Monitoring Changes in NF- kB Pathway Regulation Using Highly Sensitive Multiplex Bioluminescent Reporter AssaysDouglas Hughes, Ph.D.; Jae Choi, Ph.D.; Megan Dobbs; Janaki Narahari, Ph.D.; and Brian Webb, Ph.D.Thermo Fisher Scientific • Rockford, IL

AbstractThe study of complex cellular signaling pathways requires powerful and specific tools to monitor changes in gene activation or repression. In order to accurately monitor these processes, reporter gene assays are commonly used. We have developed a series of next generation multiplexed luciferase reporters for studying gene regulation. These reporters were developed to improve the sensitivity and convenience of conventional luciferase reporter systems. First, we have used two naturally secreted luciferase genes, Gaussia luciferase from the Marine copepod Gaussia princeps and Cypridina luciferase from the Marine ostracod Cypridina noctiluca to develop a multiplex assay system. This Gaussia/Cypridina dual system enables monitoring transcriptional regulation of two promoters within tissue culture media without the need for cell lysis. Importantly, both Gaussia luciferase and Cypridina luciferase are considerably brighter than traditional Firefly luciferase reporters. Second, we have utilized a mutant form of the Japanese Firefly Luciferase from Luciola cruciata that has a red-shifted emission spectrum to develop a dual luciferase assay with Cypridina luciferase in which the light output of the two luciferases are spectrally resolvable. This Cypridina/Red Firefly dual spectral assay allows simultaneous monitoring of two promoters in a single read assay through addition of both substrates and then spectral interrogation of the resulting light output. In the present study, we utilized both techniques, multiplexing by spectral separation using Cypridina/Red Firefly, and multiplex assays using the two secreted luciferases, Gaussia/Cypridina, to monitor changes in NFkB and CRE promoter activity in response to small molecule agonists. Our results demonstrate the utility of dual secreted luciferase assays for sensitive real time monitoring of NFkB and CRE reporter activity in the media and simultaneous detection of spectrally resolvable luciferases using filter based detection.

IntroductionReporter gene expression can be measured using bioluminescent signals formed through substrate catalysis and the natural luciferase enzyme.Our study showed the beneficial use of sensitive bioluminescent enzymes in reporter gene assays with wide dynamic ranges and flexibility in signal identification.Mutations in the Luciola cruciata firefly gene cause a spectral red shift of the reaction, which allows for spectrally resolving Cypridina and firefly light emissions in a single reaction.Naturally secreted reporter assays using Gaussia and Cypridina genes capture time course data by sampling the media of transfected cells; sampling the media prevents destruction of the cells.Our study used Gaussia/Red Firefly spectral separation and secreted Gaussia/Cypridina multiplex assays to monitor changes in NFkB and CREB activity in response to small-molecule agonists.Our results demonstrate the usefulness of multiplexed secreted luciferase assays for sensitive real-time monitoring of NFkB and CREB activity in media. Furthermore, these assays enable simultaneous detection of spectrally resolvable luciferases using filter-based detection.

Figure 1. Luciferase plasmid constructs. Multiple tandem copies of an NFkB- or CREB-binding DNA sequence were cloned into the Thermo Scientific pMCS-Cypridina or pMCS-Gaussia Mammalian Expression Vectors. The Thermo Scientific pCMV-Red Firefly Mam-malian Expression Vector was used as a constitutive luciferase signal for normalizing the Cypridina and Gaussia signals.

Figure 2. Spectrally resolvable luciferase reactions allow simultaneous detection of report-er enzymes. Chemical reaction of luciferin and red firefly luciferase. Light, with an emis-sion maximum of 613nm, is produced from the oxidation of D-luciferin by red firefly lucifer-ase in an ATP-dependent reaction. Chemical reaction of vargulin and Cypridina luciferase. Light, with an emission maximum of 463nm, is produced from the oxidation of vargulin by Cypridina luciferase (A). Thermo Scientific Pierce Cypridina-Firefly Luciferase Dual Assay emission spectra profile. The shaded boxes represent the filter ranges used (B). See Table 1 for the filter details.

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Table 1. Filter requirement for the Thermo Scientific Pierce Cypridina-Firefly Luciferase Dual Assay Kit.

Luciferase Emission Range (nm)

Emission Maximum(nm)

Recommended Filter* (nm)

Cypridina 400-600 463 480±20 BP

Red Firefly 560-700 613 640 LP

*The 480±20nm bandpass (BP) filter is designed to capture light wavelengths ranging from 460 to 500nm (Figure 3). Similarly, the 640nm longpass (LP) filter collects wavelengths above 640nm. Each luminometer requires specific filters; specifications and filters are available from Omega Optical, Inc. or Chroma Technology Corp.

