Bioluminescent Reporters

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, 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 protein 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 allows 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 or by activating the trans-acting factors through a treatment of the samples. 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 an increase of reporter gene protein activity.

Reporter proteins can be assayed by detecting inherent 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 detectable levels of reaction products. 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, a reporter assay is a means to translate 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 photons 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 and yield a lower rate of photon emission. The greater brightness of fluorescence would appear to correlate with better assay sensitivity, but commonly this is 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. Fluorescent assays tend to have much higher backgrounds, leading to lower relative signals.

Fluorescent assays have higher backgrounds 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 emitted photons typically travel a different path than excitation photons. However, optical filters are not perfect in their ability to differentiate between wavelengths, and photons can 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 also can be limited by 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 for drug discovery, living cells are increasingly used in high-throughput screening. Unfortunately, cells are enormously complex in their chemical constitutions and can exhibit substantial inherent fluorescence. Screening compound libraries also is inherently complex; although each 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 also may 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 to image cellular structures are relatively inefficient at light gathering. Thus the low background inherent in chemiluminescence is of little advantage, since the signal 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 an analyte. Edge detection relies heavily on signal strength and suffers less from uniform background noise.

In macroscopic measurements (such as in a plate well), which require accurate quantification with high sensitivity, chemiluminescent assays often outperform analogous fluorescence-based assays. 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 provide high sample throughput. However, fluorescence is more likely to be hindered by light contamination (from the excitation beam) or the chemical compositions of 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 allowing new bioluminescent techniques for high-throughput screening.

Bioluminescent Reporters

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, chemiluminescent assays often incorporate hydrophobic compounds such as micelles to protect the excited state from water or rely on energy transfer to fluorophores that are less sensitive to solvent quenching. Another difficulty with chemiluminescence is efficient coupling of the reaction to the creation of excited-state orbitals.

While chemiluminescence has relied on the ingenuity of chemists, bioluminescence, a form of chemiluminescence, has instead relied on the processes of natural evolution. 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 luminogenic substrate (Figure 8.1). The luciferases that are used most widely in high-throughput screening are beetle luciferases (including firefly luciferase), Renilla luciferase and aequorin. Beetle luciferases are the most versatile of this group, and the number of new applications is expanding rapidly. 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 is used primarily for reporter gene applications, although its use also is expanding. Aequorin is used almost exclusively to monitor intracellular calcium concentrations.

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 on a box. The enzyme would 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 to both generate and protect 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. 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 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 where it will not interfere over the time required to measure multiple samples. 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 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 some systems, a second reporter is used, expressed from a "control" vector, to normalize results of the experimental reporter. For example, the second reporter can control for variation between cell number or transfection efficiency. Typically, the control reporter gene is driven by a constitutive promoter, and control vector is cotransfected with the "experimental" vector. Different reporter genes are used for the the control and experimental vectors so that the relative activities of the two reporter products can be assayed individually. Control vectors also can be used to optimize transfection methods. Gene-transfer efficiency can be assessed relatively in cell lysates from different conditions by comparing reporter activity or assessed absolutely by estimating the percentage of cells expressing the transferred gene by in situ staining.

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

Applications

Basic research into bioluminescence has led to practical applications, particularly in molecular biology, where bioluminescent enzymes are widely used as genetic reporters. Moreover, the value of these applications 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 is 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 effects on coupled expression of a reporter gene are quantitated. However, the broader aspect of gene expression entails much more than transcription alone, and reporter genes also can be used to study other cellular events.

Some examples of analytical methodologies that use luciferase include:

• Assays and biosensors that monitor cell-signaling pathways. For example, the GloResponse™ Cell Lines facilitate rapid and convenient analysis of cell signaling through the nuclear factor of activated T-cells (NFAT) pathway or cyclic AMP (cAMP) response pathways via activation of a luciferase reporter gene. The GloSensor™ biosensor is a genetically modified form of firefly luciferase into which a cAMP-binding protein moiety has been inserted. cAMP binding induces a conformational change, leading to increased light output.
• RNA interference to study how double-stranded RNA (dsRNA) suppresses expression of a target protein by stimulating specific degradation of the target mRNA. Luciferase reporters can be used to quantitatively evaluate microRNA (miRNA) activity by inserting miRNA target sites downstream or 3′ of the firefly luciferase gene.
• 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 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 also is used in mammalian cells.
• Bioluminescence resonance energy transfer (BRET) to monitor protein-protein interactions, where two fusion proteins are made, one using the bioluminescent Renilla luciferase and another protein fused to 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).
• Live-cell and in vivo imaging. Luciferase genes are commonly used as light-emitting reporters in cellular and animal models. Visualization of reporter expression using live-cell luciferase substrates allows nondestructive, quantitative assays and repeat measures of the same samples without perturbation.

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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. Luciferase genes have been cloned from bacteria, beetles (e.g., firefly and click beetle), Renilla, Aequorea, Vargula and Gonyaulax (a dinoflagellate). Of these, only 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. Diagram of firefly and Renilla luciferase reactions with their respective substrates, beetle luciferin and coelenterazine, to yield light.

Various strategies have been tried to generate stable luminescence signal and 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 concentrations over a 100-millionfold range. The assay sensitivity allows quantitation of fewer than 10^–20 moles of enzyme.

The popularity of native firefly luciferase as a genetic reporter is due to the sensitivity and convenience of the enzyme assay and tight coupling of protein synthesis with enzyme activity. Firefly luciferase, which is encoded by the luc gene, is a monomer that does not require any post-translational modifications; it is available as a mature enzyme directly upon translation of its mRNA. Catalytic competence is attained immediately after release from the ribosome. Also, luciferase has a very short half-life in cells (approximately 3 hours). Combined, these properties make luciferase an extremely responsive reporter, far more so than other commonly used reporters.

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 and represents a natural example of BRET.

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, Renilla luciferase does not require calcium in the luminescent reaction. As a reporter molecule, Renilla luciferase, which is encoded by the Rluc gene, 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 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. Genes cloned from the ventral light organ of a luminous click beetle, Pyrophorus plagiophthalamus encode four luciferases capable of emitting luminescence ranging from green to orange (544–593nm). The Chroma-Luc™ luciferases were developed from these naturally occurring luciferase genes 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 luciferase genes generate very similar luciferase proteins; however, one is maximally similar to (~98%) the DNA sequence for the red luciferase, while the other is divergent (~68%) . Therefore experimental and control reporter genes and proteins within an experiment can be almost identical. Under circumstances where genetic recombination is a concern, the divergent luciferase gene pair may be useful.

Gene Optimization

An ideal genetic reporter should: i) express uniformly and optimally in the host cells; ii) only generate responses to effectors that the assay intends to monitor (avoid anomalous expression); and iii) have a low intrinsic stability to quickly reflect transcriptional dynamics. Despite their biology and enzymology, native luciferases are not necessarily optimal as genetic reporters. In the past decade, Promega scientists 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

Normally, in the firefly light organ, luciferase is located in specialized peroxisomes of photocytic cells. When the enzyme is expressed in foreign hosts, a conserved translocation signal causes luciferase to accumulate in peroxisomes and glyoxysomes. With 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 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 localization of luciferase also may interfere with the performance of the genetic reporter. For instance, luciferase accumulation in the cell might be differentially affected if the enzyme is distributed into two different subcellular compartments.

The stability of luciferase in peroxisomes is not known but could be different than its stability in the cytosol. If so, luciferase expression could be affected by changes in the distribution of luciferase between peroxisomes and the cytosol. Measurements of in vivo luminescence also might 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, Promega scientists followed two strategies: i) design a new C-terminal tripeptide sequence based on available data to minimize peroxisomal import, -Gly-Lys-Thr; and ii) apply random mutagenesis to the C-terminal region and select 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 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 increased substantially by adjusting the codon usage frequency while 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).

Figure 8.2. The synthetic Renilla luciferase gene supports higher expression than the native gene in mammalian cells. CHO and HeLa cells were transfected with pGL3-Control Vector (containing the SV40 enhancer/promoter) harboring either the synthetic hRluc or native Rluc gene. Cells were harvested 24 hours after transfection and Renilla luciferase activity assayed using the Dual-Luciferase^® Reporter Assay System.

