Gene reporter assays

Gene reporter assays are widely used by researchers to investigate gene expression and regulation. They find many uses in the life sciences from studying promoters and enhancers to screening for drugs or investigating signal transduction pathways.

Gene reporter techniques rely on the use of a reporter gene typically placed downstream of a regulatory sequence. If the regulatory sequence is activated or repressed the reporter gene is expressed at different levels and the product can be measured quantitatively using different detection techniques. 

In this blog, we look at the different types of gene reporter assays, provide examples of applications, and look at some of the benefits of using microplate readers for this type of measurement.

First steps towards gene reporter assays

The lacZ reporter assay was first developed in 1972 ¹ and went on to become one of the most universally used reporters of gene expression in the life sciences. Its origins depended on earlier findings that revealed the arrangement and role of crucial genes for the expression of the lacZ gene. In 1961, François Jacob and Jacques Monod published a paper describing the discovery of the lac operon in Escherichia coli (Fig.1). ² The study provided the first insights into how gene expression is regulated in bacterial cells and laid the foundation for understanding gene regulation in all organisms. In 1965, Jacob and Monod were awarded the Nobel Prize in Physiology or Medicine together with André Lwoff “for their discoveries concerning genetic control of enzyme and virus synthesis.” The proof of principle of lacZ gene and β-galactosidase as a reporter assay led to the development of further gene reporter assays where genes encoding easily measurable proteins (like green fluorescent protein and luciferase) are placed under the control of a promoter or regulatory element of interest.

Green fluorescent protein

Green fluorescent protein was first reported in the early 1960s, but it was not until 1994 that it was outlined as a marker for gene expression. ³ A complementary DNA for the Aequorea victoria green fluorescent protein was demonstrated to produce a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Unlike other gene reporter systems (β-galactosidase, firefly luciferase), exogenous substrates and cofactors are not required for this detection output which make it ideally suited to measuring gene expression and protein localization in living cells.

Luciferase gene reporter assays

The first report of using firefly luciferase as a reporter gene was published in 1987 by de Wet et al.⁴ In this study, the authors described the structure and expression of the firefly luciferase gene in mammalian cells and revealed how it could be used as a reporter gene. The luciferase assay became a significant addition to the array of genes that were available to monitor promotor activity and offered major improvements in sensitivity. The firefly luciferase assay was estimated to be 30- to 1000-fold more sensitive than chloramphenicol acetyltransferase assays which were widely in use at the time.

Dual reporter bioluminescent assays soon followed. ⁵ These are based on the use of two different luciferase reporters such as for example the Dual-Luciferase^® Reporter Assay System developed in 1996, which sequentially measures the activities of firefly and Renilla luciferases from a single sample. The assay provides rapid quantitation of both reporters either in transfected cells or in cell-free transcription/translation reactions. One reporter can readily be used for experimental measurements of the gene under study while the other functions as a control reporter. The control reporter serves to normalize the experimental data accounting for variations in transfection efficiency and cell viability. Dual reporter bioluminescent gene reporter assays continue to have many applications in the life sciences today and are a stalwart of many experiments looking at gene expression in biological systems including cell-based assays.

Today it is known that many other bacterial species control different processes with LuxI-LuxR-like systems. This includes for example activities like gene conjugation, exoenzyme production, and antibiotic synthesis.² 

Applications of gene reporter assays

The use of gene reporter assays has grown exponentially in the life sciences in recent years. They find many applications in molecular biology as well as applied research relevant to drug discovery. 

Gene reporter assays may be used in the environmental sciences in the development of biosensors for the detection of contaminants, pathogens or toxins. They also find uses in a host of cell-based assays and quality control processes. In virology gene reporter assays can be used to study how viruses infect cells and how viral and cellular genes are expressed after infection. In biotechnology and microbiology, gene reporter assays serve as fundamental tools to provide real-time quantitative data on gene activity and the control of biological processes.

