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Bioluminescence Reporters

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Chapter Sections

Introduction

Luciferase Genes and Vectors

Luciferase Reporter Assays and Protocols

References

All Chapters

Nucleic Acid Amplification

RNA Interference

Apoptosis

Cell Viability

Protein Expression

Expression Analysis

Cell Signaling

Bioluminescence Reporters

DNA Purification

Cell Imaging

Protein Purification and Analysis

Transfection

Cloning

DNA-Based Human Identification

Introduction

Genetic reporter systems have contributed greatly to the study of eukaryotic gene expression and regulation. Although reporter genes have played a significant role in numerous applications, both in vitro and in vivo, they are most frequently used as indicators of transcriptional activity in cells.

Typically, a reporter gene is joined to a promoter sequence in an expression vector that is transferred into cells. Following transfer, the cells are assayed for the presence of the reporter by directly measuring the reporter protein itself or the enzymatic activity of the reporter protein. An ideal reporter gene is one that is not endogenously expressed in the cell of interest and is amenable to assays that are sensitive, quantitative, rapid, easy, reproducible and safe.

Analysis of cis-acting transcriptional elements is a frequent application for reporter genes. Reporter vectors allow functional identification and characterization of promoter and enhancer elements because expression of the reporter correlates with transcriptional activity of the reporter gene. For these types of studies, promoter regions are cloned upstream or downstream of the gene. The promoter-gene fusion is introduced into cultured cells by standard transfection methods or into a germ cell to produce transgenic organisms. The use of reporter gene technology has allowed characterization of promoter and enhancer elements that regulate cell, tissue and development-defined gene expression.

Trans-acting factors can be assayed by co-transfer of the promoter-reporter gene fusion DNA with another cloned DNA expressing a trans-acting protein or RNA of interest. The protein could be a transcription factor that binds to the promoter region of interest cloned upstream of the reporter gene. For example, when tat protein is expressed from one vector in a transfected cell, the activity of different HIV-1 LTR sequences linked to a reporter gene increases and the activity increase is reflected in the increase of reporter gene protein activity.

Reporters can be assayed by detecting endogenous characteristics, such as enzymatic activity or spectrophotometric characteristics, or indirectly with antibody-based assays. In general, enzymatic assays are quite sensitive due to the small amount of reporter enzyme required to generate the products of the reaction. A potential limitation of enzymatic assays is high background if there is endogenous enzymatic activity in the cell (e.g., β-galactosidase). Antibody-based assays are generally less sensitive, but will detect the reporter protein whether it is enzymatically active or not.

Fundamentally, an assay is a means for translating a biomolecular effect into an observable parameter. While there are theoretically many strategies by which this can be achieved, in practice the reporter assays capable of delivering the speed, accuracy and sensitivity necessary for effective screening are based on photon production.

Luminescence versus Fluorescence

Photon production is realized primarily through fluorescence and chemiluminescence. Both processes yield photons as a consequence of energy transitions from excited-state molecular orbitals to lower energy orbitals. However, they differ in how the excited-state orbitals are created. In chemiluminescence, the excited states are the product of exothermic chemical reactions, whereas in fluorescence the excited states are created by absorption of light.

This distinction of how the excited states are created greatly affects the character of the photometric assay. For instance, fluorescence-based assays tend to be much brighter, since the photon used to create the excited states can be pumped into a sample at a very high rate. In chemiluminescence assays, the chemical reactions required to generate excited states usually proceed at a much lower rate, so yield a lower rate of photon emission. The greater brightness of fluorescence would appear to correlate with better assay sensitivity, but this is commonly not the case. Assay sensitivity is determined by a statistical analysis of signal relative to background or "noise", where the signal represents a sample measurement minus the background measurement. The limitation of fluorescence is that it tends to have much higher backgrounds, leading to lower relative signals.

The reason fluorescence assays have higher backgrounds is primarily because fluorometers must discriminate between the very high influx of photons into the sample and the much smaller emission of photons from the analytical fluorophores. This discrimination is accomplished largely by optical filtration, since emitted photons have longer wavelengths than excitation photons, and by geometry, since the emitted photons typically travel a different path than the excitation photons. But optical filters are not perfect in their ability to differentiate between wavelengths, and photons can also change directions through scattering. Chemiluminescence has the advantage that, since photons are not required to create the excited states, they do not constitute an inherent background when measuring photon efflux from a sample. The resulting low background permits precise measurement of very small changes in light.

Fluorescence assays can also be limited by the presence of interfering fluorophores within the samples. This is especially problematic in biological samples, which can be replete with a variety of heterocyclic compounds that fluoresce, typically in concentrations much above the analytical fluorophores of interest. The problem is minimized in simple samples, such as purified proteins. But in drug discovery, living cells are increasingly used for high-throughput screening. Unfortunately, cells are enormously complex in their chemical constitutions, which can exhibit substantial inherent fluorescence. Screening compound libraries is also inherently complex, since, although each assay sample may contain only one or a few compounds, the data set from which the drug leads are sifted is cumulated from many thousands of compounds. These compounds may also present problems with fluorescence interference, since drug-like molecules typically have heterocyclic structures.

For image analysis of microscopic structure, fluorescence is almost universally preferred over chemiluminescence. Brightness counts because the optics required for imaging cellular structures are relatively inefficient at light gathering. Thus the low background inherent in chemiluminescence is of little advantage since it is usually far below the detection capabilities of imaging devices. Furthermore, imaging is largely a matter of edge detection, which has different signal-to-noise characteristics than simply detecting the presence of an analyte. Edge detection relies heavily on signal strength and suffers less from uniform background noise.

But in macroscopic measurements (such as in a plate well) requiring accurate quantification with high sensitivity, chemiluminescent assays often outperform analogous assays based on fluorescence. Macroscopic measurements are the foundation for most high-throughput screening, which relies heavily on the use of multiwell plates, typically with 96-, 384- or 1536-wells, to measure a single parameter in a large number of samples as quickly as possible. Assays based on fluorescence or chemiluminescence can yield high sample throughput. However, fluorescence is more likely to be hindered by light contamination (from the excitation beam) or the chemical compositions of the samples and compound libraries. The use of chemiluminescence in high-throughput screening has been limited largely by the lack of available assay methods. Due to its long history, fluorescence has been more commonly used. But new capabilities in chemiluminescence, particularly in bioluminescence, are now adding new bioluminescence techniques to high-throughput screening.

Bioluminescence Reporters

Bioluminescence is a form of chemiluminescence that has developed through natural selection. Although most people are aware of bioluminescence primarily through the nighttime displays of fireflies, there are many distinct classes of bioluminescence derived through separate evolutionary histories. These classes are widely divergent in their chemical properties, yet they all undergo similar chemical reactions, namely the formation and destruction of a dioxetane structure. The classes are all based on the interaction of the enzyme luciferase with a luminescent substrate luciferin (Figure 8.1). The luciferases that have been used most widely in high-throughput screening are beetle luciferases (including firefly luciferase), Renilla luciferase and aequorin. The beetle luciferases are the most versatile of this group, and the number of new applications is rapidly expanding. Click beetle luciferases, which also belong to the beetle group, are becoming better known and offer a range of new luminescence color options. Renilla luciferase has been used primarily for reporter gene applications, although its use has also recently expanded. Aequorin has been used almost exclusively for monitoring intracellular calcium concentrations.

In nature, achieving efficient chemiluminescence is not a trivial matter, as evidenced by the lack of this phenomenon in daily life. The large energy transitions required for visible luminescence generally are disfavored over smaller ones that dissipate energy as heat, normally through interactions with surrounding molecules, especially water molecules in aqueous solutions. Because energy can be lost through these interactions, chemiluminescence depends strongly on environmental conditions. Thus chemiluminescence assays are often designed to incorporate hydrophobic compounds such as micelles to protect the excited state from water, or to rely on energy transfer to fluorophores that are less sensitive to solvent quenching. Another difficulty with chemiluminescence is efficient coupling of the reaction pathway to the creation of excited-state orbitals.

While chemiluminescence has relied on the ingenuity of chemists, bioluminescence has instead relied on the processes of natural evolution. As luminous organisms through the eons were selected by the brightness of their light, their luciferases have evolved both to maximize chemical couplings to generate the excited states and to protect the excited states from water. In firefly luciferase, the enzyme appears to exclude water by wrapping around the substrate, so that the excited-state reaction products are completely secluded. The enzyme structure shows two domains connected by a single polypeptide, which may act as a hinge. It is likely that the substrates bind between the domains, causing them to close like a lid onto a box. The enzyme would thus act as an insulator between the excited-state products and the environment around them. This strongly contrasts with synthetic forms of chemiluminescence, where the excited states are exposed to the solvent. In effect, a distinctive feature of bioluminescence is that the luciferase serves as a box that both generates and protects excited states.

Intracellular luciferase is typically quantified by adding a buffered solution containing detergent to lyse the cells and luciferase substrates to initiate the luminescent reaction. The luminescence will slowly decay due to side reactions, causing irreversible inactivation of the enzyme. The nature of these side reactions is not well understood, but they are probably due to the formation of damaging free radicals. To maintain a steady luminescence over an extended period of time, ranging from minutes to hours, it is often necessary to inhibit the luminescent reaction to various degrees. This reduces the rate of luminescence decay to the point that it will not interfere over the time required to measure multiple samples. However, even under these conditions the luciferase may be quantified to as few as 10^–20 moles per sample or less, which corresponds to roughly 10 molecules per cell. These assays are convenient for reporter gene applications because sample processing is not necessary prior to reagent addition. Simply add the reagent and read the resulting luminescence.

In a system where a second reporter is used, a "control" vector can be used to normalize for transfection efficiency or cell lysate recovery between treatments or transfection experiments. Typically, the control reporter gene is driven by a constitutive promoter and is cotransfected with "experimental" vectors. The experimental regulatory sequences are linked to a different reporter gene so that the relative activities of the two reporter gene products can be assayed individually. Control vectors can also be used to optimize transfection methods. Gene transfer efficiency is typically monitored by assaying reporter activity in cell lysates or by staining the cells in situ to estimate the percentage of cells expressing the transferred gene.

In general, bioluminescence reporters are preferred when experiments require high sensitivity, accurate quantitation or rapid analysis of multiple samples. Dual-bioluminescence assays can be particularly useful for efficiently extracting information.

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Luciferase Genes and Vectors

Biology and Enzymology

Bioluminescence as a natural phenomenon is widely experienced with amazement at the prospect of living organisms creating their own light. Basic research into this phenomenon has also led to practical applications, particularly in molecular biology where bioluminescence enzymes have been widely used as genetic reporters. Moreover, the value of this application has grown considerably over the past decade as the traditional use of reporter genes has broadened to cover wide ranging aspects of cell physiology.

The conventional use of reporter genes has been largely to analyze 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 consequential effects on coupled expression of a reporter gene are then quantitated. However, the broader aspect of gene expression entails much more than transcription alone, and reporter genes can also be used to study these other cellular events.

Some examples of analytical methodologies that use luciferase include:

• Stable cell lines that integrate the reporter gene of interest into the chromosome can be selected and propagated when a selectable marker is included in a transfection vector. These types of engineered cell lines have been used for drug screening and to monitor the effect of exogenous agents and stimuli upon gene expression.
• Identification of interacting pairs of proteins in vivo using a system known as the two-hybrid system (Fields et al. 1989). The interacting proteins of interest are brought together as fusion partners—one is fused with a specific DNA binding domain, and the other protein is fused with a transcriptional activation domain. The physical interaction of the two fusion partners is necessary for the functional activation of a reporter gene driven by a basal promoter and the DNA motif recognized by the DNA binding protein. This system was originally developed with yeast but has also been used in mammalian cells.
• Bioluminescence resonance energy transfer (BRET) for monitoring protein-protein interactions, where a fusion protein is made using the bioluminescent Renilla luciferase and another protein fused with a fluorescent molecule. Interaction of the two fusion proteins results in energy transfer from the bioluminescent molecule to the fluorescent molecule, with a concomitant change from blue light to green light (Angers et al. 2000).

Luciferase genes have been cloned from bacteria, beetles (e.g., firefly and click beetle), Renilla, Aequorea, Vargula and Gonyaulax (a dinoflagellate). Of these, only the luciferases from bacteria, beetles and Renilla have found general use as indicators of gene expression. Bacterial luciferase, although the first luciferase to be used as a reporter, is generally used to provide autonomous luminescence in bacterial systems through expression of the lux operon. Ordinarily it is not useful for analysis in mammalian cells.

Firefly Luciferase

Firefly luciferase is by far the most commonly used bioluminescent reporter. This monomeric enzyme of 61kDa catalyzes a two-step oxidation reaction to yield light, usually in the green to yellow region, typically 550–570nm (Figure 8.1). The first step is activation of the luciferyl carboxylate by ATP to yield a reactive mixed anhydride. In the second step, this activated intermediate reacts with oxygen to create a transient dioxetane that breaks down to the oxidized products, oxyluciferin and CO₂. Upon mixing with substrates, firefly luciferase produces an initial burst of light that decays over about 15 seconds to a low level of sustained luminescence. This kinetic profile reflects the slow release of the enzymatic product, thus limiting catalytic turnover after the initial reaction (Figure 8.1).

Various strategies to generate a stable luminescence signal have been tried to make the assay more convenient for routine laboratory use. The most successful of these incorporates coenzyme A to yield maximal luminescence intensity that slowly decays over several minutes. The mechanism of action for coenzyme A in the luminescent reaction is unclear, although it probably stems from the evolutionary ancestry of firefly luciferase. The amino acid sequence of firefly luciferase is related to a diverse family of acyl-CoA synthetases. By analogy to the catalytic mechanism of these related enzymes, formation of a thiol ester between CoA and luciferin seems likely. An optimized assay containing coenzyme A generates relatively stable luminescence in less than 0.3 seconds with linearity to enzyme concentration over a 100-millionfold range. The assay sensitivity allows quantitation to fewer than 10^–20 moles of enzyme.

The popularity of native firefly luciferase as a genetic reporter is due both to the sensitivity and convenience of the enzyme assay and to the tight coupling of protein synthesis with enzyme activity. The gene encoding firefly luciferase, luc, is a monomer that does not require any post-translational modifications; it is available as a mature enzyme directly upon translation from its mRNA. It has been shown that catalytic competence is attained immediately after release from the ribosome. Hence, the luciferase assay provides a nearly instantaneous measure of total reporter expression in the cell.

Renilla Luciferase

Renilla luciferase is a 36kDa monomeric enzyme that catalyzes the oxidation of coelenterazine to yield coelenteramide and blue light of 480nm (Figure 8.1). The host organism, Renilla reniformis (sea pansy), is a coelenterate that creates bright green flashes upon tactile stimulation, apparently to ward off potential predators. The green light is created through association of the luciferase with a green fluorescent protein.

Although Renilla and Aequorea are both luminous coelenterates based on coelenterazine oxidation, and both have a green fluorescent protein, their respective luciferases are structurally unrelated. In particular, the Renilla luciferase does not require calcium in the luminescent reaction. The reporter gene for Renilla luciferase, Rluc, like that for firefly luciferase, is a cDNA. As a reporter molecule, Renilla luciferase provides many of the same benefits as firefly luciferase. Historically, the presence of nonenzymatic luminescence, termed autoluminescence, reduced assay sensitivity; however, improvements in assay chemistry have nearly eliminated this problem. In addition, the simplicity of the Renilla luciferase chemistry and, more recently, improvements to the luciferase substrate have enabled the quantitation of Renilla luciferase from living cells, in situ or in vivo.

Click Beetle Luciferase

Click beetle and firefly luciferase belong to the same beetle luciferase family. Hence, the size and enzymatic mechanism of click beetle luciferase are similar to those of firefly luciferase. What makes the click beetle unique is the variety of luminescence colors they emit. The Chroma-Luc™ genes were developed from naturally occurring luciferase genes of a luminous click beetle, Pyrophorus plagiophthalamus. Complementary DNAs cloned from the ventral light organ encode four luciferases capable of emitting luminescence ranging from green to orange (544–593nm). The Chroma-Luc™ luciferases were developed to generate luminescence colors as different as possible; a red luciferase (611nm) and two green luciferases (544nm each). These luciferase genes were codon optimized for mammalian cells and are nearly identical to one another, with a maximum of 8 amino acids difference between any two of these genes. The two green luciferases generate very similar luciferase proteins; however, one is maximally similar to (~98%) while the other is divergent from (~68%) the DNA sequence for the red luciferase. Experimental and control reporter genes and proteins within an experiment can therefore be almost identical. Under circumstances where genetic recombination is a concern, the divergent luciferase pair may also be useful.

Gene Optimization

An ideal genetic reporter should: i) express uniformly and optimally in the host cells; ii) only generate responses to the effectors that the assay intends to monitor (avoid anomalous expression); and iii) have a low intrinsic stability to quickly reflect the transcriptional dynamics. Despite the biology and enzymology of the native luciferases, they are not necessarily optimal as genetic reporters. In the past decade, we have made significant improvements in expression, reducing the risk of anomalous expression and destabilizing these reporters. The key strategies to achieve these improvements are described here.

Peroxisomal Targeting Site Removal for the Beetle luc Gene

Normally, in the firefly light organ, luciferase is located in specialized peroxisomes of the photocytic cells. When expressed in foreign hosts, a conserved translocation signal within the enzyme structure causes it to accumulate in peroxisomes and glyoxysomes. In moderate to high levels of expression, the peroxisomes typically become saturated with luciferase and much of the reporter is found in the cytoplasm. Localization to the peroxisomes, however, might interfere with normal cellular physiology in two ways. First, large amounts of a foreign protein in the peroxisomes could impair their normal function. Second, many other peroxisomal proteins use the same translocation signals, so saturation with luciferase implies competition for the import of other peroxisomal proteins. Peroxisomal and glyoxysomal location of luciferase may also interfere with the performance of the genetic reporter. For instance, the luciferase accumulation in the cell might be differentially affected if it is distributed into two different subcellular compartments.

The stability of luciferase in peroxisomes is not known, but could well be different than its stability in the cytosol. If so, expression of luciferase could be affected by changes in the distribution of luciferase between peroxisomes and the cytosol. Measurements of in vivo luminescence might also be affected, since the availability of ATP, O₂, and luciferin within peroxisomes is not known.

The peroxisomal translocation signal in firefly and click beetle luciferases has been identified as the C-terminal tripeptide sequence, -Ser-Lys-Leu. Removal of this sequence abolishes import into peroxisomes. However, the relative specific activity of this modified luciferase has not been determined. To develop an optimal cytoplasmic form of the luciferase gene, we followed two strategies: i) we designed a new C-terminal tripeptide sequence based on the available data to minimize peroxisomal import, -Gly-Lys-Thr; and ii) we applied random mutagenesis to the C-terminal region and selected brightly luminescent colonies of E. coli transformed with the mutagenized luciferase genes. From sequence data of these selected mutants, we chose a clone with the sequence -Ile-Ala-Val. Consistently, both modified luciferases yielded about 4- to 5-fold greater luminescence than the native enzyme when expressed in NIH/3T3 cells. We chose the luciferase containing an -Ile-Ala-Val sequence for the cytoplasmic form because it usually yielded slightly greater luminescence than the luciferase with -Gly-Lys-Thr. Renilla luciferase does not contain a targeting sequence and so is not affected by peroxisomal targeting.

Codon Optimization

Although redundancy in the genetic code allows amino acids to be encoded by multiple codons, different organisms favor some codons over others. The efficiency of protein translation in a non-native host cell can be substantially increased by adjusting the codon usage frequency but maintaining the same gene product. The native luciferase genes cloned from beetles (firefly or click beetle) or sea pansy (Renilla reniformis) use codons that are not optimal for expression in mammalian cells. Therefore, we systematically altered the codons to the preferred ones while removing inappropriate or unintended transcription regulatory sequences used in mammalian cells. As a result, a significant increase in luciferase expression levels was achieved, up to several hundredfold in some cases (Figures 8.2 and 8.3).

Cryptic Regulatory Sequence Removal

Anomalous expression is defined as departure from normal or expected levels of expression. The presence of cryptic regulatory sequences in the reporter gene may adversely effect transcription, resulting in anomalous expression of the reporter gene. Removal of these sequences has been shown to reduce the risk of anomalous expression. A cryptic regulatory sequence can be a transcription factor binding site and/or a promoter module (defined as two transcription factor binding sites separated by a spacer; Klingenhoff et al. 1999). Transcription factor binding sites located downstream from a promoter are believed to affect promoter activity. Additionally, it is not uncommon for an enhancer element to exert activity and result in elevated levels of DNA transcription in the absence of a promoter sequence or in the presence of transcription regulatory sequences to increase the basal levels of gene expression in the absence of a promoter sequence. Promoter modules can exhibit synergistic or antagonistic functions (Klingenhoff et al. 1999).

We removed these cryptic regulatory sequences in the luc genes without changing the encoded amino acids. In addition, sequences resembling splice sites, poly(A) addition sequences, Kozak sequence (translation start for mammalian cells), E. coli promoters or E. coli ribosome binding sites were also removed wherever possible. This process has led to a greatly reduced number of cryptic regulatory sequences (Figure 8.4) in the luc genes and therefore a reduced risk of anomalous transcription.

Degradation Signal Addition

When performing reporter assays, measurements are made on the total accumulated reporter protein within cells. This accumulation occurs over the intracellular lifetime of the reporter, which is determined by both protein and mRNA stability. If transcription is changing during this lifetime, then the resulting accumulation of reporter will reflect a collection of different transcriptional rates. The longer the lifetime, the greater the collection of different transcriptional rates pooled into the reporter assay. This pooling process has a "dampening effect" on the representation of transcriptional dynamics, making changes in the transcriptional rate more difficult to detect. This can be remedied by reducing the lifetime of the reporter, thus reducing the pooling of different transcriptional rates into each reporter measurement. The resulting improvement in reporter dynamics is applicable to both upregulation and downregulation of gene expression.

Ideally, the reporter lifetime would be reduced to zero, completely eliminating the pooling of different transcriptional rates in each assay measurement. Only the transcription rate at the instant of the assay would be represented by the accumulation of reporter protein within the cells. Unfortunately, a zero lifetime would also yield zero accumulation, and thus no reporter could be measured. A compromise must be reached since, as lifetime reduces, so does the amount of reporter available for detection in the assay. This is where the high sensitivity of luminescent assays is useful. Relative to other reporter technologies, the intracellular stability of luciferase reporters may be greatly reduced without losing measurable signals. Thus, the high sensitivity of luciferase assays permits greater dynamics in the luciferase reporters.

The speed by which a genetic reporter can respond to changes in the transcriptional rate is correlated to the stability of the reporter within cells. Highly stable reporters accumulate to greater levels in cells, but their concentrations change slowly with changes in transcription. Conversely, lower stability yields less accumulation but a much faster rate of response. To provide reporters designed to meet different experimental needs, families of luciferase genes have been developed yielding different intracellular stabilities. The genes conferring lower stabilities are referred to as the Rapid Response™ Reporters.

Beetle and Renilla luciferase reporters have an intrinsic protein half-life of ~3 hours. However, reporter response may still lag behind the underlying transcriptional events by several hours. To further improve reporter performance, we have developed destabilized luciferase reporters by genetically fusing a protein degradation sequence to the luciferase genes (Li et al. 1998). After evaluation of many degradation sequences for their effect on response rate and signal magnitude, two sequences were chosen, one composed of the PEST protein degradation sequence and a second composed of two protein (CL1 and PEST) degradation sequences. Due to their increased rate of degradation, these destabilized reporters respond faster and often display a greater magnitude of response to rapid transcriptional events and are therefore called the Rapid Response™ Reporters.

Vector Backbone Design

Vectors that are used to deliver the reporter gene to the host cells are also critical for the overall performance of the reporter assay. Cryptic regulatory sequences such as transcription factor binding sites and/or promoter modules found on the vector backbone could lead to high background and anomalous responses. This is a common issue for mammalian reporter vectors including our pGL3 Luciferase Reporter Vectors, which have recently been improved. We have extended our successful "cleaning" strategy for reporter genes to the entire pGL3 Vector backbone, removing cryptic regulatory sequences wherever possible, while maintaining functionality. Other modifications include a redesigned multiple cloning region to facilitate easy transfer of the DNA element of interest, removal of the f1 origin of replication and deletion of an intronic sequence. In addition, a synthetic poly(A) signal/transcriptional pause site was placed upstream of either the multiple cloning region (in promoterless vectors) or the HSV-TK, CMV or SV40 promoter (in promoter-containing vectors). This extensive effort resulted in the totally redesigned and unique vector backbone of the pGL4 Luciferase Reporter Vectors.

The pGL4 family of luciferase vectors incorporates a variety of features such as a choice of luciferases, Rapid Response™ versions, mammalian-selectable markers, basic vectors without promoters and promoter-containing control vectors (Figure 8.5).

By manipulating luciferase genes we’ve developed a series of optimized reporter genes featuring additional luminescence colors and improved codon usage, while deleting cryptic regulatory sequences such as transcription factor binding sites that could decrease protein expression in mammalian cells. The following section provides information about specific bioluminescence reporters and assays, including how to choose the correct reporter genes and vectors to suit your research needs.

Advantages of the pGL4 Luciferase Reporter Vectors

1. Improved sensitivity and biological relevance due to:
   • Increased reporter gene expression: Codon optimization of synthetic genes for mammalian expression
   • Reduced background and risk of expression artifacts: Removal of cryptic DNA regulatory elements and transcription factor binding sites
   • Improved temporal response: Rapid Response™ technology available using destabilized luciferase genes
2. Additional advantages include:
   • Flexible detection options: Choice of reporter genes
   • Easy transition from transient to stable cells: Choice of mammalian selectable markers
   • Easy transfer from one vector to another: Common multiple cloning site and a unique SfiI transfer scheme

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Luciferase Reporter Assays and Protocols

The challenge for designing bioluminescence assays is harnessing this efficient light-emitting chemistry into analytical methodologies. Most commonly this is done by holding the reaction component concentrations constant, except for one component that is allowed to vary in relation to a biomolecular process of interest. When the reaction is configured properly, the resultant light is directly proportional to the variable component, thus coupling an observable parameter to the reaction outcome. In assays using luciferase, the variable component may be the luciferase itself or its substrates or cofactors. Because of very low backgrounds in bioluminescence, the linear range of this proportionality can be enormous, typically extending 10⁴- to 10⁸-fold over the concentration of the variable component.

Choosing the assay appropriate for your research needs is assisted by the following considerations and Tables 8.1 and 8.2, showing available luciferase genes, assays and reagents.

Single-Reporter Assays

Assays based on a single reporter provide the quickest and least expensive means for acquiring gene expression data from cells. However, because cells are inherently complex, the quantity of information gleaned from a single-reporter assay may be insufficient for achieving detailed and accurate results. Thus one of the first considerations in choosing a reporter methodology is deciding whether the speed and depth of information from a single reporter is sufficient or whether a greater density of information is desired. If a greater density of information is required, see the Dual-Reporter (and dual-color) Assays, Section III.B.

Using an assay reagent that produces stable luminescence is more convenient when performing assays in multiwell plates. Unfortunately, because bright reactions fade relatively quickly, a trade-off is necessary between luminescence intensity and duration. The Bright-Glo™ Reagent is designed for firefly luciferase to yield maximal luminescence intensity and sufficient duration for analysis in a multiwell plate. The Steady-Glo^® Reagent provides even greater luminescence duration but with lower intensity. Both reagents are designed to work directly in culture medium for mammalian cells, so prior cell lysis is not necessary. This allows the user to grow cells in multiwell plates and then measure expression with a single step.

Dual-Reporter Assays

The most commonly used dual-reporter assay is based on combining the chemistries for firefly and Renilla luciferases. These luciferases use different substrates and thus can be differentiated by their enzymatic specificities. The method comprises adding two reagents to each sample, with a measurement of luminescence following each addition. Addition of the first reagent activates the firefly luciferase reaction; addition of the second reagent extinguishes firefly luciferase and initiates the Renilla luciferase reaction. The Dual-Luciferase^® Assay Reagent relies on cell lysis prior to performing the assay and thus requires the use of reagent injectors if used with multiwell plates. The Dual-Glo™ Reagent is optimized for multiwell plates, providing longer luminescence duration (in other words, a longer luciferase half-life). As with other reagents designed for use in multiwell plates, the Dual-Glo™ Assay works directly in the culture medium for mammalian cells without the prior cell lysis.

Generally the benefits of a dual assay can improve experimental 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.

Reducing Variability

Because cells are complex micro-environments, significant variability may occur between samples within an experiment and between experiments performed at different times. Challenges include trying to maintain uniform cell density and viability between samples and accomplishing reproducible transfection of exogenous DNA. Multiwell plates introduce variables such as edge effects, brought about by differences in heat capacity and humidity across a plate. Dual assays can control for much of this variability, leading to more accurate and meaningful comparisons between samples (Hawkins et al. 2002; Hannah et al. 1998; Wood, 1998; Faridi et al. 2003).

Dual-Color Assays

In some cases, researchers may prefer to activate both luciferase assays simultaneously by adding a single reagent. This reduces total assay volume and liquid handling requirements. The light emission of the two luciferases can be differentiated by the color of the luminescence. We have developed click beetle luciferases, which are related to firefly luciferase, to yield red and green luminescence. The structures of these luciferases are nearly identical, containing only a few amino acid substitutions necessary to create the different colors. This structural similarity means that both the control reporter and the experimental reporter are likely to respond similarly to biochemical changes within the cell, resulting in even more accurate normalization to the control.

The genes encoding these reporters, the Chroma-Luc™ genes, are codon optimized for mammalian cells. We developed two genes encoding the green-emitting reporter, one which is nearly identical to the gene encoding red luminescence, and one that is maximally divergent from it but that encodes the same protein. The divergent gene may be useful under circumstances where genetic recombination is a concern.

The Chroma-Glo™ Assay reagent is designed for use in multiwell plates. Its formulation supports optimal reaction kinetics for both reporters simultaneously, and it works directly in culture medium. Because color differentiation is required for the Chroma-Glo™ Assay, a luminometer capable of using colored optical filters is required. Since the light is transmitted through the optical filters, sensitivity relative to other assay methods is reduced. Both luciferases may be detectable using optical filters when the relative concentrations differ by approximately 100-fold. This is less than dual-luciferase assays using chemical differentiation, where the relative concentration may differ by over 1,000-fold.

Distinguishing among the Dual Assays

Dual-reporter and dual-color assays allow the user either to measure expression of two different reporter genes or one reporter gene and cell viability. In all cases, the assays allow both measurements to be made sequentially from each sample. Most dual assays are optimized for use in multiwell plates.

Live Cell Substrates

Researchers strive to monitor cellular activities with as little impact on the cell as possible. The endpoint of an experiment, however, sometimes requires complete disruption of the cells so that the environment surrounding the reporter enzyme can be carefully controlled. Recently, nondestructive live cell substrates were developed, which allow monitoring of Renilla luciferase without cell lysis. Renilla luciferase requires only oxygen and coelenterazine to generate luminescence, providing a simple luciferase system with which to measure luminescence from living cells. Unfortunately, coelenterazine is unstable in aqueous solutions and so it has been difficult and inconvenient to measure Renilla luciferase. EnduRen™ and ViviRen™ Live Cell Substrates have been designed to overcome this difficulty and to easily generate luminescence from live cells expressing Renilla luciferase. Because the Renilla luciferase luminescence is generated from living cells, these substrates are also ideal for multiplexing with assays that determine cell number.

Normalizing Interfering Phenomena

When making correlations between experimental conditions and the expression of a reporter gene, other events associated with cell physiology may affect reporter gene expression. Of particular concern is the effect of cytotoxicity, which can appear as genetic downregulation when using a single-reporter assay. Live cell assays that can be multiplexed with a cell viability assay allow independent monitoring of both reporter expression and cell viability to avoid misinterpretation of the data (Farfan et al. 2004). The use of multiplexed assays allow correlation of events within cells, such as the coupling of target suppression by RNAi to its consequences on cellular physiology (Hirose et al. 2002).

The CellTiter-Glo^® Luminescent Cell Viability Assay provides a rapid and sensitive assay of cell viability based on luminescent detection of cellular ATP. Because the CellTiter-Glo^® Assay uses a stabilized firefly luciferase in its formulation, it cannot be directly combined with a reporter assay for expression of firefly luciferase. However, it can be readily combined with nondestructive reporter assays of Renilla luciferase.

Expression of Renilla luciferase may be quantitated, or continuously monitored, by adding EnduRen™ Substrate to the culture medium. When reporter measurements are completed, CellTiter-Glo^® Reagent is added to the sample to inactivate the Renilla luminescence and initiate the ATP-dependent luminescence. Because the CellTiter-Glo^® Assay is an endpoint assay, further sample monitoring after measuring cell viability is not possible.

Luciferase Reporter Cell Lines

Luciferase reporter assays have been widely used to investigate cellular signaling pathways and as high-throughput screening tools for drug discovery (Brasier et al. 1992, Zhuang et al. 2006) The GloResponse™ Cell Lines contain optimized, state-of-the-art luciferase reporter technology integrated into a cell line. The GloResponse™ NFAT-RE-luc2P HEK293 Cell Line and CRE-luc2P HEK293 Cell Line are designed for rapid and convenient analysis of cell signaling through the nuclear factor of activated T-cells (NFAT) pathway or cAMP response pathways via activation of a luciferase reporter gene. Activity of non-native activators of these pathways (including GPCRs) can be studied after they have been introduced by transfection.

The GPCR assays configured using the GloResponse™ Cell Lines are amenable for high-throughput screening. These assays typically have greater response dynamics (fold of induction) than other assay formats and good quality as indicated by the high Z´ values (Zhang et al. 1999).

GPCRs regulate a wide-range of biological functions and are one of the most important target classes for drug discovery (Klabunde et al. 2002). GPCR signaling pathways can be categorized into three classes based on the G protein α-subunit involved: Gs, Gi/o and Gq. The GloResponse™ CRE-luc2P HEK293 Cell Line can be used to study and configure screening assays for Gs- and Gi/o-coupled GPCRs, which signal through cAMP and CRE. For Gq-coupled GPCRs, which signal through calcium ion and NFAT-RE, the GloResponse™ NFAT-RE-luc2P HEK293 Cell Line should be used.

The GloResponse™ Cell Lines were generated by clonal selection of HEK293 cells stably transfected with pGL4-based vectors carrying specific response elements for the pathway of interest. These cell lines incorporate the improvements developed for the pGL4 family of reporter vectors for enhanced performance. The destabilized luc2P luciferase reporter is used for improved responsiveness to transcriptional dynamics. The luc2P gene is codon optimized for enhanced expression in mammalian cells, and the pGL4 plasmid backbone has been engineered to reduce background reporter expression. The result is a cell line with very high induction levels when the pathway of interest is activated.

Selecting a Reporter Gene and Assay: Tables 8.1–8.3

The tables in this section are designed to show the varous features of reporter vectors (Tables 8.1 and 8.2), including the reporter gene, whether the vector contains a multiple cloning region or not, what gene promoter and protein degradation sequence the vector has, and it's mammalian selectable marker, as well as the features of our reporter assays (Table 8.3). In addition to the tables, the following tools will help in your choice of pGL4 Vector or a reporter assay.

For a step-by-step guide to help you choose the best pGL4 Vector for your studies use the pGL4 Vector Selector. To go to the tool, click the link, then select the "Solution Finder" tab, and choose "pGL4 Vector Selector".

The Introduction to Reporter Gene Assays animation demonstrates the basic design of a reporter gene assay using the Dual-Luciferase^® Assay to study promoter structure, gene regulation and signaling pathways.

¹Do not use this product/reagent with automated reagent injectors, available on certain luminometers.

²Use with plate assays only when luminometer has a reagent injector.

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References

1. Angers, S. et al. (2000) Detection of β2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc. Natl. Acad. Sci. USA 97, 3684–9.
2. Farfan, A. et al. (2004) Multiplexing homogeneous cell-based assays Cell Notes 10, 15–8.
3. Faridi, J. et al. (2003) Expression of constitutively active Akt-3 in MCF-7 breast cancer cells reverses the estrogen and tamoxifen responsivity of these cells in vivo. Clin. Can. Res. 9, 2933–9.
4. Fields, S. et al. (1989) A novel genetic system to detect protein-protein interactions. Nature 340, 245–6.
5. Hannah, R. et al. (1998) Rapid luciferase reporter assay systems for high-throughput studies. Promega Notes 65, 9–14.
6. Hawkins, E.H. et al. (2002) Dual-Glo™ Luciferase Assay System: Convenient dual-reporter measurements in 96- and 384-well plates. Promega Notes 81, 22–6.
7. Hirose, F. et al. (2002) Drosophila Mi-2 negatively regulates dDREF by inhibiting its DNA-binding activity. Mol. Cell. Biol. 22, 5182–93.
8. Klingenhoff, A. et al. (1999) Functional promoter modules can be detected by formal models independent of overall nucleotide sequence similarity. Bioinformatics 15, 180–6.
9. Li, X. et al. (1998) Generation of destabilized green fluorescent protein as a transcription reporter. J. Biol. Chem. 273, 34970–5.
10. Wood, K.V. (1998) The chemistry of bioluminescent reporter assays. Promega Notes 65, 14–20.

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CellTiter-Glo, Dual-Luciferase, Steady-Glo and TNT are registered trademarks of Promega Corporation. Bright-Glo, Chroma-Glo, Chroma-Luc, Dual-Glo, EnduRen, GloResponse, Rapid Response and ViviRen are trademarks of Promega Corporation.

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