Figure 8.3. The firefly luc2 gene displays higher expression than the luc+ gene. The luc2 gene was cloned into the pGL3-Control Vector (Cat.# E1741), replacing the luc+ gene. Thus both firefly luciferase genes were in the same pGL3-Control Vector backbone. The two vectors containing the firefly luciferase genes were co-transfected into NIH/3T3, CHO, HEK 293 and HeLa cells using the phRL-TK Vector as a transfection control. Twenty-four hours post-transfection the cells were lysed with Passive Lysis 5X Buffer (Cat.# E1941), and luminescence was measured using the Dual-Luciferase^® Reporter Assay System (Cat.# E1910). Relative light units were normalized to Renilla luciferase expression from the phRL-TK Vector. The fold increase in expression is listed above each pair of bars. A repeat of this experiment yielded similar results.

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 affect transcription, resulting in anomalous expression of the reporter gene. Removal of these sequences reduces 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, resulting in elevated levels of transcription in the absence of a promoter sequence or increased basal levels of gene expression in the presence of transcription regulatory sequences. 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 amino acid sequence to create the luc2 gene. In addition, sequences resembling splice sites, poly(A) addition sequences, Kozak sequences (translation start sites for mammalian cells), E. coli promoters or E. coli ribosome-binding sites also were removed wherever possible. This process has led to a greatly reduced number of cryptic regulatory sequences (Figure 8.4) and therefore a reduced risk of anomalous transcription.

Figure 8.4. Reduced number of consensus transcription factor-binding sites for the luc2 gene. The number of consensus transcription factor-binding sites identified in the luc+ gene is greatly reduced in the luc2 gene.

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 of the reporter protein, the greater the collection of different transcriptional rates pooled into the reporter assay. This pooling process has a "dampening effect" on the apparent transcriptional dynamics, making changes in the transcriptional rate more difficult to detect. This can be remedied by reducing the reporter lifetime, 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 reporter protein accumulation within the cells. Unfortunately, a zero lifetime also would yield zero accumulation, and thus no reporter could be measured. A compromise must be reached since, as lifetime decreases, so does the amount of reporter available for detection. 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 signal. Thus, the high sensitivity of luciferase assays permits greater dynamics of the luciferase reporters.

The speed by which a genetic reporter can respond to changes in transcriptional rate correlates to reporter stability within cells. Highly stable reporters accumulate to greater levels in cells, but their concentrations change slowly relative to changes in transcription. Conversely, lower stability yields less accumulation but a much faster rate of response. To provide reporters that meet different experimental needs, the Rapid Response™ Reporter family of luciferase genes were developed with different intracellular stabilities.

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, Promega scientists developed destabilized luciferase reporters by genetically fusing a protein degradation sequence to the luciferase gene products (Li et al. 1998). After evaluating many degradation sequences for their effect on response rate and signal magnitude, we chose two sequences: the PEST protein degradation sequence and a second sequence composed of two protein degradation sequences (CL1 and PEST). Due to an 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 used to deliver the reporter gene to host cells are critical for overall reporter assay performance. Cryptic regulatory sequences such as transcription factor-binding sites and/or promoter modules within the vector backbone can lead to high background and anomalous responses. This is a common issue for mammalian reporter vectors, including the pGL3 Luciferase Reporter Vectors. Promega scientists extended the 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 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 Vectors.

pGL4 Luciferase Reporter Vectors

By manipulating luciferase genes Promega scientists have 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 pGL4 family of luciferase vectors incorporates a variety of features such as your choice of firefly or Renilla luciferase, Rapid Response™ versions, mammalian-selectable markers, basic vectors without promoters, promoter-containing control vectors and predesigned vectors with your choice of several response elements (Figure 8.5).

Figure 8.5. The family of pGL4 Luciferase Reporter Vectors incorporates a variety of additional features, such as a choice of luciferase genes, Rapid Response™ versions, a variety of mammalian selectable markers, and vectors with or without promoters and response elements.