As mentioned earlier, researchers often use gene reporter assays to study the function of promoters and enhancers. For example, gene reporter assays can serve as important indicators of what might be the most effective promoter to drive high level gene expression. In genome editing, they can help verify the success of CRISPR-Cas9 mediated modifications or the efficiency of other gene editing protocols. In the development of gene therapies, gene reporter assays can be used to assess how effectively a gene is delivered to a target cell or how well it is expressed in vitro or in vivo. 

In addition, gene reporter assays are routinely used in high throughput drug screening of small molecules, proteins or other compounds. In this context, they can help to identify molecules that affect gene expression and provide information on the impact of these compounds on genes linked to disease. 

Gene reporter assays also offer ways to analyse signal transduction pathways by looking at how external signals like hormones or cytokines influence gene expression. They also find many uses in functional genomics (gene identification) and protein interaction studies. An overview of the different types of gene reporter assays is given in Table 1.

Table 1: Overview of different types of gene reporters

* In dual luciferase assays, a second reporter is often expressed from a “control” vector to normalize the results of the experimental reporter.

BMG solutions for gene reporter assays

Today researchers have several detection options for gene reporter assays. Fluorescence intensity and luminescence assays are most commonly used, each with distinct benefits. Colorimetric assays, for example for β-galactosidase activity measurements, are also used and depend in most cases on absorbance detection.

Here we briefly consider the different detection methods for gene reporter assays on microplates and highlight distinctive technological features of BMG LABTECH solutions that facilitate these applications.

Fluorescence

Fluorescence intensity (FI) measurements are widely used to study gene expression in biological systems including live cells and reconstituted systems. They offer many different solutions that can be readily adapted for the applications mentioned in the previous section. For example, cells are often transfected with exogenous DNA to study the regulation of gene and protein expression. To monitor transfection efficiency, a reporter gene is often attached to the gene of interest as a tag to keep track of insertion of the gene into a cell’s genome. A portfolio of fluorescent protein reporters is available for these types of measurements including for example green fluorescent protein, red fluorescent protein, or yellow fluorescent proteins. 

Well-scanning capabilities
In the application note Transfection efficiency determined with fluorescence-based bottom reading for GFP and mcherry, gene reporters are readily used to measure the success of transfection experiments (Fig. 2). Several features of BMG LABTECH microplate readers are highlighted in this application note including well scanning that facilitates these types of assays. Both matrix scanning and spiral averaging help deliver accurate results. Spiral averaging has advantages for the speed of measurements while matrix scans provide a high level of local resolution in reporter-gene-expressing cells. Furthermore, the use of red-shifted dyes such as mCherry to determine transfection efficiency helps circumvent autofluorescence that can arise from media and cell-derived components. The bottom reading capabilities of BMG LABTECH microplate readers deliver considerable benefits in measuring the fluorescence signal of these cell-based assays for the same reasons. In these experiments, BMG LABTECH microplate readers reliably detected cells expressing a fluorescent marker down to around 600 cells/well in a 96-well plate and represent a valuable alternative to the use of microscopes to monitor transfection efficiency.

The application note Studying the molecular mechanism of viral replication in real time using the CLARIOstar Plus with ACU describes a fluorescent-based gene reporter approach for near real-time measurements of viral gene expression. The assay involves expression of the red fluorescent mCherry protein which serves as a proxy for viral gene expression and can be measured at regular intervals throughout viral infection. The atmospheric control unit permitted the incubation of the cells used in the study at 37^oC and 5% CO₂.

The CLARIOstar Plus provides a further example of how the expression of red fluorescent proteins can be used to monitor viral replication in cells. In the application note Antiviral assay based on expression of fluorescent proteins by the viruses replication of respiratory syncytial virus (RSV) results in the expression of a monomeric red fluorescent protein mKate2. Use of the well-scan mode in the microplate reader allowed for detection of the signal over the entire well. The results were comparable to data obtained from the use of an imager.

Other fluorescence technologies can also be used in gene reporter assays. Fluorescence resonance energy transfer (FRET) for example can be applied to measure the signals from pairs of fluorescent proteins. FRET offers advantages for studies that involve close-range molecular interactions, for example certain protein-protein interactions or measurements looking at conformational changes. Traditional fluorescence is better suited for measuring overall gene expression or localizing a single protein but cannot provide the interaction-based insights that techniques like FRET offer.

