title: Demethylation using the epigenetic modifier, 5-azacytidine, increases the efficiency of transient transfection of macrophages.
abstract: 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-1alpha 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.
authors.affiliation: Baker Heart Research Institute, Melbourne, Victoria, 8008, Australia.
authors.forename: Geneviève
authors.initials: G
authors.lastname: Escher
authors.forename: Anh
authors.initials: A
authors.lastname: Hoang
authors.forename: Suzan
authors.initials: S
authors.lastname: Georges
authors.forename: Urbain
authors.initials: U
authors.lastname: Tchoua
authors.forename: Assam
authors.initials: A
authors.lastname: El-Osta
authors.forename: Zygmunt
authors.initials: Z
authors.lastname: Krozowski
authors.forename: Dmitri
authors.initials: D
authors.lastname: Sviridov
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001374:Azacitidine / Q000494:pharmacology*; D002460:Cell Line; D053493:Cholestanetriol 26-Monooxygenase; D002784:Cholesterol / Q000378:metabolism; D003587:Cytomegalovirus / Q000235:genetics; D003637:DEAE-Dextran / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D019175:DNA Methylation; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008055:Lipids / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D008745:Methylation; D051379:Mice; D018613:Microscopy, Confocal; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013250:Steroid Hydroxylases / Q000235:genetics; D014162:Transfection / Q000379:methods*
chemical_list: C498175:Effectene; D004791:Enzyme Inhibitors; C411955:FuGene; D008055:Lipids; C086724:Lipofectamine; D049452:Green Fluorescent Proteins; D004247:DNA; D003637:DEAE-Dextran; D002784:Cholesterol; D013250:Steroid Hydroxylases; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase; C506832:Cyp27a1 protein, mouse; D001374:Azacitidine