Advantages of the pGL4 Vectors include:

1. Improved sensitivity and biological relevance due to:
2. Increased reporter gene expression: Codon optimization of synthetic genes for mammalian expression
3. Reduced background and risk of expression artifacts: Removal of cryptic DNA regulatory elements and transcription factor-binding sites
4. Improved temporal response: Rapid Response™ technology using destabilized luciferase genes
5. Additional advantages include:
6. Flexible detection options: Choice of reporter genes
7. Easy transition from transient to stable cells: Choice of mammalian selectable markers
8. 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 when designing bioluminescent assays is harnessing this efficient light-emitting chemistry for 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 the 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, 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.

The following section provides information about specific bioluminescent reporters and assays, including how to choose the correct reporter genes to suit your research needs. Tables 8.2 and 8.3 show available luciferase genes, assays and reagents.

Single-Reporter Assays

Assays based on a single reporter provide the quickest and least expensive means to acquire gene expression data from cells. However, because cells are inherently complex, the quantity of 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 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 Assays section.

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™ Luciferase Assay System yields maximal firefly luciferase luminescence intensity and sufficient signal duration for analysis in a multiwell plate. The Steady-Glo^® Luciferase Assay System provides even greater luminescence duration but with lower intensity. Both reagents work directly in culture medium for mammalian cells, so prior cell lysis is not necessary. This allows you to grow cells in multiwell plates, then measure expression in a single step.

Dual-Reporter Assays

The most commonly used dual-reporter assays measure both firefly and Renilla luciferase activities. These luciferases use different substrates and thus can be differentiated by their enzymatic specificities. The method involves adding two reagents to each sample and measuring 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^® Reporter Assay System requires cell lysis prior to performing the assay and requires the use of reagent injectors with multiwell plates. The Dual-Glo™ Reagent is optimized for multiwell plates, providing longer luminescence duration (in other words, a longer luciferase signal half-life). As with other reagents designed for use in multiwell plates, the Dual-Glo™ Assay works directly in mammalian cell culture medium without prior cell lysis.

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.

Reducing Variability

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. Multiwell plates introduce variables such as edge effects, 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. 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 luminescence. Promega scientists 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, with only a few amino acid substitutions necessary to create the different colors. This structural similarity means that both the control and experimental reporters are likely to respond similarly to biochemical changes within the cell, resulting in even more accurate normalization to the control reporter.

The genes encoding these reporters, the Chroma-Luc™ genes, are codon-optimized for mammalian cells. Promega scientists developed two genes encoding the green-emitting reporter: one which is nearly identical to the luciferase emitting red luminescence, and one that is maximally divergent from it. These genes encode reporter proteins that have nearly identical sequences. The divergent gene may be useful in situations where genetic recombination is a concern.

The Chroma-Glo™ Luciferase Assay System measures Chroma-Luc™ activity in multiwell plates. The Chroma-Glo™ Reagent 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 optical filters, sensitivity relative to other assay methods is reduced. Both red- and green-emitting Chroma-Luc™ luciferase activities are detectable using optical filters when the relative concentrations differ by up to 100-fold. This is less than dual-luciferase assays that use chemical differentiation, where the relative concentrations may differ by over 1,000-fold.

Distinguishing among the Dual Assays

Dual-reporter and dual-color assays allow you to measure expression of two different reporter genes or, when using a luciferase-based cell viability assay, one reporter gene and cell health. 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 cells so that the environment surrounding the reporter enzyme can be carefully controlled. Recently, Promega scientists developed a variety of live-cell substrates to monitor Renilla and firefly luciferase activity without disrupting cells.

Renilla luciferase requires only oxygen and coelenterazine to generate luminescence, providing a simple luciferase system to measure luminescence from living cells. Unfortunately, coelenterazine is unstable in aqueous solutions, so it has been difficult and inconvenient to measure Renilla luciferase. EnduRen™ and ViviRen™ Live Cell Substrates overcome this difficulty and easily generate luminescence from live cells expressing Renilla luciferase. Because luminescence is generated from living cells, these substrates are ideal for multiplexing with assays that determine cell number.

