Transfection of adherent murine peritoneal macrophages with a reporter gene using DEAE-dextran.
We have sought a transfection strategy which preserves cell viability and achieves sufficient efficiency to perform reporter gene assays in primary cultures of thioglycollate-elicited murine peritoneal macrophages. Murine peritoneal macrophages were transfected with an eukaryotic expression vector containing the Rous sarcoma virus enhancer-promoter upstream of the bacterial chloramphenicol acetyl-transferase reporter gene (FC4-CAT). Transfection using DEAE-dextran followed by 10% DMSO provided much higher CAT activity than either calcium-phosphate or lipofection. The transfected macrophages increased CAT activity (1.9-7.6-fold) following stimulation with 10% serum. DEAE-dextran/DMSO-mediated transfection provides a simple, inexpensive method to transfect primary cultures of adherent macrophages with heterologous plasmid DNA. Transfection of macrophages with a CAT reporter gene using this method permits the characterization of gene regulation in primary macrophage cultures in vitro.
Rupprecht
A P
AP
Department of Internal Medicine, Yale University School of Medicine, West Haven, CT 06516.
Coleman
D L
DL
D000818:Animals; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000235:genetics*; D003637:DEAE-Dextran / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C; D010529:Peritoneal Cavity / Q000166:cytology; D014162:Transfection
D003637:DEAE-Dextran; D015500:Chloramphenicol O-Acetyltransferase