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

Abstract

Macrophages play an important role in host defense pathways and are also involved in a variety of diseases, including atherosclerosis (1, 2). The key role of macrophages in the development of atherosclerosis has made this cell type a versatile in vitro model of this disease (3).Transfection of macrophages is a powerful tool to study their function, and a number of methods have been described to achieve high levels of expression of different genes through transient transfection (4-6). These levels of expression are sufficiently high to study synthetic processes, when proteins are tagged or otherwise distinguished from host proteins. However, studying cell functions such as growth, lipoprotein binding, lipid uptake, and efflux requires not only high levels of gene expression but also for the gene to be expressed in a majority of cells, a highefficiency transfection. High efficiency of transfection is also critical for a multiple gene transfection, as it requires that all transfected genes be expressed in the same population of cells. Viral and stable transfections offer adequate efficiency of DNA transfer; however, they are often laborintensive and time-consuming. High-efficiency transient transfection of macrophages has proved to be difficult.Here, we describe a method for high-efficiency transient transfection of RAW 264.7 mouse macrophages. We fortuitously found that the low efficiency of expression of transfected genes in macrophages is a consequence of methylation-mediated silencing of transfected genes rather than of low uptake of DNA into cells. To maximize the efficiency of macrophage transfection, we evaluated the DNA methylation inhibitor, 5-azacytidine, an epigenetic modifier often used to reactivate methylation-dependent transcriptionally silent genes (7). We demonstrated by methylationspecific PCR that 5-azacytidine prevents methylation of the promoter of transfected genes, and for the first time we achieved transient expression of a reporter protein in 80-100% of macrophage cells. The method was then used for the high-efficiency transient transfection of RAW 264.7 macrophages with sterol 27-hydroxylase (CYP27A1), which led to the stimulation of cholesterol efflux from these cells.Abbreviations: apoA-I, apolipoprotein A-I; CMV, cytomegalovirus; CYP27A1, sterol 27-hydroxylase; GFP, green fluorescent protein; LXR, liver X receptor.

Advanced glycation of apolipoprotein A-I impairs its anti-atherogenic properties

“…Cells were treated for 18 h with 1 μmol/l 5′-azacytidine to prevent methylation of the cytomegalovirus (CMV) promoter [17].…”

Functional Analysis of a Potassium-Chloride Co-Transporter 3 (SLC12A6) Promoter Polymorphism Leading to an Additional DNA Methylation Site

“…It could be demonstrated that the less frequent allele of the investigated SNP can lead to DNA methylation at a CpG site, and was associated with differences in the regulation of gene expression in our test system. As shown by Escher et al (2005), plasmids can be methylated after transfection. Therefore, we tested SLC12A6 promoter activity comparing G vs A alleles.…”

Increased maternal and fetal cholesterol efflux capacity and placental CYP27A1 expression in preeclampsia

“…RCT is defined as the transport of cholesterol from peripheral organs/macrophages to an acceptor in plasma, followed by its transfer to the liver in order to be eliminated/recycled. Overexpression of CYP27A1, as seen in Chinese hamster ovary cells, expressing polyoma LT antigen (CHOP) cells and macrophages (8,9) results in enhanced cholesterol efflux. Indeed, the appearance of 27-OHC may produce conformal changes in the plasma membrane and facilitate the release of cholesterol and 27-OHC to an acceptor in plasma.…”