Potential problems inherent in cell-based stable NF-kappaB-GFP reporter systems.
The nuclear factor-kappaB (NF-kappaB) family of transcription factors plays a central role in numerous physiological processes including development, cell survival, immunity, and inflammation. We generated a series of stable clonal lines in mouse embryonic fibroblasts carrying NF-kappaB-GFP plasmid as a reporter. These cell lines were selected by flow cytometry for their high responsiveness to tumor necrosis factor (TNFalpha) or lipopolysaccharide (LPS), two classic NF-kappaB-inducing stimuli. Although all clones were generated from the same parental cell line, they each had a distinctive pattern of response to NF-kappaB stimuli. While exhibiting distinct profiles with regard to the GFP reporter, analysis of endogenous NF-kappaB downstream targets did not always show the same variability. This suggests that in the absence of confirmation of the signaling outcomes using endogenous outputs, considerable caution must be exercised in the interpretation of data using stable reporter systems.
El-Guendy
Nadia
N
Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, 800 Rose St., Lexington, KY 40536, USA.
Sinai
Anthony P
AP
D000818:Animals; D002478:Cells, Cultured; D002999:Clone Cells; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics*; D020672:I-kappa B Proteins / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection / Q000379:methods*
C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D016328:NF-kappa B; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases