Establishment of a newly improved detection system for NF-kappaB activity.
The transcription factor nuclear factor-kappaB (NF-kappaB) plays roles in apoptosis, inflammation and oncogenesis. It is important for biological and medical research to understand when proteins of interest are activated in cells, leading to the establishment of a luciferase/EGFP assay to monitor the activation of transcription factors. Here, we describe an improved reporter system for NF-kappaB, the NF-kappaB-activated transgene (NAT) system that can detect NF-kappaB signalling with high sensitivity and specificity. The NAT system consists of large copy numbers of NF-kappaB consensus sequence and a minimal promoter derived from the mouse interleukin-2 (IL-2) gene. Furthermore, we generated NAT systems with stable or unstable luciferase/EGFP proteins. Stable and unstable types of luciferase/EGFP are suitable for analyzing the accumulation of and the real-time activity of NF-kappaB signal, respectively. Our findings suggest that the NAT system is effective for in vivo imaging of NF-kappaB signalling using cells or animals.
Matsuda
Mayuko
M
Department of Molecular Biochemistry, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido, Japan.
Tsukiyama
Tadasuke
T
Bohgaki
Miyuki
M
Nonomura
Katsuya
K
Hatakeyama
Shigetsugu
S
D000818:Animals; D002460:Cell Line; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics; D053308:Lymphotoxin-beta / Q000276:immunology; D051379:Mice; D016328:NF-kappa B / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection / Q000379:methods; D019076:Transgenes; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology
D053308:Lymphotoxin-beta; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008156:Luciferases