Assessment of usefulness of J774A.1 macrophages for the assay of IL-1beta promoter activity.
Existing data indicate that the increase of il-1beta gene expression can be a promising marker of Langerhans cells activation after exposure to contact sensitizers. In this study, we were interested in development of an alternative in vitro screening test detecting such sensitizers. Two IL-1beta reporter constructs containing the enhanced green fluorescent protein (GFP) gene and mouse IL-1beta promoter fragments of varying lengths (-500 bp and -4093 bp) were used for transient transfections of J771A.1 murine monocyte-macrophage cells. As a result of the transfections performed using Lipofectamine reagent we did not observe any GFP fluorescence after stimulation of the cells with LPS as well as known sensitizers (potassium tetrachloroplatinate, dinitrochlorobenzene and nickel sulfate). Low transfection efficiency of J774A.1 cells (less than 0.1%) was confirmed using control plasmid containing GFP gene under the control of cytomegalovirus promoter. The fact that, using the same conditions, we were able to transfect murine fibroblasts 3T3-L1 with the control plasmid very efficiently, may support the theory of high metabolic activity of macrophages being responsible for the extremely low transfection efficiency. These data suggest limited suitability of J774A.1 cell line for transient transfections using cationic liposomes.
Arkusz
J
J
Nofer Institute of Occupational Medicine, 8 Sw. Teresy St., 91-348 Łódź, Poland.
Stepnik
M
M
Trzaska
D
D
Dastych
J
J
Rydzyński
K
K
D041721:3T3-L1 Cells; D000485:Allergens / Q000633:toxicity; D000818:Animals; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007375:Interleukin-1 / Q000235:genetics* / Q000378:metabolism; D007509:Irritants / Q000633:toxicity; D008055:Lipids; D008081:Liposomes; D008264:Macrophages; D051379:Mice; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D018675:Toxicity Tests / Q000379:methods*; D014162:Transfection / Q000379:methods*
D000485:Allergens; D007375:Interleukin-1; D007509:Irritants; D008055:Lipids; C086724:Lipofectamine; D008081:Liposomes; D012333:RNA, Messenger; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA