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I. Introduction 1 A. Luminescence versus Fluorescence 1 B. Bioluminescent Reporters 2 C. Applications 3 II. Luciferase Genes and Vectors 4 A. Biology and Enzymology 4 B. Gene Optimization 6 C. pGL4 Luciferase Reporter Vectors 8 D. pNL Vectors 8 III. Luciferase Reporter Assays and Protocols 8 A. Single-Reporter Assays 9 B. Dual-Reporter Assays 10 C. Live-Cell Substrates 12 D. Bioluminescent Reporters to Normalize for Changes in Cell Physiology 12 E. Bioluminescent Reporters to Monitor RNA Interference 13 F. Bioluminescent Reporters in Cell-Signaling Assays 13 G. Luciferase Reporter Cell Lines 15 H. Bioluminescent Reporters to Study Protein Dynamics 15 I. Promega Reporter Vectors and Assays 16 IV. Getting the Most Out of Your Genetic Reporter Assays 16 A. Introduction 16 B. Choice of Reporter Gene and Assay 16 C. Reporter Construct Design 16 D. Controls 17 E. Transfection Parameters 17 F. Assay Timing 17 G. Cells and Cell Culture Conditions 18 H. Special Considerations for High-Throughput Assays 18 V. References 19 VI. Vector Tables 20 Protocols & Applications Guide www.promega.com rev. 4/15 |||||||| 8Bioluminescent Reporter Gene Assays CONTENTS PROTOCOLS & APPLICATIONS GUIDE
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Bioluminescent Reporter Gene Assays Protocols and Applications ...

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Page 1: Bioluminescent Reporter Gene Assays Protocols and Applications ...

I. Introduction 1A. Luminescence versus Fluorescence 1

B. Bioluminescent Reporters 2

C. Applications 3

II. Luciferase Genes and Vectors 4A. Biology and Enzymology 4

B. Gene Optimization 6

C. pGL4 Luciferase Reporter Vectors 8

D. pNL Vectors 8

III. Luciferase Reporter Assays and Protocols 8A. Single-Reporter Assays 9

B. Dual-Reporter Assays 10

C. Live-Cell Substrates 12

D. Bioluminescent Reporters to Normalize for Changes inCell Physiology 12

E. Bioluminescent Reporters to Monitor RNAInterference 13

F. Bioluminescent Reporters in Cell-Signaling Assays 13

G. Luciferase Reporter Cell Lines 15

H. Bioluminescent Reporters to Study ProteinDynamics 15

I. Promega Reporter Vectors and Assays 16

IV. Getting the Most Out of Your Genetic ReporterAssays 16A. Introduction 16

B. Choice of Reporter Gene and Assay 16

C. Reporter Construct Design 16

D. Controls 17

E. Transfection Parameters 17

F. Assay Timing 17

G. Cells and Cell Culture Conditions 18

H. Special Considerations for High-ThroughputAssays 18

V. References 19VI. Vector Tables 20

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CONTENTSPROTOCOLS & APPLICATIONS GUIDE

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I. IntroductionGenetic reporter systems have contributed greatly to thestudy of eukaryotic gene expression and regulation.Although reporter genes have played a significant role innumerous applications, both in vitro and in vivo, they aremost frequently used as indicators of transcriptional activityin cells.Typically, a reporter gene is joined to a promoter sequencein an expression vector that is transferred into cells.Following transfer, cells are assayed for the presence of thereporter by directly measuring the reporter protein itselfor the enzymatic activity of the reporter protein. An idealreporter gene is one that is not endogenously expressed inthe cell of interest and is amenable to assays that aresensitive, quantitative, rapid, easy, reproducible and safe.Analysis of cis-acting transcriptional elements is a frequentapplication for reporter genes. Reporter vectors allowfunctional identification and characterization of promoterand enhancer elements because expression of the reporterprotein correlates with transcriptional activity of thereporter gene. For these types of studies, promoter regionsare cloned upstream or downstream of the gene. Thepromoter-reporter gene fusion is introduced into culturedcells by standard transfection methods or into a germ cellto produce transgenic organisms. The use of reporter genetechnology allows characterization of promoter andenhancer elements that regulate cell-, tissue- anddevelopment-defined gene expression.Trans-acting factors can be assayed by co-transfer of thepromoter-reporter gene fusion DNA with another clonedDNA expressing a trans-acting protein or RNA of interestor by activation of the trans-acting factors through treatmentof the samples. The protein could be a transcription factorthat binds to the promoter region of interest clonedupstream of the reporter gene. For example, when tatprotein is expressed from one vector in a transfected cell,the activity of different HIV-1 LTR sequences linked to areporter gene increases, and the activity increase is reflectedin an increase of reporter gene protein activity.In addition to assessing promoter activity, reporters arecommonly used to study the effect of untranslated regions(UTRs) on mRNA stability, protein localization andtranslation efficiency. For example, microRNA (miRNA)target sites can be inserted downstream or 3´ of the reportergene to study how microRNAs affect RNA stability.Reporter proteins can be assayed by detecting inherentcharacteristics, such as enzymatic activity orspectrophotometric characteristics, or indirectly withantibody-based assays. In general, enzymatic assays aremore sensitive due to the small amount of reporter enzymerequired to generate detectable levels of reaction products.A potential limitation of enzymatic assays is highbackground if there is endogenous enzymatic activity inthe cell (e.g., β-galactosidase). Antibody-based assays aregenerally less sensitive but will detect the reporter proteinwhether it is enzymatically active or not.

