abbreviations

  1. apoA-I, apolipoprotein A-I

  2. CMV, cytomegalovirus

  3. CYP27A1, sterol 27-hydroxylase

  4. GFP, green fluorescent protein

  5. LXR, liver X receptor

Published, JLR Papers in Press, November 1, 2004. DOI 10.1194/jlr.D400014-JLR200