Promega also offers a number of live-cell firefly luciferase substrates, including VivoGlo™ Luciferin, the potassium salt of D-luciferin; VivoGlo™ Caspase-3/7 Substrate (Z-DEVD-Aminoluciferin, Sodium Salt), a firefly luciferase prosubstrate containing the DEVD tetrapeptide sequence recognized by caspase-3 and -7; and VivoGlo™ Luciferin-β-Galactoside Substrate (6-O-β-galactopyranosyl-luciferin), a substrate for the reporter enzyme β-galactosidase. These substrates are useful for imaging firefly luciferase in live cells and organisms.

Normalizing Interfering Phenomena

When correlations between experimental conditions and reporter gene expression are examined, other 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 assay allow independent monitoring of both reporter expression and cell viability to avoid data misinterpretation (Farfan et al. 2004). 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. 2002).

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

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

Fluorescent cell viability assays also are available to monitor cell health and normalize single-reporter assay results to live cell number. For example, the CellTiter-Fluor™ Cell Viability Assay is a nonlytic, fluorescence assay that measures the relative number of viable cells in a population. The CellTiter-Fluor™ Substrate enters intact cells, where it is cleaved by a live-cell protease that is restricted to intact cells to generate a fluorescent signal proportional to the number of living cells. The number of nonviable cells does not affect fluorescence because the live-cell protease becomes inactive upon loss of membrane integrity and leakage into the culture medium. This assay is well-suited for multiplexing with homogeneous luciferase assay reagents such as Bright-Glo™, Steady-Glo™ and ONE-Glo™ Luciferase Assay Systems (Zakowicz, H. et al. 2008) because it exhibits no intrinsic color quenching, which can limit luminescent assay sensitivity. Viability assays using older resazurin-based substrates can quench up to 82% of luminescence due to color intensity of the dye.

Bioluminescent Reporters to Monitor RNA Interference

Bioluminescent reporters have been harnessed to study RNA interference (RNAi), a phenomenon by which double-stranded RNA complementary to a target mRNA can specifically inactivate a gene by stimulating degradation of the target mRNA. As such, RNAi has emerged as a powerful tool to analyze gene function. Since its report in C. elegans (Fire et al. 1998), RNAi has been reported in a variety of organisms, including zebrafish, planaria, hydra, fungi, Drosophila and plant and mammalian systems. These phenomena have been collectively termed RNA silencing and appear to use a common set of proteins and short RNAs. These processes are mechanistically similar but not identical. For more information about the RNAi process and technologies and products that can be used to design, synthesize and evaluate siRNAs and shRNAs, refer to the RNA Interference chapter.

Bioluminescent Reporters in Cell-Signaling Assays

Luciferase reporter assays are widely used to investigate cellular signaling pathways and as high-throughput screening tools for drug discovery (Brasier et al. 1992, Zhuang et al. 2006). 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, subjecting the transfected cells to a particular treatment, then measuring reporter activity, researchers can determine what REs are used, and thus, what signaling pathways are involved. The use of inhibitors and short interfering RNAs (siRNAs) can be used to confirm what factors are involved in this response.

To speed this type of research, Promega scientists have designed several convenient pGL4 Vectors with your choice of a number of response elements and regulatory sequences to take advantage of the benefits of the pGL4 Vector backbone and luc2P gene. See Tables 8.1 and 8.2. Many of these vectors encode the hygromycin-resistance gene to allow selection of stably transfected cell lines. Alternatively, Promega offers cell lines that are already stably transfected with pGL4-based vectors with specific response elements. See Section III.E, Luciferase Reporter Cell Lines.

Bioluminescent reporters also enable characterization of 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, which is similar in many ways to the two-hybrid assay. In a two-hybrid assay, two proteins that are thought to interact are expressed as fusion proteins, one fused with the DNA-binding domain (DBD) of the yeast GAL4 transcription factor and the other fused to the VP16 activation domain. Protein:protein interactions bring the two domains together to yield expression of a reporter gene downstream of tandem GAL4-binding sites and a minimal promoter. The universal nuclear reporter assay can be thought of as a "one-hybrid" assay, where the ligand-binding domain (LBD) of a nuclear receptor replaces the bait and prey proteins and VP16 activation domain (Figure 8.6).

