Highly Sensitive Multiplex Luciferase Reporter Assays

Monitor changes in NFkB and CRE promoter activity using bioluminescent reporter assays

by Douglas Hughes, Ph.D.; Jae Choi, Ph.D.; Megan Dobbs, B.S.; Janaki Narahari, Ph.D.; Brian Webb, Ph.D. - 02/28/12

The study of complex cellular signaling pathways requires powerful and specific tools to monitor changes in gene activation or repression. To accurately monitor these processes, reporter gene assays are commonly used. Our multiplexed luciferase reporters for studying gene regulation are more sensitive and convenient than luciferase reporter systems from other suppliers. We use two naturally secreted luciferase genes: Gaussia luciferase from the marine copepod Gaussia princepsand Cypridina luciferase from the marine ostracod Cypridina noctiluca. This Gaussia-Cypridina dual system enables the monitoring of transcriptional regulation of two promoters within tissue culture media without the need for cell lysis. Furthermore, both Gaussia and Cypridina luciferases are considerably brighter than traditional firefly luciferase reporters.

We also use a mutant form of the Japanese firefly luciferase from Luciola cruciata, which has a red-shifted emission spectrum, and Cypridina luciferase for a dual-spectral luciferase assay, which has spectrally resolvable light outputs. This Cypridina/red firefly dual-spectrum assay enables simultaneous monitoring of two promoters in a single-read assay by adding both substrates and performing spectral interrogation of the light output. This reporter and control combination enables simultaneous monitoring of experimental reporter and control luciferase activity without the need for two-step addition of substrate reagents or quenching. The bioluminescent signal produced from the luciferase reaction is captured using a luminometer. The resulting signal is proportional to the amount of luciferase protein produced, which is proportional to the transcriptional activity of the promoter driving the luciferase expression.

To demonstrate the usefulness of the Thermo Scientific Pierce Luciferase Assay Kits, we monitored changes in NF-kappa B (NFkB) and CRE promoter activity in response to small molecule agonists. We used Cypridina/red firefly spectral separation multiplexing, and Gaussia/Cypridina multiplex assays. The dual-secreted Gaussia/Cypridina luciferase assays were sensitive for real-time monitoring of NFkB and CRE reporter activity in the media and simultaneous detection of spectrally resolvable luciferases using filter-based detection.

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 (Figure 1). The Thermo Scientific pCMV-Red Firefly Mammalian Expression Vector was used as a constitutive luciferase signal for normalizing the Cypridina and Gaussia signals. The ATP-dependent oxidation of luciferin by red firefly luciferase produces light with an emission maximum of 613nm, and the oxidation of vargulin by Cypridina luciferase produces light with an emission maximum of 463nm (Figure 2; Table 1). The activity of Cypridina and red firefly luciferases is simultaneously assessed using a filter-based assay. In this assay, Cypridina luciferase plasmid acts as an experimental reporter coupled with red firefly expression plasmid (CMV-red firefly) as a normalization control.

Figure 1. Plasmid constructs for Cypridina (Part No. 16146) and Red Firefly (Part No. 16156).

Figure 2. Spectrally resolvable luciferase reactions enable simultaneous detection of reporter enzymes. The Pierce Cypridina-Firefly Luciferase Dual Assay emission spectra have separate Cypridina and red firefly signals. The shaded boxes represent the filter ranges that were used. 

When expressed in mammalian cells, high levels of Gaussia luciferase are secreted into the cell culture media, enabling live-cell monitoring of reporter activity. The activity of Cypridina luciferase expressed from the NFkB-Cypridina reporter plasmid increased in response to increasing TNFα concentration (Figure 3A). Similarly, the activity of Gaussia luciferase expressed from the CRE-Gaussia reporter plasmid increased with increasing concentrations of forskolin (Figure 3B). These results demonstrate the ability to simultaneously detect two distinct pathway activations within the same cells.

Figure 3. Simultaneous detection of multiple signal transduction pathways using spectrally resolved luciferase reporters. HEK293 cells were treated with forskolin or TNFα alone or in combination to activate the cAMP-mediated CREB pathway or the NFkB-mediated cell survival pathway. Luciferase activities were determined using the Pierce Luciferase Assay Kits, according to the instructions. The luminescence signal was normalized to the activity of red firefly luciferase from the CMV-red firefly control plasmid.

We performed real-time monitoring of CREB and NFkB reporter activity by measuring the secreted luciferase activity in the cell media. After 3 hours of adding forskolin, cells were treated with TNFα to activate the NFkB-Cypridina Luc reporter. Cell culture media was sampled at 2 hour intervals for the duration of the experiment. The secreted Gaussia and Cypridina luciferases in the culture media were monitored. Fold inductions for both reporters increased with time (Figure 4). Distinct activation of NFkB and CRE reporters was observed in real time without destroying the cells.

Figure 4. Real-time detection of multiple signal transduction pathways using secreted luciferase reporters. Three hours after of adding forskolin, cells were treated with TNFα. The culture media was sampled to measure luciferase activity using the Pierce Luciferase Assay Kits. The luminescence signal was normalized to the activity of red firefly luciferase from the CMV-red firefly control plasmid.

The Pierce Luciferase Assay Kits provide a highly sensitive system for detecting intracellular or secreted luciferase activity from promoter or pathway activation in mammalian cell cultures. These assays enable simultaneous, real-time detection of NFkB and CREB signal transduction pathways using spectrally resolved luciferase reporters without destroying the cells.

Cell Culture: HEK293 cells were cultured in DMEM/high glucose media (Thermo Scientific HyClone) supplemented with 10% fetal bovine serum (FBS) and antibiotics. Cells were plated at 10,000 cells/well in 96-well tissue culture plates and incubated in a humidified cell culture incubator at 37°C for 24 hours before transfection.

Transfections: Plasmids containing promoter gene constructs of CRE-Gaussia, NFkB-Cypridina and CMV-red firefly (Figure 1) were co-transfected into HEK293 cells using Thermo Scientific TurboFect Transfection Reagent (Part No.R0533). After 24 hours of transfection, cells were treated with forskolin or TNFα alone or in combination to activate the cAMP-mediated CREB pathway or the NFkB-mediated cell survival pathway.

Luciferase Assays: Activities of the Cypridina and red firefly luciferases were determined using the Pierce Cypridina-Firefly Luciferase Dual Assay Kit (Part No. 16183), according to the instructions. Activity of Gaussia luciferase was determined using the Pierce Gaussia Luciferase Flash Assay Kit (Part No. 16158). 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 activity 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.

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.

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.

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.

Widder, E.A. (2010). Bioluminescence in the ocean: Origins of biological, chemical and ecological diversity. Science 328:704-8.