How does reporter gene work

He, M. Matta, H. Development and characterization of a novel luciferase based cytotoxicity assay. Scientific Reports, 8. Min, J. Journal of Clinical Medicine, 9 8 , Nakajima, Y.

Multicolor luciferase assay system: one-step monitoring of multiple gene expressions with a single substrate. BioTechniques, 38 6 , — Smale, S. Luciferase assay. Cold Spring Harbor Protocols, 5 , pdb. What is a Luciferase Reporter Assay? Some applications of luciferase reporter assays are: Gene expression analysis Promoter structure analysis SNPs analysis Antiviral research and therapy Cytotoxicity assay Drug discovery such as drugs targeting G-protein-coupled receptors How Does Luciferase Reporter Assay Work?

How to Choose a Cell Lysis Buffer for the Luciferase Assay When choosing a buffer to lyse the cells for the luciferase assay, use ingredients that are compatible with the bioluminescent reagents. How to Make Luciferin Solution Common substrates for the luciferase assay are luciferin and coelenterazine. Preparing a Coelenterazine Stock Solution 1. How is Luciferase Activity Measured?

Fireflies are able to emit light via a chemical reaction in which luciferin is converted to oxyluciferin by the luciferase enzyme. Some of the energy released by this reaction is in the form of light. This reaction is highly energetically efficient, meaning nearly all the energy put into the reaction is rapidly converted to light. This makes it extremely sensitive, which is great for a reporter assay! As an interesting aside… bioluminescence serves a variety of different purposes in nature.

Some of these functions include communication, finding a mating partner, finding food, camouflage, and self-defense. In insects, it is thought to play an additional role in oxygen detoxification, a function that presumably evolved more recently. But I digress… back to the assay…. There are several commercially available luciferase reporter assay kits comprising expression vectors that contain the luciferase reporter gene or a variation of it and the reagents necessary for the reaction to occur.

To perform the reporter assay, you clone the regulatory region of your gene-of-interest X upstream of the luciferase gene in one of these expression vectors, introduce that resulting vector DNA into cells, and let the cells grow for a period of time to allow transcription and translation to occur. You then collect the cells, break them open to release all the proteins including the luciferase , add luciferin and all the necessary cofactors, and measure the enzymatic activity using a luminometer an instrument that measures light emission from samples and gives you a quantitative reading.

Since X controls the expression of the luciferase reporter gene, the luciferase activity can be directly correlated with the activity of X.

However, because cells are inherently complex, the information gleaned from a single-reporter assay may be insufficient to achieve detailed and accurate results. Thus, one of the first considerations when choosing a reporter methodology is deciding if the information from a single reporter is sufficient or if you would benefit from the additional information that can be gleaned from a second reporter e.

If more information is required, see the section below that covers Dual-Reporter Assays. When choosing a luciferase assay, a trade-off between luminescence intensity and duration is often necessary because bright reactions fade relatively quickly.

Using a firefly or Renilla luciferase assay that yields maximum luminescence results in higher sensitivity, but using an assay with a longer signal half-life and a more stable luminescent signal is more convenient when performing assays in multiwell plates.

Figure 7. Example data that illustrates the luminescent signal stability of various firefly luciferase reporter assays. This allows you to grow cells in multiwell plates, and then measure reporter expression in a single step. Renilla luciferase assays with different signal intensities and half-lives are also available. N provides a simple, single-addition reagent that generates a glow-type signal with a half-life of approximately minutes in commonly used tissue culture media.

Measuring two reporters in a single assay is called a dual-reporter assay or, if both reporters are luciferases, a dual-luciferase assay. While the most commonly used dual-reporter assays measure both firefly and Renilla luciferase activities, the next-generation dual-luciferase assay uses NanoLuc and firefly luciferases. These pairs of luciferases use different substrates and thus can be differentiated by their enzymatic specificities.

Performing most dual-luciferase assays involves adding two reagents to each sample and measuring luminescence following each addition. Adding the first reagent activates the first luciferase reporter reaction; adding the second reagent extinguishes first luciferase reporter activity and initiates the second luciferase reaction.

E , which measures both firefly and Renilla luciferase activities sequentially from a single sample. This system requires cell lysis prior to performing the assay and requires the use of reagent injectors with multiwell plates. In general, dual-reporter assays improve experimental accuracy and efficiency by: i reducing variability that can obscure meaningful correlations; ii normalizing interfering phenomena that may be inherent in the experimental system; and iii normalizing differences in transfection efficiencies between samples.

In addition, dual-reporter assays can reduce the number of nonrelevant hits i. The use of co-incidence reporters—reporters that have dissimilar profiles of compound interference—can help differentiate compounds that modulate the biological pathway of interest from those that affect the stability or activity of the reporter enzyme.