Fundamentally, a reporter assay is a means to translate abiomolecular effect into an observable parameter. Whilethere are theoretically many strategies by which this canbe achieved, in practice the reporter assays capable ofdelivering the speed, accuracy and sensitivity necessaryfor effective screening are based on photon production.

A. Luminescence versus FluorescencePhoton production is realized primarily throughchemiluminescence and fluorescence. Both processes yieldphotons as a consequence of energy transitions fromexcited-state molecular orbitals to lower energy orbitals.However, they differ in how the excited-state orbitals arecreated. In chemiluminescence, the excited states are theproduct of exothermic chemical reactions, whereas influorescence the excited states are created by absorption oflight.This difference in how the excited states are created greatlyaffects the character of the photometric assay. For instance,fluorescence-based assays tend to be much brighter becausehigh-intensity light can be used to generate the excitedstate. In chemiluminescent assays, the chemical reactionsrequired to generate excited states usually yield lower ratesof photon emission. The greater brightness of fluorescencewould appear to correlate with better assay sensitivity, butcommonly this is not the case. Assay sensitivity isdetermined by a statistical analysis of signal relative tobackground, where the relative signal represents a samplemeasurement minus the background measurement.Fluorescent assays tend to have much higher backgrounds,leading to lower relative signals.Fluorescent assays have higher backgrounds primarilybecause fluorometers cannot discriminate perfectly betweenthe very high influx of photons into the sample and themuch smaller emission of photons from the analyticalfluorophores. This discrimination is accomplished largelyby optical filtration—emitted photons have longerwavelengths than excitation photons—and by geometrybecause emitted photons typically travel a different paththan excitation photons. However, optical filters are notperfect in their ability to differentiate between wavelengths,and photons can change directions through scattering.Chemiluminescence has the advantage that, becausephotons are not required to create the excited states, theydo not constitute an inherent background when measuringphoton efflux from a sample. The resulting low backgroundpermits precise measurement of very small changes in light.Fluorescent assays also can be limited by interferingfluorophores within the samples. This is especiallyproblematic in biological samples, which can be repletewith a variety of heterocyclic compounds that fluoresce,typically in concentrations much above the analyticalfluorophores of interest. The problem is minimized insimple samples, such as purified proteins, but for drugdiscovery, living cells are increasingly used inhigh-throughput screening. Unfortunately, cells areenormously complex in their chemical constitutions andcan exhibit substantial inherent fluorescence. Screening

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compound libraries also is inherently complex. Althougheach sample may contain only one or a few compounds,the data set from which the drug leads are sifted iscumulated from many thousands of compounds. Thesecompounds also may present problems with fluorescenceinterference because drug-like molecules typically haveheterocyclic structures.For image analysis of microscopic structure, fluorescenceis almost universally preferred over chemiluminescence.Brightness counts because the optics required to imagecellular structures are relatively inefficient at lightgathering. Thus the low background inherent inchemiluminescence is of little advantage because the signalis usually far below the detection capabilities of imagingdevices. Furthermore, imaging is largely a matter of edgedetection, which has different signal-to-noise characteristicsthan simply detecting an analyte. Edge detection reliesheavily on signal strength and suffers less from uniformbackground noise.In macroscopic measurements (such as in a plate well),which require accurate quantification with high sensitivity,chemiluminescent assays often outperform analogousfluorescence-based assays. Macroscopic measurements arethe foundation for most high-throughput screening, whichrelies heavily on the use of multiwell plates, typically with96, 384 or 1536 wells, to measure a single parameter in alarge number of samples as quickly as possible. Assaysbased on fluorescence or chemiluminescence can providehigh sample throughput. However, fluorescence is morelikely to be hindered by light contamination (from theexcitation beam) or the chemical compositions of samplesand compound libraries. The use of chemiluminescence inhigh-throughput screening has been limited largely by thelack of available assay methods. Due to its long history,fluorescence has been more commonly used. But newcapabilities in chemiluminescence, particularly inbioluminescence, are now allowing new bioluminescenttechniques for high-throughput screening.

B. Bioluminescent ReportersIn nature, achieving efficient chemiluminescence is not atrivial matter, as evidenced by the lack of this phenomenonin daily life. The large energy transitions required for visibleluminescence generally are disfavored over smaller onesthat dissipate energy as heat, normally through interactionswith surrounding molecules, especially water moleculesin aqueous solutions. Because energy can be lost throughthese interactions, chemiluminescence depends stronglyon environmental conditions. Thus, chemiluminescentassays often incorporate hydrophobic compounds such asmicelles to protect the excited state from water or rely onenergy transfer to fluorophores that are less sensitive tosolvent quenching. Another difficulty withchemiluminescence is efficient coupling of the reaction tothe creation of excited-state orbitals. Whilechemiluminescence has relied on the ingenuity of chemists,bioluminescence, a form of chemiluminescence, has reliedmore on the processes of natural evolution. As luminous

organisms through the eons were selected by the brightnessof their light, their light-producing enzymes (i.e.,luciferases) have evolved both to maximize chemicalcouplings to generate the excited states and to protect theexcited states from water.Although most people are aware of bioluminescenceprimarily through the nighttime displays of fireflies, thereare many distinct classes of bioluminescence derivedthrough separate evolutionary histories. These classes arewidely divergent in their chemical properties, yet they allundergo similar chemical reactions. The classes are all basedon the interaction of the enzyme luciferase with aluminogenic substrate to produce light (Figure 8.1). Theluciferases that are used most widely in high-throughputscreening are beetle luciferases (including firefly luciferase),Renilla luciferase and a modified deep sea shrimp luciferase(NanoLuc® luciferase). Beetle luciferases are the mostcommon of this group and are used in a variety of reporterapplications. Renilla and NanoLuc® luciferases are usedprimarily for reporter gene applications, although theiruses are expanding. Click beetle luciferases are becomingbetter known and offer a range of luminescence coloroptions. Although not considered a luciferase enzyme, thephotoprotein aequorin also is used as a luminescentreporter almost exclusively to monitor intracellular calciumconcentrations.Intracellular luciferase is typically quantified by adding abuffered solution containing detergent to lyse the cells anda luciferase substrate to initiate the luminescent reaction.Luminescence will slowly decay due to side reactions,causing irreversible inactivation of the enzyme. The natureof these side reactions is not well understood, but they areprobably due to formation of damaging free radicals. Tomaintain steady luminescence of firefly and Renillaluciferase assays over an extended period of time, rangingfrom minutes to hours, it is often necessary to inhibit theluminescent reaction to various degrees. This inhibitioncan reduce the rate of luminescence decay to the pointwhere the reduction in signal is insignificant over the timerequired to measure multiple samples. Even under theseconditions, as few as 10–20 moles of luciferase or less persample may be quantified. This corresponds to roughly 10molecules per cell.

Unlike firefly and Renilla luciferases, NanoLuc® luciferasedoes not require enzyme inhibition for extended half-lifeassays. NanoLuc®-based assays, which use a novelfurimazine substrate to measure NanoLuc® luciferaseactivity, exhibit a much brighter signal and a much slowerrate of luminescence decay, with a signal half-life ofapproximately 120 minutes. These extended half-life assaysare convenient for reporter gene applications becausesample processing is not required prior to reagent addition.Simply add the reagent, and measure the resultingluminescence.

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Most reporter gene assays involve either one reporter geneor two. Most often, the second reporter is expressed froma "control" vector to normalize results of the experimentalreporter. For example, the second reporter can control forvariation in cell number or transfection efficiency. Typically,the control reporter gene is driven by a constitutivepromoter, and the control vector is cotransfected with the"experimental" vector. Different reporter genes are usedfor the control and experimental vectors so that the relativeactivities of the two reporter products can be assayedindividually.Alternatively, you can design dual-reporterbioluminescence assays in which both reporter genes areused as experimental reporters. Such dual-reporter assayscan be particularly useful for efficiently extracting moreinformation in a single experiment. For more information,see the Special Considerations for High-Throughput Assayssection.The Introduction to Reporter Gene Assays animationdemonstrates the basic design of a reporter assay using theDual-Luciferase® Reporter Assay System as an example tostudy promoter structure, gene regulation and signalingpathways.

C. ApplicationsBasic research into bioluminescence has led to practicalapplications, particularly in molecular biology, wherebioluminescent enzymes are widely used as geneticreporters. Moreover, the value of these applications hasgrown considerably over the past decade as the traditionaluse of reporter genes has broadened to cover wide-rangingaspects of cell physiology.The conventional use of reporter genes is largely to analyzegene expression and dissect the function of cis-actinggenetic elements such as promoters and enhancers(so-called "promoter bashing"). In typical experiments,deletions or mutations are made in a promoter region, andtheir effects on coupled expression of a reporter gene arequantitated. However, the broader aspect of geneexpression entails much more than transcription alone, andreporter genes also can be used to study other cellularevents, including events that are not related to geneexpression.Some examples of analytical methodologies that useluciferase include:• Assays and biosensors that monitor cell-signaling

pathways. For example, the GloResponse™ Cell Linesfacilitate rapid and convenient analysis of cell signalingthrough the nuclear factor of activated T-cells (NFAT)pathway or cyclic AMP (cAMP) response pathways viaactivation of a luciferase reporter gene. The GloSensor™biosensor is a genetically modified form of fireflyluciferase into which a cAMP-binding protein moietyhas been inserted such that cAMP binding induces aconformational change, leading to increased lightoutput.

• RNA interference to study how double-stranded RNA(dsRNA) suppresses expression of a target protein bystimulating specific degradation of the target mRNA.Luciferase reporters can be used to quantitativelyevaluate microRNA activity by inserting miRNA targetsites downstream or 3′ of the firefly luciferase gene. Forexample, the pmirGLO Dual-Luciferase miRNA TargetExpression Vector (Cat.# E1330) is based ondual-luciferase technology, with firefly luciferase as theprimary reporter to monitor mRNA regulation andRenilla luciferase as a control reporter for normalization.

• Identification of interacting pairs of proteins in vivousing a system known as the two-hybrid system (Fieldset al. 1989). The interacting proteins of interest arebrought together as fusion partners—one is fused witha specific DNA-binding domain, and the other proteinis fused with a transcriptional activation domain. Thephysical interaction of the two fusion partners isnecessary for functional activation of a reporter genedriven by a basal promoter and the DNA motifrecognized by the DNA-binding protein. This systemwas originally developed with yeast but also is used inmammalian cells.

• Reporters of protein abundance and stability. The rateof protein turnover is tightly regulated for manysignaling proteins. Protein stabilization and subsequentaccumulation can occur in response to cell signalingevents and changing cellular conditions and result inactivation of downstream transcriptional events. TheNanoLuc® Stability Sensor Vectors (Cat.# N1381 andCat.# N1391) enable stability studies of two keysignaling proteins, HIF1A and NRF2, and provide amethod to directly measure the cellular effects ofhypoxia and oxidative stress, respectively (Robers et al.2014).

• Bioluminescence resonance energy transfer (BRET) tomonitor protein:protein interactions, where two fusionproteins are made: one fused to the bioluminescentRenilla or NanoLuc® luciferase and another proteinfused to a fluorescent molecule. Interaction of the twofusion proteins results in energy transfer from thebioluminescent molecule to the fluorescent molecule,with a concomitant change from blue light to greenlight (Angers et al. 2000).

• Live-cell and in vivo imaging. Luciferase genes can beused as light-emitting reporters in cellular and animalmodels. Visualization of reporter expression usinglive-cell luciferase substrates or secreted forms ofluciferase allows nondestructive, quantitative assaysand multiple measures of the same samples withoutperturbation.

For more information, view the Introduction toBioluminescent Assays animation.