To perform the universal nuclear receptor assay, simply cotransfect the cell line of interest with a construct encoding the LBD-GAL4 DBD fusion protein and a suitable reporter vector with multiple copies of the GAL4 UAS upstream of the promoter and reporter gene. Two to three days posttransfection, 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, which you can use to identify receptor agonists, antagonist, 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. An example of a suitable reporter construct is the pGL4.35[luc2P/9XGAL4UAS/Hygro] Vector (Cat.# E1370), which contains nine copies of the GAL4 UAS immediately upstream of a minimal promoter driving expression of luc2P reporter gene. For added convenience, Promega offers HEK293 cells that are stable transfected with the pGL4.35[luc2P/9XGAL4UAS/Hygro] Vector: the GloResponse™ 9XGAL4UAS-luc2P HEK293 cells. For more information, see the Luciferase Reporter Cell Lines section below.

Figure 8.6. The two-hybrid assay and universal nuclear receptor assay. Panel A. The traditional two-hybrid assay. The pG5luc Vector contains five GAL4 upstream activator sequences (UAS) upstream of a minimal TATA box, which in turn is upstream of the firefly luciferase gene. Interaction between the two test proteins, expressed as GAL4-X and VP16-Y fusion proteins, results in an increase in luciferase transcription and expression. Panel B. The universal nuclear receptor assay. The ligand-binding domain of the nuclear receptor replaces the bait and prey proteins and VP16 activation domain. 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.

Promega offers a number of additional reagents to simplify universal nuclear receptor assays. The pBIND-ERα Vector (Cat.# E1390) contains the yeast Gal4 DBD and an estrogen receptor-ligand binding domain (ER-LBD) gene fusion, and the pBIND-GR Vector (Cat.# E1581) contains the yeast Gal4 DBD and glucocorticoid receptor-ligand binding domain (GR-LBD) gene fusion. Promega also offers the pFN26A (BIND) hRluc-neo Flexi^® Vector (Cat.# E1380), which allows expression of 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. You simply clone the DNA fragment encoding the ligand-binding domain of your receptor into SgfI and PmeI sites at the 5′ and 3′ ends of a lethal barnase gene, which acts as a positive selection for successful ligation of the insert. Each BIND vector contains a Renilla luciferase/neomycin resistance co-reporter for normalization of transfection efficiency or construction of a double-stable cell line without the need for additional cloning.

Bioluminescent reporters also are useful for 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. 2002). The firefly luciferase-based GloSensor™ cAMP assay provides a sensitive and easy-to-use format to interrogate overexpressed or endogenous GPCRs that signal via changes in intracellular cAMP concentration. The assay uses genetically encoded biosensor variants comprised of cAMP-binding domains fused to mutant forms of Photinus pyralis luciferase. cAMP binding induces conformational changes that promote large increases in light output. Following pre-equilibration with a luciferase substrate, cells transiently or stably expressing the biosensor variant can be used to assay GPCR function using a nonlytic assay format, enabling kinetic measurements of cAMP accumulation or turnover in living cells. Moreover, the assay offers a broad dynamic range, with up to 500-fold changes in light output. Extreme sensitivity allows detection of G_i-coupled receptor activation or inverse agonist activity in the absence of artificial stimulation by compounds such as forskolin. For more information, visit: www.promega.com/glosensor/

Luciferase Reporter Cell Lines

The GloResponse™ Cell Lines contain optimized, state-of-the-art luciferase reporter technology integrated into a cell line. These cells use the destabilized and optimized luc2P gene, allowing greater sensitivity and shorter induction times than native reporter enzymes. The GloResponse™ NFAT-RE-luc2P HEK293 Cell Line, NFκB-RE-luc2P HEK293 Cell Line and CRE-luc2P HEK293 Cell Line allow rapid and convenient analysis of cell signaling through the NFAT, NF-κB or cAMP response pathways via activation of a luciferase reporter gene. Non-native activators of these pathways, including GPCRs, can be studied after the appropriate proteins are introduced by transfection.

GPCR signaling pathways can be categorized into three classes based on the G protein α-subunit involved: G_s, G_i/o and G_q. The GloResponse™ CRE-luc2P HEK293 Cell Line can be used to study and configure screening assays for G_s- and G_i/o-coupled GPCRs, which signal through cAMP and the cAMP reponse element (CRE). For G_q-coupled GPCRs, which signal through calcium ions and NFAT-RE, the GloResponse™ NFAT-RE-luc2P HEK293 Cell Line should be used. GPCR assays that use the GloResponse™ Cell Lines are amenable to high-throughput screening. These assays typically have greater response dynamics (fold of induction) than other assay formats and generate high-quality data as indicated by the high Z′ values.

