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Gene Regulation

LightSwitch™ Luciferase Assay System

everything needed for luciferase reporter assays

Active Motif’s LightSwitch™ Luciferase Assay System is a complete solution for performing gene regulation studies in living mammalian cells. It includes over 30,000 regulatory elements that have been cloned from the human genome, which are assayed using optimized reagents and protocols that make every step of the procedure faster and simpler. LightSwitch lets you bypass time-consuming cloning and assay development and go immediately to the production of meaningful data.

Brief summaries of each part of the LightSwitch System are listed below under the 'LightSwitch Luciferase Assay Overview' tab.

To search the GoClone™ Collections on the website of our affiliate, SwitchGear Genomics, click one of these buttons:

Button to jump to the LightSwitch GoClone Search page     Button to jump to the LightSwitch GoClone Search page

LightSwitch™ Promoter Reporter GoClone™ Collection

The Promoter Reporter Collection includes over 18,000 promoters cloned from the human genome and available as transfection-ready GoClone constructs. So, you don't need to spend time and effort cloning the promoters needed for your transcriptional regulation studies. Simply use our Search Tool to find vectors with your promoters of interest, then transfect and assay their activity immediately.

LightSwitch Promoter Reporter vector
LightSwitch™ Promoter
Reporter Vector.

LightSwitch™ 3´UTR Reporter GoClone™ Collection &
miRNA Mimics and Inhibitors

The 3´UTR Reporter Collection includes over 12,000 3´UTRs cloned from the human genome into ready-to-transfect LightSwitch reporter vectors. Combined with our large collection of miRNA Mimics and Inhibitors, you have everything needed to study miRNA-3´UTR interactions, validate miRNA targets, measure RNA stability, translation efficiency and the functional impact of miRNAs on a gene-by-gene basis.

LightSwitch 3´UTR Reporter vector
LightSwitch™ 3´UTR
Reporter Vector.

LightSwitch™ lncRNA Promoter Reporter Collection

While long noncoding RNAs (lncRNAs) have been shown to play important roles in the regulation of both transcription and chromatin organization, the mechanisms by which their own expression is regulated seem to differ from those of protein-coding genes. The LightSwitch lncRNA Promoter Reporter Collection was designed to make it fast & easy to study the regulation of lncRNA promoters.

LightSwitch Promoter Reporter vector
LightSwitch™ Promoter
Reporter Vector.

LightSwitch™ Validated
Pathway Collections

The Validated Pathway Collections are subsets of our LightSwitch Promoter constructs that were selected based on sequence motif analysis and published information, followed by in-house testing using our LightSwitch Luciferase Assay. All members have been experimentally validated to show significant activation or repression in response to pathway-specific induction conditions, so they are ideal when studying pathways such as inflammation, hypoxia, DNA damage, heat shock, etc.

LightSwitch Validated Pathway Collections
LightSwitch™ Validated
Pathway Collections.

Synthetic Response Elements &
Synthetic miRNA Targets

Because they have been engineered for increased sensitivity, the use of synthetic regulatory elements can improve some regulation experiments greatly. The stronger responses provided by our optimized LightSwitch Synthetic Response Elements and LightSwitch Synthetic miRNA Target Reporters can yield more meaningful data than that from native, endogenous promoter and 3´UTR sequences.

LightSwitch Synthetic Enhancers
Synthetic Enhancers &
miRNA Targets.

LightSwitch™ Custom Services

In addition to the many different products that make up the LightSwitch Luciferase Assay System, we offer a wide variety of custom services, including:

LightSwitch Custom Services
Our Custom Services Lab Can
Accelerate Your Research.

LightSwitch™ Pathway Reporter
Stable Cell Lines

Each LightSwitch Pathway Reporter Stable Cell Line contains a stably integrated, human regulatory element driving the expression of our optimized RenSP luciferase gene. This makes it far easier to measure the activity of specific pathways in response to various treatments and growth conditions. Each cell line has been validated using a variety of different plate-based formats, making them particularly useful for high-throughput screening experiments.

LightSwitch Pathway Reporter Stable Cell Lines
LightSwitch™ Pathway Reporter
Stable Cell Lines.

Transfection Reagents

After selecting a LightSwitch reporter construct, you must get it into your cell line. For plasmid transfections and co-transfections, we offer FuGENE® HD Transfection Reagent because of its consistent, high-quality performance across a wide variety of cell lines. For co-transfecting plasmids and miRNAs, we offer DharmaFECT® Duo Transfection Reagent, which provides more efficient co-transfection of those two distinct types of molecules. We also offer the LightSwitch Transfection Optimization Kit to quickly determine the conditions needed for high-efficiency transfections of your specific cell lines.

