- Proteins and Peptides
- Lysates and Cell Lines
Reporter cell lines provide a simple, rapid and reliable method of monitoring the activity of signaling pathways. Cells are engineered to express a reporter gene downstream of the promoter or regulatory sequence of the gene of interest (Figure 1). The encoded reporter protein is then quantified in response to an appropriate stimulus.
Figure 1. The SEAPorterTM cell lines express the reporter gene for SEAP under the transcriptional control of an NF-κB response element.
A good reporter protein should be easily detectable and not normally be present in the organism being investigated. Well-studied reporter proteins include luciferase, secreted alkaline phosphatase (SEAP), β-galactosidase and Green Fluorescent Protein (GFP). The reporter protein can itself be directly measured; alternatively its enzymatic activity can be quantified. Novus Biologicals specializes in SEAPorterTM reporter cell lines for studying inflammation, autoimmune disease and cancer-related signaling pathways including NF-κB, TLR, AP-1, NFAT, GATA3 and STAT pathways.
SEAPorterTM cell lines
SEAPorterTM cells are also stably transfected HEK293 cells, however they rely on secreted alkaline phosphatase (SEAP) as the reporter gene. SEAP catalyses the hydrolysis of pNitrophenyl phosphate (pNPP), converting it to a colored product, the absorbance of which can then be read in a spectrophotometer at 405nm. Since SEAP is secreted by cells, lysis is not required for its detection. This enables kinetic experiments to be performed through sequential sampling of the media, and also allows the cells to be used for additional purposes such as Western blotting or RNA extraction.
Novus Biologicals currently offers more than 10 SEAPorterTM cell lines, which have been stably co-transfected with TLR and SEAP genes that are under the transcriptional control of an NF-κB response element (Figure 3). The SEAPorterTM cell lines can be used to screen for TLR agonists and antagonists. SEAP activity is measured with the Secreted Alkaline Phosphatase Reporter Assay Kit (catalog no. NBP-25285), which is optimized to pair with all of the SEAPorterTM cell lines. Additionally, Novus Biologicals offers a unique NF-kB Secreted Alkaline Phosphatase Assay Kit (NBP2-25286), that includes an NF-kB/SEAP plasmid for researchers who want to construct their own SEAP reporter cell lines.
|Target Gene||Promoter||Catalog Number|
|TLR4, MD2, CD14||NF-κB RE||NBP2-26503|
Figure 2. SEAPorterTM cell lines. RE = Response element
Reporter cell line applications - summary
Reporter cell lines are powerful tools for evaluating gene activity, especially when studying complex signaling such as that exhibited by the TLR pathway. The SEAPorterTM cell lines are designed for the analysis of cellular responses that result in modulation of NF-κB activities, and enable the TLR specificity of agonists, or antagonists, to be investigated.
Homogeneous bioluminescent assays can rapidly be developed using reporter cell lines, and are easily amenable to multiwell plate formats and automation. Reporter cell line assays also provide a simple and sensitive screening platform for the evaluation of agonistic or antagonistic effects and, in the case of secreted reporter proteins such as SEAP, are an extremely powerful tool for kinetic analysis. Unlike conventional antibody-based analyses, reporter gene assays are quantifiable, have a large dynamic range, and are amenable to kinetic analysis.
Toll-like receptors (TLRs) play a pivotal role in initiating innate immune responses following exposure to conserved microbial components such as lipopolysaccharide and dsRNA. Thirteen mammalian TLRs are currently known; these bind and become activated by various different ligands, resulting in signaling cascades that ultimately trigger activation of transcription factors and expression of genes involved in the immune response (1).
NF-κB is involved in controlling the immune response to infection, and incorrect regulation of this protein is implicated in cancer, inflammatory disorders and autoimmune conditions. In resting cells, NF-κB is sequestered by inhibitor of κB (I-κB). Activation of TLR signaling pathways results in phosphorylation of I-κB, leading to its ubiquitination and degradation, allowing the released NF-κB to translocate to the nucleus, where it binds to NF-κB response elements and recruits other proteins to activate the transcription of target genes (2).
|Download the PDF version of the whitepaper Engineered cell lines which stably express SEAP reporter proteins|
Also, don’t miss our webinar recording - Engineered reporter cell lines: Advanced tools for profiling cell signal on YouTube.