Evaluation of methods for transient transfection of a murine macrophage cell line, RAW 264.7

Monocyte/macrophage cell lines are fastidious cells commonly used in transi ent transfection experiments. In the course (of a study of gene regulation by lipopolysaccharide (LPS), lye have compared several methods for DNA-medi ated cell transfection to determine which would be optimally applicable to the macrophage line, RAW 264.7. Both the response level (LPS inducibility) and the degree of inter-assay variation were evaluated for each transfectio n technique The following methods were compared: LIPOFECTIN(R), LIPOFECTAMI NE(TM), LIPOFECTAMINE PLUS(TM), SyperFect(TM), Ca-3(PO4)(2) DNA co-precipit ation, DEAE dextran-mediated transfection and electroporation. The transfec ted plasmid DNA included a luciferase reporter construct containing the jun B minimal promoter under the control of an LPS-inducible 1300-bp regulatory fragment downstream of junB 5'-flanking sequence, as well as a beta-galact osidase reporter construct under the adenovirus promoter and enhancer used as an internal control. Electroporation, followed by a resting period of 16 -24 h before stimulation with LPS, had the highest inducibility of all meth ods. DEAE dextran and Ca-3(PO4)(2) precipitation showed the least and the g reatest inter-assay variation, respectively. For all other methods, inter-a ssay variability fell within this range. The results presented may serve as both a general reference and a guide for reporter gene studies in this or other macrophage cell lines.