Active Motif,
Tools to analyze nuclear function,
Your CartYour Cart 0 items
    
Functional Genomics - LightSwitch™

LightSwitch™ Reporter System

With over 30,000 regulatory elements that have been cloned from the human genome, our LightSwitch™ Luciferase Assay System is a complete solution for performing gene regulation studies in living mammalian cells. In addition to the product details listed below, brief summaries of each part of the LightSwitch System are listed below under the 'LightSwitch Luciferase Assay Overview' tab.

Searching the GoClone® Collections

        

LightSwitch™ Promoter & Distal Element Collection


LightSwitch™ Promoter Reprter GoClone Collection
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.


LightSwitch™ lncRNA Promoter Products
lncRNA Promoter Reporter Collection

The lncRNA Promoter Reporter Collection was designed to make it
fast & easy to study
the regulation of
lncRNA promoters.
 


LightSwitch™ Promoter Controls
Promoter Reporter GoClone Controls

The LightSwitch System contains many positive and negative Promoter Controls to optimize experimental conditions and normalize for non-specific effects.


LightSwitch™ Synthetic Response Elements
Synthetic Response Element Collection

The Synthetic Response Elements are useful positive controls due to their stronger response compared to endogenous promoter and enhancer sequences.


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

These validated stable cell lines enables the fast and easy quantification of pathway activity in response to various treatments and growth conditions.


LightSwitch™ Validated Pathway Collections
Validated Pathway Biomarker Kits

Pathway Promoters that were selected based on sequence motif analysis & published data. They show significant activation or repression in response to pathway-specific induction.

LightSwitch™ miRNA & 3´UTR Collection


LightSwitch™ 3´UTR Reporter GoClone Collection
3´UTR Reporter GoClone Collection

Our 3´UTR Reporter Collection includes
over 12,000 3´UTRs
cloned from the
human genome into
ready-to-transfect LightSwitch™ reporter vectors.


ChIP Normalization
Synthetic
miRNA Targets

The LightSwitch Synthetic miRNA Target Reporters are useful positive controls due to their strong response compared to endogenous 3`UTRs sequences.


LightSwitch™ 3´UTR Control Vectors
3´UTR Reporter GoClone Controls

The LightSwitch System contains a number of positive and negative 3´UTR Controls to optimize experimental conditions and
normalize for
non-specific effects


LightSwitch™ miRNA Mimics & Inhibitors
miRNA Mimics & Inhibitors

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™ System Companion / Assay Reagents


LightSwitch™ Luciferase Assay Kit

Luciferase Assay Kit

The novel substrate and optimized lysis buffer included in the Luciferase Assay Kit were formulated to provide high sensitivity over a broad dynamic range when used with our engineered RenSP Luciferase.


LightSwitch™ Dual Assay Kit

Dual Assay Kit

Intended for those experiments that utilize co-transfection for normalization, although our unique RenSP luciferase technology eliminates the need for co-transfection of a normalizing control in most cases.


LightSwitch™ Empty Vector Suite
LightSwitch™ Empty
Vector Suite

Complete list of all LightSwitch™ vector options together with sequence and annotation files and insert sequencing primers.


DharmaFECT® Duo Transfection Reagent
DharmaFECT Duo
Transfection Reagent

For co-transfecting plasmids and miRNAs, DharmaFECT DUO provides more efficient co-transfection of these two distinct types of molecules.


FuGENE® Transfection Reagent
Fugene HD Promoter
Transfection Reagent

For plasmid transfections and co-transfections, FugeneHD provides consistent, high-quality performance across a wide variety of cell lines.

 

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.