Because cells are complex micro-environments, significant variability can occur between samples within an experiment and between experiments performed at different times. Challenges include maintaining uniform cell density and viability between samples and accomplishing reproducible transfection of exogenous DNA. The use of multiwell plates introduce variables such as edge effects, which are brought about by differences in heat distribution and humidity across a plate.

Dual-reporter assays can control for much of this variability, leading to more accurate and meaningful comparisons between samples Hawkins et al. Researchers strive to monitor cellular activities with as little effect on the cell as possible. Most reporter activity assays use an endpoint lytic method to disrupt cells so that the environment surrounding the reporter enzyme can be carefully controlled.

However, there are advantages to using a nonlytic assay to measure reporter gene activity, including continuous monitoring of expression changes over time and multiplexing with assays that assess cell health.

Promega scientists have developed a variety of live-cell substrates to monitor luciferase activity without disrupting cells. These live-cell detection reagents can provide kinetic measurements of reporter expression for investigating protein interaction and simplifying time course studies.

Renilla luciferase requires only oxygen and coelenterazine to generate luminescence, providing a simple luciferase system to measure luminescence from living cells. Because the cells are still alive, you can determine viable cell number by multiplexing with another assay.

An alternative to live-cell substrates is a secreted form of reporter protein, which can be quantified by measuring reporter activity in the cell culture medium. Promega reporter assays provide a wide range of choices for single or dual-reporter formats. The conventional use of reporter genes is largely to analyze gene expression and dissect the function of cis-acting genetic elements such as promoters and enhancers so-called "promoter bashing".

In typical experiments, deletions or mutations are made in a promoter region, and their effects on coupled expression of a reporter gene are quantitated. However, reporter genes also can be used to study other cellular events, including events that are not related to gene expression such as cell health and signaling pathways.

For more information, view the following Introduction to Bioluminescent Assays animation. Events associated with cell physiology can affect reporter gene expression. Of particular concern is the effect of cytotoxicity, which can mimic genetic downregulation when using a single-reporter assay.

Reporter assays that can be multiplexed with a cell viability or cytotoxicity assay for independent monitoring of both reporter expression and cell viability to avoid data misinterpretation Farfan et al. The use of multiplexed assays allows correlation of events within cells, such as the coupling of target suppression by RNAi, to the consequences on cellular physiology Hirose et al. G , use a stabilized firefly luciferase to generate a luminescent signal that indicate cell health.

Because these assays contain firefly luciferase, they cannot be directly combined with a firefly luciferase reporter assay. However, the assays can be readily combined with nondestructive luciferase assays.

Alternatively, you can multiplex a luminescent reporter assay with fluorescent cell viability and cytotoxicity assays to monitor cell health and normalize single-reporter assay results. G is a nonlytic, fluorescence assay that measures the relative number of viable cells in a population.

G uses a proprietary dye that is excluded from viable cells but preferentially binds to DNA from dead cells. Upon DNA binding, fluorescence of the dye is substantially enhanced, and the resulting fluorescence is proportional to the level of cytotoxicity. Bioluminescent reporters have been harnessed to study RNAi. E is based on dual-luciferase technology, with firefly luciferase as the primary reporter to monitor mRNA regulation and Renilla luciferase as a control reporter for normalization.

Reduced firefly luciferase expression indicates binding of endogenous or introduced miRNAs to the cloned miRNA target sequence. Luciferase reporter assays are widely used to investigate cellular signaling pathways and as high-throughput screening tools for drug discovery Brasier et al.

Synthetic constructs with cloned regulatory elements directing reporter gene expression can be used to monitor signal transduction and identify the signaling pathways involved.

By linking luciferase expression to specific response elements REs within the reporter construct, transfecting cells with this construct, adding a particular treatment, and then measuring reporter activity, researchers can determine what REs are used, and thus, what signaling pathways are involved. The use of inhibitors and siRNAs can be used to confirm what factors are involved in this response.

There are a variety of firefly luciferase pGL4 Vectors with your choice of a number of response elements and regulatory sequences for use in characterizing and modulating signaling pathways. See Table 1 for a complete list. Many of these vectors encode the hygromycin-resistance gene to allow selection of stably transfected cell lines. Bioluminescent reporter genes can also characterize nuclear receptors, a class of ligand-regulated transcription factors that sense the presence of steroids and other molecules inside the cell.

Nuclear receptors typically reside in the cytoplasm and are often complexed with associated regulatory proteins. Ligand binding triggers translocation into the nucleus, where the receptors bind specific response elements via the DNA-binding domain, leading to upregulation of the adjacent gene. Bioluminescent reporters can be harnessed to identify and characterize nuclear receptor agonists, antagonists, co-repressors and co-activators using a universal receptor assay.

