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.
Highlights
This study was aimed at developing a method for high-efficiency transient transfection of macrophages
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 [4,5,6]. These levels of expression are sufficiently high to study synthetic processes, when proteins are tagged or otherwise distinguished from host proteins. Studying cell functions such as growth, lipoprotein binding, lipid uptake, and efflux requires high levels of gene expression and for the gene to be expressed in a majority of cells, a highefficiency transfection
The method was 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
Summary
This study was aimed at developing a method for high-efficiency transient transfection of macrophages. 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 [4,5,6]. Studying cell functions such as growth, lipoprotein binding, lipid uptake, and efflux requires high levels of gene expression and for the gene to be expressed in a majority of cells, a highefficiency transfection. 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 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
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