Mammalian cell transfection
Mammalian cell transfection.

LightSwitch Assay Reagents

After transfection, the amount of luciferase produced is measured using our LightSwitch Luciferase Assay Kit. Because RenSP catalyzes oxidation of its substrate in a reaction that produces light, gene expression can be quantified using a luminometer. The novel, proprietary substrate and optimized lysis buffer included in the LightSwitch Luciferase Assay Kit were formulated to provide high sensitivity over a broad dynamic range when used with our engineered RenSP. For those experiments that utilize co-transfection for normalization, we offer our LightSwitch Dual Assay Kit.

LightSwitch Luciferase Assay Kits
Light produced by luciferase.

LightSwitch Controls & Vectors

To help ensure you get the best results possible, we have developed panels of LightSwitch Promoter Control Vectors and LightSwitch 3´UTR Control Vectors. These positive & negative control constructs make it easier to interpret your data and to troubleshoot your work should things not go as expected. In addition, we offer Empty LightSwitch Reporter Vectors, which are useful as controls and for cloning your own regulatory elements.

Positive & negative LightSwitch Promoter Control Vectors
Positive & negative promoter controls.
 

LightSwitch Luciferase Assay Overview

Graphic depicting an overview of the LightSwitch Luciferase Assay System

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

Chemical reaction showing the oxidation of coelenterazine into coelenteramide that is catalyzed by the RenSP luciferase protein
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

Graph comparing the relative brightness of Active Motif's RenSP compared to hRlucP, another humanized form of the Renilla luciferase
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.

Graph comparing the relative brightness of Active Motif's RenSP compared to hRlucP, another humanized form of the Renilla luciferase
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.


LightSwitch™ References

  1. Lorenz, W., Cormier, M., O’Kane, D., Hua, D., Escher, A., and Szalay, A. (1996) Expression of the Renilla reniformis luciferase gene in mammalian cells. J. Biolumin. Chemilumin. 11:31-37.
  2. Zhuang, Y., Butler, B., Hawkins, E., Paguio, A., Orr, L., Wood, M., and Wood, K. (2001) A new age of enlightenment. Promega Notes. 79:6-11.
  3. Hori, K., Wampler, J., Matthews, J., and Cormier, M. (1973) Identification of the product excited states during the chemiluminescent and bioluminescent oxidation of Renilla (sea pansy) luciferin and certain of its analogs. Biochemistry. 12:4463-4468.
  4. Haddock, S., Moline, M., and Case, J. (2010) Bioluminescence in the sea. Ann. Rev. Mar. Sci. 2:293-343.
  5. Matthews, J., Hori, K., and Cormier, M. (1977) Purification and properties of Renilla reniformis luciferase. Biochemistry. 16:85-91.
  6. De Wet, J., Wood, K., Helsinki, D., and DeLuca, M. (1985) Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli. Proc. Natl. Acad. Sci. 82:7870-7873.
  7. De Wet, J., Wood, K., DeLuca, M., Helsinki, D., and Subramani, S. (1987) Firefly luciferase gene: structure and expression in mammalian cells. Mol. Cell. Biol. 7:725-737.
  8. Lorenz, W., McCann, R., Longiaru, M., and Cormier, M. (1991) Isolation and expression of a cDNA encoding Renilla reniformis luciferase. Proc. Nat. Acad. Sci. 88:4438-4442.
  9. Zhuang, Y., Butler, B., Hawkins, E., Paguio, A., Orr, L., Wood, M., and Wood, K. (2001) A new age of enlightenment. Promega Notes. 79:6-11.
  10. Rogers, S., Wells, R., and Rechsteiner, M. (1986) Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Science. 234:364-368.
  11. Loetscher, P., Pratt, G., and Rechsteiner, M. (1991) The C terminus of mouse ornithine decarboxylase confers rapid degradation on dihydrofolate reductase. Support for the PEST hypothesis. J. Biol. Chem. 266:11213-11220.
  12. Ghoda, L., Sidney, D., Macrae, M., and Coffino, P. (1992) Structural elements of ornithine decarboxylase required for intracellular degradation and polyamine-dependent regulation. Mol. Cell. Biol. 12:2178-2185.
  13. pGL4 Luciferase Reporter Vectors Technical Manual. #TM259. Promega Corporation.