The reporter protein's activity or fluorescence within a transfected cell population is approximately proportional to the steady-state mRNA level. A commonly used reporter gene is the luciferase gene from the firefly Photinus pyralis.
Including coenzyme A in the reaction enhances the sensitivity of the assay and provides a sustained light reaction. This bioluminescence directly corresponds with the effect of the protein on expression of the target gene. Examples of Bioluminescent Signal Measurements. When a protein of interest activates transcription, a luminometer reads a bright bioluminescent signal. However, if the protein represses transcription, the luminometer will detect no signal.
To perform a luciferase reporter assay, you will need a DNA plasmid to express the protein that you hypothesize could affect transcription. You will also need to use another DNA plasmid with the regulatory element or a target promoter region fused with the DNA coding sequence for a luciferase enzyme.
When activated, this system produces luciferase. After cells are transfected with the luciferase reporter plasmid and allowed to grow for few days, the next steps are to lyse the cells, add a substrate to the cell lysate, and measure the luciferase activity based on the amount of bioluminescent signal.
A Luciferase Reporter Assay. The luciferase assay is useful to study whether a protein of interest regulates a particular gene at the transcription level. Another DNA construct introduced into the cells consists of a coding region of the protein of interest. When this protein activates transcription, the cell will produce luciferase enzyme.
After the addition of a lysis buffer and a substrate, a luminometer quantifies the luciferase activity. If your protein activates the expression of the target gene, the amount of signal produced increases. However, if it blocks the gene expression, the cells produce less luciferase. As a result, these samples generate a lower signal than the positive control. When choosing a buffer to lyse the cells for the luciferase assay, use ingredients that are compatible with the bioluminescent reagents.
For Firefly luciferase reporter assays, use a luciferase lysis buffer , whereas for the Renilla luciferase reporter assays, choose a passive lysis buffer PLB. Email address is unverified. Account is locked. Password Incorrect password. Password reset is required. Account is invalid.
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Check your inbox to complete email verification. By holding the concentration of each reaction component constant, except for one component that varies in relation to the process being studied, the resulting light is directly proportional to the variable component. This couples an observable parameter to the reaction outcome. In assays using luciferase, the variable component may be the luciferase itself, its substrates or cofactors. Due to the very low backgrounds in bioluminescence, the proportional linear range can be enormous, typically extending 10 4 - to 10 8 -fold over the concentration of the variable component.
Intracellular luciferase is typically quantified by adding a buffered solution containing detergent to lyse the cells and a luciferase substrate to initiate the luminescent reaction. The luminescence slowly decays, decreasing the signal over time. To maintain steady luminescence of firefly and Renilla luciferase assays over an extended period of time, ranging from minutes to hours, luminescent reaction needs to be inhibited to various degrees.
Even under these conditions, as few as 10 —20 moles of luciferase or less per sample may be quantified. This corresponds to roughly 10 molecules per cell. These extended half-life assays are convenient for reporter gene applications. Simply add the reagent, and measure the resulting luminescence.
Most reporter gene assays involve either a one reporter gene or two. Most often, the second reporter is expressed from a "control" vector to normalize results of the experimental reporter. For example, the second reporter can control for variation in cell number or transfection efficiency. Typically, the control reporter gene is driven by a constitutive promoter, and the control vector is cotransfected with the "experimental" vector.
Different reporter genes are used for the control and experimental vectors so that the relative activities of the two reporter products can be assayed individually. Alternatively, you can design dual-reporter assays in which both reporter genes are used as experimental reporters.
Such dual-reporter assays can be particularly useful for efficiently extracting more information in a single experiment. The following section provides information about specific bioluminescent reporter assays with information to help you choose the reporter gene and assay that suit your research needs.
Tables 1 , 2 and 3 summarize available luciferase genes, assays and reagents. Assays based on a single reporter provide the quickest means to acquire gene expression data from cells. 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.
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