Figure 3. Thermo Scientific Pierce Cypridina-Firefly Luciferase Dual Assay Kit procedure summary. The activity of two spectrally resolvable luciferases, Cypridina and red firefly lu-ciferase, is simultaneously assessed using a filter-based assay (Figure 2). The Cypridina luciferase produces an emission spectrum ranging from 400-600nm (λmax= 463nm). The red firefly luciferase produces a red-shifted emission spectrum ranging from 560-700nm (λmax= 613nm). Cypridina luciferase plasmid acts as an experimental reporter coupled with red firefly expression plasmid (CMV-Red Firefly) as a normalization control. This reporter and control combination enables simultaneous monitoring of experimental reporter and control luciferase activity in a single-read assay without the need for two-step addition of substrate reagents or quenching. The bioluminescent signals produced from the luciferase reaction can be captured using a luminometer equipped with filters. The resulting signal is proportional to the amount of luciferase protein produced, which is then proportional to the transcriptional activity of the promoter driving the luciferase expression.

Figure 4. Thermo Scientific Pierce Gaussia Luciferase Flash Assay Kit procedure summary. Gaussia luciferase is a ~22kDa protein, which is highly secreted into the cell culture me-dia, allowing for live cell monitoring of reporter activity. The Gaussia luciferase biolumines-cent signal originates from the oxidative decarboxylation of its coelenterazine substrate. Gaussia luciferase gives high levels of bioluminescent signal when transfected into mam-malian cells. The bioluminescent signal produced from Gaussia luciferase reaction can be captured using a luminometer and is proportional to the amount of Gaussia luciferase pro-tein produced, which in turn is proportional to the transcriptional activity of the promoter driving Gaussia expression.

Figure 5. Simultaneous detection of multiple signal transduction pathways using spec-trally resolved luciferase reporters. Plasmids containing promoter gene constructs of CRE-Gaussia, NFkB-Cypridina and CMV-Red Firefly were co-transfected into HEK293 cells using Thermo Scientific TurboFect Transfection Reagent. After 24 hours of transfection, cells were treated with Forskolin or TNFα alone, or in combination, for 3 hours to activate the cAMP-mediated CREB pathway or the NFkB-mediated cell survival pathway. Activity of the Cypridina and Red Firefly luciferases was determined using the Thermo Scientific Pierce Cypridina-Red Firefly Luciferase Dual Assay Kit (A). Activity of Gaussia luciferase was determined using the Pierce Gaussia Luciferase Flash Assay Kit (B). Bioluminescence signals were detected using a Thermo Scientific Varioskan Flash Luminometer equipped with reagent injectors, a 480±20nm band pass filter and a 640nm long pass filter. The activ-ity of Cypridina or Gaussia luciferase expressed from the NFkB-Cypridina or CRE-Gaussia reporter plasmid was normalized to the activity of red firefly luciferase from the CMV-Red Firefly control plasmid. The combined results of A and B expressed as a percentage of maximum induction demonstrates the simultaneous detection of two distinct pathway activations (C).

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Figure 6. Real-time detection of multiple signal transduction pathways using secreted lu-ciferase reporters. Real-time monitoring of CREB and NFkB reporter activity over time (A). Fold inductions for each reporter increase over time as the cells continue to secrete Cypridina and Gaussia luciferase into the cell culture media. Plasmids containing the pro-moter gene constructs of CRE-Gaussia, NFkB -Cypridina and CMV-Red Firefly were co-transfected into HEK293 cells as described in Figure 5. After 24 hours of transfection, cells were treated with Forskolin to increase intracellular cAMP levels resulting in activation of the CRE-Gaussia Luc reporter. After 3 hours of Forskolin addition, cells were treated with TNFα to activate the NFkB -Cypridina Luc reporter. For the duration of the experiment, cell culture media was sampled at 2 hour intervals and stored on ice. The secreted Gaussia present in the culture media was measured using the Thermo Scientific Pierce Gaussia Flash Assay Kit; the secreted Cypridina present in the culture media was measured using the Pierce Cypridina Flash Assay Kit. After 7 hours of Forskolin addition, the cells were lysed and CMV-Red Firefly activity was measured using the Pierce Firefly Flash Assay Kit. Signals obtained were used to normalize the Cypridina and Gaussia luciferase signals.

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Summary• The Thermo Scientific Pierce Luciferase Assay Kits provide a highly sensitive system for

detecting intracellular or secreted luciferase activity from promoter or pathway activation in mammalian cell culture experiments.

• Assay systems allow simultaneous detection of NFkB and CREB signal transduction pathways using spectrally resolved luciferase reporters.

• We achieved real-time detection of NFkB and CREB signal transduction pathways using secreted luciferase reporters without destruction of the cells.

References1. Widder, E. A. (2010). Bioluminescence in the ocean: origins of biological, chemical and

ecological diversity. Science. 328:704-708.2. Szent-Gyorgyi, C., et al. (1999). Cloning and characterization of new bioluminescent

proteins. Part of the SPIE Conference on Molecular Imaging: Reporters, Dyes, Markers, and Instrumentation. San Jose, CA. Proc SPIE. 3600:4-11.

3. Tannous, B. A., et al. (2005). Codon-optimized gaussia luciferase cDNA for mammalian gene expression in culture and in vivo. Molecular Therapy. 11:435-43.

4. Nakajima, Y., et al. (2004). cDNA cloning and characterization of a secreted luciferase from the luminous Japanese ostracod, Cypridina noctiluca. Biosci Biotechnol Biochem. 68(3):565-70.

For more information please visit http://www.thermoscientific.com/pierce, or dial 1-800-874-3723 in the U.S.A and +1-815-968-0747 for International.

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