One example of using FRET for reporter gene applications is GeneBlazer^®. GeneBlazer is often used for cell-based target validation, pathway analysis and compound screening in drug discovery. It can be used to look at surface and intracellular reporters (such as nuclear and cytokine receptors, orphan and known G-protein coupled receptors), a wide range of signal transduction pathways, ion channels, other transporters, and transcriptional regulators (including kinases and intracellular processes). You can read more about the uses of GeneBlazer^® in the BMG LABTECH blog GeneBLAzer technology overview which provides access to further information resources for this technology.

Luminescence

Luminescence offers several distinct advantages for gene reporter assays that often make it a preferred choice in many applications. It offers high sensitivity, lower background signals, and is less prone to experimental artefacts like autofluorescence and photobleaching. These features make it ideal for high sensitivity detection and high-throughput gene reporter assays. 

Fluorescence may be the go-to detection technology where multi-colour assays are required but luminescence offers superior performance for quantitative measurements of gene expression or protein activity. In addition, luminescent assays are often easier to set up since they do not require choices of sometimes complex optics or filter selection. Cells, media and plastics do not produce luminescence which avoids the problems associated with autofluorescence in fluorescent-based gene reporter assays.

Simultaneous dual emission
In the application note Simultaneous dual emission detection of luciferase reporter assays BMG LABTECH microplate readers were used to perform dual-luciferase assays. Spectral resolution allowed detection of two luciferases in one sample. Light from both reporters was measured either using the simultaneous dual emission option which uses two photomultiplier tubes or could be measured sequentially one emission after the other (Fig. 3).

Cross-talk reduction

Cross-talk reduction can help reduce unwanted effects in luminescence measurements if light strays into different wells (Fig. 4). The application note Establishing luminescence-based assays: miniaturised experiments help to optimise results demonstrates how BMG LABTECH’s automatic cross-talk reduction can significantly reduce luminescence cross-talk effects. In addition, the choice of the right microplate can affect the quality of the results. White microplates provide the best signal output for luminescence assays but cross-talk may increase when they are used. It is therefore beneficial to have a microplate reader that can automatically minimize these effects. This can include selecting the right layout of samples for measurement in the microplate to prevent carry-over of signal between neighboring wells. In certain cases, it may be useful to use grey or black plates which will mean less signal intensity but will also reduce the contribution of cross-talk to measurements in different wells.

Colorimetric assays

Colorimetric gene reporter assays work by means of reporter genes that encode for enzymes that convert substrates into colored products. Examples include β-galactosidase, alkaline phosphatase, or β-glucuronidase. These types of assays are relatively easy to use, inexpensive and suitable for large-scale screening. They may deploy absorbance or fluorescence for detection and can be used to screen transgenic plants or animals, to study gene regulation, or quantify gene expression.

Gene reporter assays on microplate readers

Overall, microplate readers offer several key advantages for the performance of gene reporter assays (Table 2). In addition to sensitivity and reliability, they offer high-throughput capabilities and versatility for many different applications. 

Table 2: Summary of advantages of using microplate readers for gene reporter assays

Dual gene reporter assays

Over the years, BMG LABTECH’s microplate readers have demonstrated their capabilities to perform many of the gene reporter assays chosen by scientists working in laboratories around the globe. The application note Dual Luciferase Reporter (DLR) assay certification gives some examples of assays validated for the PHERAstar^® FSX, CLARIOstar^® and Omega series readers.

As mentioned earlier, dual luciferase reporter assays are at the heart of many assays for studying gene expression in the life sciences. Many of these assays have tangible applications in looking for ways to tackle disease. Hepatitis C, to take one example, is a global health challenge affecting an estimated 50 million people worldwide (World Health Organization). In the application note Dual luciferase assay to assess the replication of the hepatitis C virus subgenomic replicon, the early stages of viral replication were successfully monitored to test the efficacy of potential inhibitors. The effect of inhibitors could simultaneously be investigated on the RNA replication of the virus as well as overall translation in the cell. The assays were performed on 96-well plates which allowed for different concentrations of inhibitors to be simultaneously tested against cells derived from a single electroporation event (Figures 5 and 6).