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1399

MF

HO S

N S

NO S

N S

NOCOOH

+ATP + O2

Recombinant FireflyLuciferase

+AMP + PPi + CO2 + Light

Beetle Luciferin Oxyluciferin

Mg2+

NH

N N

O OH

+O2

HO

N

N NH

OOH

+CO2 + Light

HO

RenillaLuciferase

Coelenterazine Coelenteramide

NH

N N

O

+ O2

N

N NH

O

O

+ CO2 + Light

NanoLuc®

Luciferase

O

Furimazine Furimamide

Figure 8.1. Diagram of firefly, Renilla and NanoLuc® luciferase reactions with their respective substrates (beetle luciferin, coelenterazineand furimazine) to yield light.

II. Luciferase Genes and VectorsA. Biology and Enzymology

Bioluminescence as a natural phenomenon is widelyexperienced with amazement at the prospect of livingorganisms creating their own light. Luciferase genes havebeen cloned from bacteria, beetles (e.g., firefly and clickbeetle), Renilla, deep sea shrimp (Oplophorus), Aequorea,Vargula and Gonyaulax (a dinoflagellate). Of these, onlyluciferases from bacteria, beetles, deep sea shrimp andRenilla have found general use as indicators of geneexpression. Bacterial luciferase is generally used to provideautonomous luminescence in bacterial systems throughexpression of the lux operon but is not often used foranalysis in mammalian cells.Firefly LuciferaseFirefly luciferase is by far the most commonly usedbioluminescent reporter. This monomeric enzyme of 61kDawas cloned from the North American firefly (Photinuspyralis) and catalyzes a two-step reaction in which oxidationof D-luciferin yields light, usually in the green to yellowregion, typically 550–570nm (Figure 8.1). The first step isactivation of luciferin with ATP to give a luciferyl-adenylateand pyrophosphate. In the second step, the

luciferyl-adenylate reacts with molecular oxygen to yieldoxyluciferin in an electronically excited state and CO2. Theexcited-state oxyluciferin then returns to the ground statewith concomitant release of light. Upon mixing withsubstrates, firefly luciferase produces an initial burst oflight that decays over about 15 seconds to a low level ofsustained luminescence. This kinetic profile reflects theaccumulation of one or more potent luciferase inhibitors,such as dehydroluciferyl-AMP, thus limiting catalyticturnover.Various strategies have been tried to generate stableluminescence and make the assay more convenient forroutine laboratory use. The most successful of theseincorporates coenzyme A (CoA) to yield maximalluminescence intensity that slowly decays over severalminutes. CoA appears to help clear the active site of potentinhibitors like dehydroluciferyl-AMP, thus sustainingluminescence over longer periods of time. An optimizedassay containing coenzyme A generates relatively stableluminescence in less than 0.3 seconds, with linearity overa 100-millionfold range of enzyme concentrations. Theassay sensitivity allows quantitation of fewer than 10–20

moles of enzyme.

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The popularity of native firefly luciferase as a geneticreporter is due to the sensitivity and convenience of theenzyme assay and tight coupling of protein synthesis withenzyme activity. Firefly luciferase, which is encoded by theluc gene, is a monomer that does not require anypost-translational modifications; it is available as a matureenzyme directly upon translation of its mRNA. Catalyticcompetence is attained immediately after release from theribosome. Also, firefly luciferase has a relatively shorthalf-life in cells compared to other commonly usedreporters.

NanoLuc® LuciferaseNanoLuc® luciferase is a small monomeric enzyme(19.1kDa, 171 amino acids) based on the luciferase fromthe deep sea shrimp Oplophorus gracilirostris (Hall et al.2012). This engineered enzyme uses a novel substrate,furimazine, to produce high-intensity, glow-typeluminescence (λmax = 465nM) in an ATP-independentreaction (Figure 8.1). The signal half-life is >2 hours, andluminescence is about 100-fold brighter than that of fireflyor Renilla luciferase (Figure 8.2). See why the brightness ofNanoLuc® Luciferase matters.

In mammalian cells, NanoLuc® luciferase shows noevidence of post-translational modifications, disulfidebonds or subcellular partitioning. The enzyme is activeover a broad pH range (pH 6–8), exhibits high physicalstability and retains activity at temperatures of up to 55°Cor in culture medium for >15 hours at 37°C.

Unlike other forms of luciferase, NanoLuc® luciferase isideally suited for both standard (lytic) and secretion-based(nonlytic) reporter gene applications. The small size of thegene (513bp) and encoded protein is ideal for viralapplications and protein fusions.

