LightSwitch Luciferase Reporter Assay System Reagents

The LightSwitch Luciferase Assay System is a unique collection of over 30,000 regulatory elements that are available in the LightSwitch Promoter and 3´UTR Collections. After selecting a pre-cloned LightSwitch Promoter or 3´UTR Luciferase Reporter Vector, it is transfected into an appropriate cell line. The cells are stimulated, if required, to induce transcription of RenSP luciferase. Gene expression is then quantified using a LightSwitch Assay Kit and a luminometer to measure the amount of luminescence produced.

Advantages of the LightSwitch Luciferase Assay System

• Quantitative – Novel RenSP luciferase technology allows you to measure promoter activity with industry-leading sensitivity and dynamic range.
• Simple, fast, complete solution – With pre-cloned LightSwitch Reporter vectors and optimized transfection & assay reagents, you can study regulation of your gene today. No cloning, DNA preparation or optimization is needed, and most studies do not require any internal transfection controls.
• Comprehensive and verified – The genome-wide LightSwitch Reporter Collections are sequence-verified, transfection-ready promoter and 3´UTR reporter vectors.
• Cost-effective – Efficiently screen for activation and/or repression using a multitude of conditions.

RenSP – maximum brightness and minimal background

Why use Renilla luciferase?

Marine luciferases have become popular alternatives to firefly luciferase as a genetic reporter based on assay simplicity, high sensitivity, and a broad linear range of signal that provides greater sensitivity over firefly luciferases.^1,2 The Renilla luciferase protein catalyzes oxidation of its coelenterazine substrate in the reaction shown below to produce light at 480 nm, easily read by standard luminometers.³

Inherent advantage of Renilla luciferase

Because marine bioluminescence has evolved independently many times, a variety of luciferases target the same substrate (coelenterazine) yet bear little resemblance to one another.⁴ Renilla reniformis is a sea pansy that responds to mechanical stimulation by generating a blue-green bioluminescence.⁴ The small size of its gene and protein (936 bp and 36 kD) and its lack of dependence on ATP provide Renilla luciferase with a distinct advantage over larger, ATP-dependent luciferases like those from fireflies (~1.6 kb and 62 kD).^5-8

Figure 1: Light is produced from coelenterazine by Renilla luciferase.

Optimizing Renilla for LightSwitch reporter assays – increased enzymatic activity and brightness

We have created an optimized Renilla luminescent reporter gene, called RenSP, by increasing its overall enzymatic activity (light output) and adding a protein destabilization domain to decrease the half-life of the RenSP protein. Starting with a base sequence of the native Renilla gene, we functionally screened thousands of synthetic gene sequence variants that included a variety of predicted improvements. We also removed transcription factor binding sites from the gene sequence as these might confound expression measurements. As a result, we created the RenSP luciferase that is 100% brighter than other humanized versions of Renilla luciferase (Figure 2).⁹

Figure 2: The absolute signal of RenSP is significantly brighter than hRlucP.

To determine the relative brightness of RenSP compared to hRlucP, another humanized form of the Renilla luciferase, the RenSP and hRlucP genes were cloned into separate vectors each containing the human RPL10 promoter. Three independent plasmid purifications were conducted for each vector, and 50 ng of each plasmid was transfected with FuGENE HD in triplicate in human HT1080 cells using a 96-well format. After 24 hours of incubation, 100 µl of LightSwitch Reagent was added to each well and incubated for 30 minutes before being read for 2 seconds on an LmaxII-384 luminometer. These results show that the optimized RenSP luciferase is significantly brighter than hRlucP.

Enhanced degradation rate of RenSP improves assay response

One limitation of reporter gene assays is that the reporter protein can accumulate in the cell; this can delay and dilute the measurable response to stimulation or repression. To eliminate this problem, the RenSP gene has been fused to a protein destabilization domain to reduce the accumulation of reporter protein. The RenSP reporter gene contains a PEST protein degradation sequence from mouse Ornithine Decarboxylase (mODC) that has been shown to increase rates of protein turnover.^10-13

The destabilized RenSP luciferase protein has a half-life of approximately 1 hour compared to the ~3 hour half-life of the native luciferase protein, the CAT reporter protein half-life of ~50 hours and the GFP half-life of 25 hours. The RenSP-PEST fusion protein therefore combines the benefits of increased signal with a short half-life reporter to provide a sensitive measure of the induction or repression of reporter gene activity. Figure 3 highlights an example in which signal knock-down of a UTR reporter after addition of a miRNA is more easily detected when the target UTR is fused to a PEST-containing reporter gene.

