title: Evaluation of methods for transient transfection of a murine macrophage cell line, RAW 264.7.
abstract: Monocyte/macrophage cell lines are fastidious cells commonly used in transient transfection experiments. In the course of a study of gene regulation by lipopolysaccharide (LPS), we have compared several methods for DNA-mediated 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 transfection technique. The following methods were compared: Lipofectin, LipofectAMINE, LipofectAMINE PLUS, SuperFect, Ca3(PO4)2 DNA co-precipitation, DEAE dextran-mediated transfection and electroporation. The transfected plasmid DNA included a luciferase reporter construct containing the junB minimal promoter under the control of an LPS-inducible 1300-bp regulatory fragment downstream of junB 5'-flanking sequence, as well as a beta-galactosidase 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 methods. DEAE dextran and Ca3(PO4)2 precipitation showed the least and the greatest inter-assay variation, respectively. For all other methods, inter-assay 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.
authors.affiliation: OTRR/CBER/FDA, Bethesda, MD, USA. thompsonc@cber.fda.gov
authors.forename: C D
authors.initials: CD
authors.lastname: Thompson
authors.forename: M R
authors.initials: MR
authors.lastname: Frazier-Jessen
authors.forename: R
authors.initials: R
authors.lastname: Rawat
authors.forename: R P
authors.initials: RP
authors.lastname: Nordan
authors.forename: R T
authors.initials: RT
authors.lastname: Brown
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002130:Calcium Phosphates; D002411:Cation Exchange Resins; D002460:Cell Line; D011232:Chemical Precipitation; D003637:DEAE-Dextran; D004247:DNA; D018274:Electroporation; D017930:Genes, Reporter; D016758:Genes, jun; D008055:Lipids; D008070:Lipopolysaccharides / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D010714:Phosphatidylethanolamines; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D002130:Calcium Phosphates; D002411:Cation Exchange Resins; D008055:Lipids; C086724:Lipofectamine; D008070:Lipopolysaccharides; D010714:Phosphatidylethanolamines; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; C020888:1,2-dielaidoylphosphatidylethanolamine; D004247:DNA; D003637:DEAE-Dextran; C020243:calcium phosphate; D008156:Luciferases; D001616:beta-Galactosidase; C485829:calcium phosphate, dibasic, anhydrous