Looking for a strong promoter for Physcomitrella patens demonstrates the use of a novel system that uses fluorescence gene reporter assays to probe the strength of different gene promoters. This type of assay is useful for example to develop effective screening methods for new inhibitors of gene expression. Green fluorescent protein and mCherry were used as reporter proteins to research promoter strength in living protoplasts in a 96-well format (Fig. 7). The LVF monochromator of the CLARIOstar allowed for detection of less than 100 protoplasts per well. This type of assay can be used as a standard method for screening transiently transfected protoplasts.

Multiplexed assays for drug discovery

Genotoxicity is an undesirable effect of certain substances where damage occurs to the genetic material within a cell. In drug development and regulatory assessment, genotoxicity testing helps identify potential risks of DNA damage that could lead to cancer or reproductive issues and helps to ensure the safety of new drugs before they are launched. The application note BlueScreen HC - a luminescence based, high-throughput, in vitro genotoxicity assay describes an example of this type of approach for a commercially available test that can be readily performed on a microplate reader. Specifically, the test makes use of a luminescence-based reporter system that exploits the proper regulation of the GADD45a gene (Fig. 8).

Genotoxicity and cytotoxicity were measured simultaneously using flash luminescence, absorbance and fluorescence on BMG LABTECH multi-mode readers and made use of reagent injection systems. The assays are suitable for testing undesirable properties of pharmaceuticals, industrial chemicals and personal care products. Table 3 provides an overview of some of the application notes covering gene reporter assays that are available on the BMG LABTECH website.

Table 3. Overview of some BMG LABTECH application notes for gene reporter assays.

Future advances in gene reporter assays

The number of studies using gene reporter assays on BMG LABTECH microplate readers continues to grow at pace. Future advances in gene reporter assays for microplate readers will proceed on several fronts. In fluorescence detection, there has been a move towards using red fluorophores like mCherry to circumvent autofluorescence from media and cell-derived components. Many gene reporter assays are performed in cell-based systems which are attractive due to their physiological relevance. The repertoire of cell-based assays is likely to grow significantly in the years ahead. Increasingly, researchers like to perform multiplex assays where they can analyze multiple reporters in one run. Multi-mode readers facilitate the use of different detection technologies in parallel in these types of assays. The use of multiplex assays is a crucial driver in the development and use of methods compatible with high-throughput screening. More advanced detection technologies and innovation in specific gene reporter assays should translate into further benefits for researchers.

Whatever your requirements in gene reporter assays, BMG LABTECH has the microplate reader for your applications.

The PHERAstar FSX was specifically conceived for screening campaigns and is your go-to reader for the fastest high-performance high-throughput screenings.

Both the VANTAstar^® and CLARIOstar Plus allow for wavelength flexibility and include Enhanced Dynamic Range technology for superior performance in a single run. They also offer increased light transmission and sensitivity courtesy of Linear Variable Filter Monochromators^TM and different filter options.

All BMG LABTECH microplate readers have exceptionally fast reading capabilities. In addition, the Omega series, CLARIOstarPlus,  and PHERAstar FSX microplate readers can be equipped with on-board injectors that can offer the very best options for detection at the time of injection.

Collectively, BMG LABTECH multi-mode readers combine high-quality measurements with miniaturised assays, short measurement times, and offer considerable savings on materials and other resources.

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3. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. Green fluorescent protein as a marker for gene expression. Science. 1994 Feb 11;263(5148):802-5. doi: 10.1126/science.8303295. 
4. de Wet JR, Wood KV, DeLuca M, Helinski DR, Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725-37. doi: 10.1128/mcb.7.2.725-737.1987. 
5. Promega, Technical Manual, Dual-Luciferase^® Reporter Assay System, 1996-2023 Promega Corporation Dual-Luciferase^® Reporter Assay System Protocol (promega.com)