Figure 3: RenSP with PEST increases the knock-down of 3´UTR targets in the presence of mir-122.

The knockdown of 3´UTR-luciferase activity in the presence of mir-122 was measured by co-transfecting a LightSwitch 3´UTR Reporter construct with a synthetic miRNA. DharmaFECT DUO was used to transfect HT1080 cells in triplicate in 96-well format with 100 ng of 3´UTR reporter and 20 nM of mimic or non-targeting control miRNA. After 24 hours of incubation, 100 µl of LightSwitch Assay reagent was added to each well, plates were incubated at room temperature for 30 minutes and read on a LmaxII-384 luminometer. The log2 ratio of the average mimic signal divided by the average signal from the non-targeting control was calculated and shows that the RenSP luciferase with PEST gives a significantly stronger knockdown than RenS without PEST.

Empty 3´UTR vector: The empty 3´UTR vector contains the RenSP luciferase reporter gene and its constitutive promoter. This construct may serve as a positive control for the transfection because of its promoter. It may also be utilized for creating additional constructs. For a vector map, annotations and primer & restriction sites, please see the Empty LightSwitch™ Reporter Vectors page.

Housekeeping gene 3´UTR vectors: Housekeeping control constructs contain the 3´UTRs for common housekeeping genes cloned downstream of the RenSP luciferase reporter gene.

Random control 3´UTR vectors: Random control constructs contain non-conserved, non-genic, and non-repetitive human genomic fragments.

Figure 1: LightSwitch assay performed using the positive and negative LightSwitch 3´UTR Controls.
Fifty ng of each 3´UTR Control Vector was transfected into HT1080 cells. Because the constitutive promoter driving the RenSP luciferase gene is the same for all vectors, it is expected that all of the vectors will give comparable signals, as this experiment does not include miRNAs to interact with the various 3´UTR regions and change the amount of luciferase produced.
The Y-axis values are shown in log scale. Absolute and relative activity will vary depending on cell line, experimental conditions and transfection reagents.

3´UTRs of housekeeping genes

Random genomic fragments in 3´UTR vector

Empty promoter vector: The empty promoter vector contains the RenSP luciferase reporter gene without a promoter. This construct serves as a measure of background signal in the experiment. This construct may also be used to create additional experimental constructs. For a vector map, annotations and primer & restriction sites, please see the Empty LightSwitch™ Reporter Vectors page.

Housekeeping gene promoter vectors: Housekeeping control constructs contain promoters for common housekeeping genes driving the RenSP luciferase reporter gene. These constructs serve as positive transfection controls and may also serve as controls for comparing signals between conditions if they are known to be unresponsive to the test condition.

Random control promoter vectors: Random control constructs contain 1 kb non-conserved, non-genic, non-repetitive fragments from the human genome cloned upstream of the RenSP luciferase reporter gene. These vectors produce slightly different signals than the empty promoter vector and are considered optimal negative or background controls.

Figure 1: LightSwitch assay performed using the positive and negative LightSwitch Promoter Controls.
The 4 housekeeping promoter controls serve as positive controls and give relative luciferase activity. The 4 random promoter constructs and the Empty LightSwitch Reporter Vector serve as negative/background controls and establish baseline luciferase activity.
The Y-axis values are shown in log scale. Control vector activity may vary depending on cell line, treatment condition and protocol changes.

Promoters of housekeeping genes (Positive controls)

Random genomic fragments in promoter vector (Negative controls)

Contents & Storage

The LightSwitch™ Luciferase Assay Kit contains:

• Lyophilized 100X LightSwitch™ Assay Substrate (-20°C)
• LightSwitch™ Substrate Solvent (room temperature)
• LightSwitch™ Assay Buffer (-20°C)
• Protocol

All reagents are guaranteed stable for 6 months when stored properly.

Figure 1: Broad, linear dynamic range of signal produced by the LightSwitch Luciferase Assay Kit.
To determine the dynamic range of LightSwitch reagents, cell lysate was prepared from HT1080 cells that had been transfected using FuGENE^® HD Transfection Reagent with a LightSwitch Promoter Reporter vector containing the ACTB to drive expression of the RenSP luciferase protein (Product ID S717678). A 12-point, 2-fold dilution series of lysate was prepared and total luminescence was measured using the LightSwitch Luciferase Assay Kit. These results show that the kit produces a linear dynamic range across 5 orders of magnitude of signal.