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Proceedings Paper

Stably transfected human cell lines as fluorescent screening assay for nuclear factor KB activation dependent gene expression
Author(s): Christine E. Hellweg; Christa Baumstark-Khan; Gerda Horneck
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Paper Abstract

Activation of the Nuclear Factor kappaB (NF-kappaB) pathway as a possible antiapoptotic route represents one important cellular stress response. For identifying conditions which are capable to modify this pathway, a screening assay for detection of NF-kappaB-dependent gene activation using the reporter proteins Enhanced Green Fluorescent Protein (EGFP) and its destabilized variant (d2EGFP) has been developed. Human Embryonic Kidney (HEK/293) cells were stably transfected with a vector carrying EGFP or d2EGFP under control of a synthetic promoter containing four 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 d2EGFP expression after treatment with TNF-alpha or phorbol ester was measured using flow cytometry. Cellular response to TNF-alpha was faster than to phorbol ester. Treatment of cells with TNF-alpha and DMSO revealed antagonistic interactions of these substances in the activation NF-kappaB dependent gene expression. 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.

Paper Details

Date Published: 14 June 2004
PDF: 11 pages
Proc. SPIE 5329, Genetically Engineered and Optical Probes for Biomedical Applications II, (14 June 2004); doi: 10.1117/12.530867
Show Author Affiliations
Christine E. Hellweg, DLR (Germany)
Christa Baumstark-Khan, DLR (Germany)
Gerda Horneck, DLR (Germany)

Published in SPIE Proceedings Vol. 5329:
Genetically Engineered and Optical Probes for Biomedical Applications II
Alexander P. Savitsky; Darryl J. Bornhop; Ramesh Raghavachari; Samuel I. Achilefu, Editor(s)

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