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 improvements developed for the pGL4 Vectors for enhanced performance. The destabilized luc2P luciferase reporter improves responsiveness to transcriptional dynamics and is codon-optimized for enhanced expression in mammalian cells. The pGL4 vector backbone was engineered to reduce background reporter expression. The result is cell lines with very high reporter induction levels when the pathway of interest is activated.

Selecting a Reporter Gene and Assay

The tables in this section show the various features of reporter vectors, including the reporter gene, presence of a multiple cloning region, gene promoter, protein degradation sequences and mammalian selectable marker (Tables 8.2 and 8.3), as well as the features of Promega reporter assays (Table 8.3). These tables and the following tools will help you choose a pGL4 Vector or 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 on the link, select the Solution Finder tab, then choose "pGL4 Vector Selector".

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

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

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

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Getting the Most Out of Your Genetic Reporter Assays

When performed properly, experiments using genetic reporters can yield tremendous amounts of information. However, there are several important considerations when designing and performing these types of experiments to ensure that the data are sound.

Reporter Design

A common question when designing reporter vectors for promoter dissection is “What sequences should I clone into my vector?”. Unfortunately, there is no one correct answer. The necessary sequences depend on the biological question you are trying to answer and the vector into which you are cloning. An advantage of transgenic reporter assays is that you can control which elements are examined. You might include the entire proximal promoter (including ~1kb upstream of the promoter), a specific promoter subsection or as little as a single response element. When generating a transcriptional fusion or using a vector with no transcription start site, you might include the +1 transcription start site. The reporter might include the 5′ UTR if you want to understand how this sequence may affect promoter activity, but keep in mind that UTR sequences also can affect post-transcriptional regulation. The reporter might include an intron and all or part of one or both flanking exons to study RNA splicing or characterize regulatory elements contained within the intron (but if the exons include coding sequence, be sure to clone the reporter gene in frame). The reporter might contain the 3′ UTR to focus on only post-transcriptional regulation through miRNA effects. You might clone any combination of sequences from your gene of interest to look at the integration of regulatory pathways; reporter assays allow this flexibility and refined experimental design.

Controls

The proper controls are an important part of reporter assays. The most important is a control using untransfected cells to define the background signal in the assay (from luminometer noise or reagent background). Generally, background luminescence is inconsequential, and the signal:background ratio is quite high in luciferase assays. Additional controls may include the parent vector used to prepare the reporter vector (minus any inserts) and a positive control vector. Measuring luciferase activity from the parent vector allows you to discount any reporter response due to the vector backbone, not the insert; experimental results can be expressed as the ratio of experimental vector response to parent vector response. The same function is generally provided by the second reporter in a dual-reporter assay when using matched vectors (i.e., pGL4 firefly and Renilla vectors). A reporter vector with a relatively strong promoter can serve as a positive control for luciferase expression and detection in the cell line of interest.

Transfection Parameters

Transfection is necessary to introduce the reporter vector into a cell. Transient transfection is the most common method, but stable transfection should be considered if you are performing the same reporter assay frequently. Both approaches have advantages and disadvantages. Transient transfection allows you to vary the reporter vectors and vector ratios, but cells must be transfected for each set of experiments and will lose the reporter vector over time. Transfection efficiency can be low for primary cells and some cell lines and can vary considerably. Often a second reporter must be cotransfected into cells to normalize for differences in transfection efficiency; this reporter also can help to determine if a response was due to cell toxicity and not a promoter-specific event. Because transfection is stressful, cells must be allowed to recover prior to experimental treatment. Stable transfection eliminates variability in transfection effiency and the stress of transfection but requires additional time and effort to select stably transfected cells.

The optimal transfection reagent and conditions depend on the cell line used and often must be determined empirically. Efficient transfection can be critical when using less sensitive reporters such as chloramphenicol acetyltransferase (CAT) or β-galactosidase) but is less of a concern with sensitive reporters such as luciferase. For a detailed discussion of transfection, see the Transfection chapter of the Protocols and Applications Guide.

