Stably transfected human cell lines as fluorescent screening assay for nuclear factor KB activation dependent gene expression

Christine E. Hellweg; Christa Baumstark-Khan; Gerda Horneck

doi:10.1117/12.530867

14 June 2004 Stably transfected human cell lines as fluorescent screening assay for nuclear factor KB activation dependent gene expression

Christine E. Hellweg, Christa Baumstark-Khan, Gerda Horneck

Author Affiliations +

Proceedings Volume 5329, Genetically Engineered and Optical Probes for Biomedical Applications II; (2004) https://doi.org/10.1117/12.530867
Event: Biomedical Optics 2004, 2004, San Jose, CA, United States

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.

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Christine E. Hellweg, Christa Baumstark-Khan, and Gerda Horneck "Stably transfected human cell lines as fluorescent screening assay for nuclear factor KB activation dependent gene expression", Proc. SPIE 5329, Genetically Engineered and Optical Probes for Biomedical Applications II, (14 June 2004); https://doi.org/10.1117/12.530867

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KEYWORDS

Luminescence

Proteins

Green fluorescent protein

Tumors

Kidney

Resistance

Flow cytometry

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