The universal nuclear reporter assay can be thought of as a "one-hybrid" assay, where the ligand-binding domain LBD of a nuclear receptor is fused to yeast GAL4 transcription factor and when a ligand binds to the nuclear receptor, firefly luciferase is expressed Figure 8. Figure 8. The universal nuclear receptor assay. The ligand-binding domain of the nuclear receptor is fused to GAL4.

Within the cell, binding of the appropriate ligand to the nuclear receptor-GAL4 fusion protein releases any co-repressors bound to the ligand-binding domain. Co-activators help recruit the transcription machinery to the luciferase reporter gene, resulting in luciferase expression and an increase in luminescence. E that has multiple copies of the GAL4 upstream activation sequence UAS upstream of a minimal promoter to drive expression of the firefly luciferase reporter gene.

Two to three days post-transfection, treat cells with the test compounds of interest, then measure luciferase activity. This approach allows you to convert any cell line into a nuclear receptor-responsive cell line for identifying receptor agonists, antagonists, co-activators and co-repressors. You can even perform mutagenesis on the ligand-binding domain to determine the effect in your responsive cell line without interference from the endogenous receptor. To simplify universal nuclear receptor assays, there are additional reagents to use.

E for expressing a fusion protein comprised of the GAL4 DBD, a linker segment and an in-frame protein-coding sequence under the control of the human cytomegalovirus CMV immediate early promoter. Studying G protein-coupled receptors GPCRs , which regulate a wide-range of biological functions and are one of the most important target classes for drug discovery Klabunde et al. The assay uses genetically encoded biosensor variants comprised of cAMP-binding domains fused to mutant forms of Photinus pyralis luciferase.

Moreover, the assay offers a broad dynamic range, with up to fold changes in light output. The sensitive assay detects G i -coupled receptor activation or inverse agonist activity in the absence of artificial stimulation by compounds such as forskolin.

These cells use the destabilized and optimized luc2P gene for greater sensitivity and shorter induction times compared to native reporter enzymes. Non-native activators of these pathways, including GPCRs, can be studied after the appropriate proteins are introduced by transfection.

These cell lines, generated by clonal selection, provide very high reporter induction levels when the pathway of interest is activated. The rate of protein turnover is tightly regulated for many signaling proteins.

Protein stabilization and subsequent accumulation can occur in response to cell signaling events and changing cellular conditions and result in activation of downstream transcriptional events. N and Cat. N enable stability studies of two key signaling proteins, HIF1A and NRF2, and provide a method to directly measure the cellular effects of hypoxia and oxidative stress, respectively Robers et al. With a constitutive promoter like CMV, changes in light output correlate to dynamic changes in protein levels.

When the two fusion proteins interact, there is an energy transfer from the bioluminescent molecule to the fluorescent molecule, with a concomitant change from blue light to green light Angers et al.

Another way to detect protein interactions is to use a structural complementation reporter system. That is, when two subunits that are each fused to target proteins of interest and expressed in cells. When the two proteins interact, the subunits come together to form an active enzyme and generate a bright luminescent signal in the presence of substrate.

Luciferase reporter genes can be used as light-emitting reporters in cellular and animal models. Visualize reporter expression using live-cell luciferase substrates or secreted forms of luciferase for nondestructive, quantitative assays and multiple measures of the same samples without perturbation. Learn more about imaging.

We use these cookies to ensure our site functions securely and properly; they are necessary for our services to function and cannot be switched off in our systems. They are usually only set in response to actions made by you which amount to a request for services, such as logging in, using a shopping cart or filling in forms.

You can set your browser to block or alert you about these cookies, but some parts of our services will not work without them.

Like the other cookies we use, strictly necessary cookies may be either first-party cookies or third - party cookies. We use these cookies to remember your settings and preferences. For example, we may use these cookies to remember your language preferences.

Allow Preference Cookies. We use these cookies to collect information about how you interact with our services and to help us measure and improve them. Research Highlights 30 January Researchers have developed the first fully genetically encodable eukaryotic bioluminescent system. Advanced search. Skip to main content Thank you for visiting nature. Research 12 August Multiplexed direct detection of barcoded protein reporters on a nanopore array Reporter proteins are easily multiplexed using protein barcodes measured with nanopores.

Nature Biotechnology , Research 26 July A 4D single-cell protein atlas of transcription factors delineates spatiotemporal patterning during embryogenesis A protein expression atlas of transcription factors charted onto cell lineage maps of C aenorhabditis elegans development that uncovers mechanisms of spatiotemporal cell fate patterning and regulators of embryogenesis.