Two important factors when transfecting cell prior to dual-reporter assays are the reporter vector ratio and relative promoter strengths in the cell line of interest. The optimal ratio often is related to promoter strength and must be determined empirically. Strong promoters, such as cytomegalovirus (CMV) and SV40 promoters, can easily interfere with transcriptional activity of weaker promoters by sequestering transcription factors and are more likely to be regulated by experimental treatments due to the high number of transcription factor-binding sites. In general, we recommend avoiding promoters with the highest activities in your cells. Vectors with weaker promoters often are a better choice,and even vectors without a promoter yield sufficient luciferase activity for normalization purposes in most cells and are less likely to be regulated by the treatment.

It is important, especially if you must use a reporter vector with a strong promoter, to transfect cells with several different ratios of reporter vectors. For assays using pGL4 Vectors with firefly and Renilla luciferases, we recommend a 20:1 ratio as a good starting point, but the ratio could be as high as 200:1 when the promoter of one vector is dramatically stronger than that of the other. For vectors with promoters of equal strengths, the ratio might be 1:1. When performing vector titrations, be sure to transfect all cells with a constant amount of DNA to minimize differences in transfection efficiency due to differing DNA amounts. The ideal ratio will provide moderate but consistently detectable Renilla luciferase signal that is not influenced by the amount of firefly luciferase vector present, and the firefly and Renilla luciferase signals will be at least 3 standard deviations above background levels, below the saturation point of the luminometer and within 4 orders of magnitude of each other.

Assay Timing

The times between plating and transfection, transfection and experimental treatment, and treatment and reporter assay need to be consistent within a set of experiments to minimize variability and improve assay precision and accuracy. When plating cells prior to transfection, take into account the growth rate of the cells so that cells reach proper confluency at the time of transfection. After transfection allow time for cells to recover and the reporter to reach steady state levels of expression. During initial assay optimization, perform a time course to determine the time of peak reporter expression. The optimal time between treatment and reporter assay depends on a number of factors, including the kinetics of your system, longevity of the change you are monitoring (i.e., the assay window) and stability of the reporter protein.

For early-responding genes, we recommend a reporter with a short protein half-life such as those found in the Rapid Response™ luciferase genes, which encode protein degradation sequences (PEST; Li et al. 1998 or CL1; Gilon et al. 1998) to destabilize the reporter protein. The Rapid Response™ luciferase genes (luc2P and luc2CP) respond more quickly and with greater magnitude to changes in transcriptional activity than their more stable counterparts (luc and luc2). The onset of the response is more tightly coupled to the induction event, and the assay window is narrower. The use of more stable luciferase versions will result in a later and longer response with a wider assay window, so assay timing is not as critical. In these cases, reporter assays are commonly performed 12–24 hours after treatment.

Reporter Assay Choice

There are many factors to consider when choosing a reporter assay, including the reporter gene used, assay format (individual tubes or multiwell plates), luminometer capabilities, and the need to maintain cell viability. A summary of luciferase reporter assay features is provided in Table 8.4.

Cells and Cell Culture Conditions

Cells and cell culture conditions used to dissect a promoter can affect assay design and results. Three types of cells are commonly used: 1) fibroblasts, which are easy to maintain and amenable to most reporter assays but may not express necessary co-factors or be as biologically relevant as other cells; 2) cancer cells, which may be more relevant and are easy to use; and 3) primary cells, which may be the most biologically relevant but often are difficult to obtain, maintain and transfect. Cell cultures should not be confluent during the experiment, since confluent cells can exhibit differences in metabolism, gene expression and physiological response compared to preconfluent cell cultures. Likewise, cells at higher passage numbers may not behave in the sameway as cells at lower passage numbers. If necessary, grow and freeze a large quantity of cells at a lower passage number to ensure that your experiments are performed with cells at the same passage number. Cells should be healthy and, ideally, easy-to-transfect. The cell culture medium should replaced several days before and after transfection. When repeating an experiment, be sure to replicate cell culture and transfection conditions as closely as possible to ensure consistency.

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