Generation of stably transfected Mammalian cell lines as fluorescent screening assay for NF-kappaB activation-dependent gene expression.
Cellular stress protection responses lead to increased transcription of several genes via modulation of transcription factors. Activation of the Nuclear Factor kappaB (NF-kappaB) pathway as a possible antiapoptotic route represents one important cellular stress response. To identify conditions that are capable of modifying this pathway, a screening assay for detection of NF-kappaB-dependent gene activation using the reporter protein Enhanced Green Fluorescent Protein (EGFP) and its destabilized variant (d2EGFP) was developed. Human Embryonic Kidney (HEK/293) cells were stably transfected with a vector carrying EGFP or d2EGFP under control of a synthetic promoter containing 4 copies of the NF-kappaB response element. Treatment with tumor necrosis factor alpha (TNF-alpha) gave rise to substantial EGFP/d2EGFP expression in up to 90% of the cells and was therefore used to screen different stably transfected clones for induction of NF-kappaB-dependent gene expression. The time course of NF-kappaB activation leading to d2EGFP expression was measured in an oligonucleotide-based NF-kappaB-ELISA. NF-kappaB binding in-creased after 15-min incubation with TNF-alpha. In parallel, d2EGFP increased after 3 h and reached its maximum at 24 h. These results show (1) the time lag between NF-kappaB activation and d2EGFP transcription, translation, and protein folding and (2) the increased reporter gene expression after treatment with TNF-alpha to be caused by the activation of NF-kappaB. The detection of d2EGFP expression required FACS analysis or fluorescence microscopy, while EGFP could also be measured in the microplate reader, rendering the assay useful for high-throughput screening.
Hellweg
Christine E
CE
Radiation Biology, Institute of Aerospace Medicine, DLR, Linder Höhe, D-51170 Köln, Germany. christine.hellweg@dlr.de
Baumstark-Khan
Christa
C
Horneck
Gerda
G
D000818:Animals; D002460:Cell Line; D004742:Enhancer Elements, Genetic; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D005453:Fluorescence; D005470:Fluorometry / Q000295:instrumentation / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008322:Mammals; D016328:NF-kappa B / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D009841:Oligonucleotides / Q000235:genetics / Q000494:pharmacology; D013997:Time Factors; D015533:Transcriptional Activation; D014162:Transfection / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D008164:Luminescent Proteins; D016328:NF-kappa B; D009841:Oligonucleotides; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins