Generation of Stably Transfected Mammalian Cell Lines as Fluorescent Screening Assay for NF-kB Activation-Dependent Gene Expression

摘要

Cellular stress protection responses lead to increased transcription of several genes via modulation of transcription factors. Activation of the Nuclear Factor kB (NF-kB) 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-kB-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-kB response element. Treatment with tumor necrosis factor α (TNF-α) 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-kB-dependent gene expression. The time course of NF-kB activation leading to d2EGFP expression was measured in an oligonucleotide-based NF-kB-ELISA. NF-kB binding increased after 15-min incubation with TNF-α. In parallel, d2EGFP increased after 3 h and reached its maximum at 24 h. These results show (1) the time lag between NF-kB activation and d2EGFP transcription, translation, and protein folding and (2) the increased reporter gene expression after treatment with TNF-α to be caused by the activation of NF-kB. 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.