1054

9MA

Log[luciferase], pM

Lum

ines

cenc

e (R

LU)

–3 –2 –1 0 1 2 3 4 5 6 71 × 102

1 × 103

1 × 104

1 × 105

1 × 106

1 × 107

1 × 108

1 × 109

1 × 1010

Firefly LuciferaseRenilla Luciferase

NanoLuc® Luciferase

Figure 8.2. A comparison of the sensitivity of NanoLuc®, fireflyand Renilla luciferase assays. Luminescence was measured fromvarying concentrations of purified luciferases after mixing thereporter enzyme with its respective detection reagent. NanoLuc®

luciferase was approximately 100-fold brighter than firefly orRenilla luciferases at equivalent concentrations. Luminescence wasmeasured using the Nano-Glo® Luciferase Assay for NanoLuc®

luciferase, ONE-Glo™ Luciferase Assay System for firefly luciferaseand Renilla-Glo™ Luciferase Assay System for Renilla luciferase.

Renilla LuciferaseRenilla luciferase is a 36kDa monomeric enzyme thatcatalyzes the oxidation of coelenterazine to yieldcoelenteramide and blue light of 480nm (Figure 8.1). Thehost organism, Renilla reniformis (sea pansy), is acoelenterate that creates bright green flashes upon tactilestimulation, apparently to ward off potential predators.The green light is created through association of theluciferase with a green fluorescent protein and representsa natural example of BRET.Although Renilla and Aequorea are both luminouscoelenterates based on coelenterazine oxidation and bothhave a green fluorescent protein, their respective luciferasesare structurally unrelated. In particular, Renilla luciferasedoes not require calcium in the luminescent reaction. As areporter molecule, Renilla luciferase, which is encoded bythe Rluc gene, provides many of the same benefits as fireflyluciferase. Historically, the presence of nonenzymaticluminescence, termed autoluminescence, reduced assaysensitivity; however, improvements in assay chemistryhave nearly eliminated this problem. In addition, thesimplicity of the Renilla luciferase chemistry and, morerecently, improvements to the luciferase substrate haveenabled quantitation of Renilla luciferase from living cellsin situ or in vivo.Click Beetle LuciferaseClick beetle and firefly luciferase belong to the same beetleluciferase family. Hence, the size and enzymatic mechanismof click beetle luciferase are similar to those of fireflyluciferase. What makes the click beetle unique is the varietyof luminescence colors it emits. Genes cloned from theventral light organ of a luminous click beetle, Pyrophorusplagiophthalamus, encode four luciferases capable of emitting

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luminescence ranging from green to orange (544–593nm).The Chroma-Luc™ luciferases were developed from thesenaturally occurring luciferase genes to generateluminescence colors as different as possible: a red luciferase(611nm) and two green luciferases (544nm each). Theseluciferase genes were codon-optimized for mammaliancells and are nearly identical to one another, with amaximum of 8 amino acids difference between any two ofthese genes. The two green luciferase genes generate verysimilar luciferase proteins; however, one is maximallysimilar (~98%) to the DNA sequence for the red luciferase,while the other is divergent (~68%). Therefore, experimentaland control reporter genes and proteins within anexperiment can be almost identical. Under circumstanceswhere genetic recombination is a concern, the divergentluciferase gene pair may be useful.

B. Gene OptimizationAn ideal genetic reporter should: i) express uniformly andoptimally in the host cells; ii) only generate responses toeffectors that the assay intends to monitor (avoid anomalousexpression); and iii) have a low intrinsic stability to quicklyreflect transcriptional dynamics. Despite their biology andenzymology, native luciferases are not necessarily optimalas genetic reporters. In the past decade, Promega scientistshave made significant improvements in luciferaseexpression, reducing the risk of anomalous expression anddestabilizing these reporters. The key strategies to achievethese improvements are described here.Peroxisomal Targeting Site RemovalNormally, in the firefly light organ, luciferase is located inspecialized peroxisomes of photocytic cells. When the fireflyor click beetle enzyme is expressed in foreign hosts, aconserved translocation signal causes luciferase toaccumulate in peroxisomes and glyoxysomes. Peroxisomaland glyoxysomal localization of luciferase may interferewith normal cellular physiology and performance of thegenetic reporter. For instance, luciferase accumulation inthe cell might be differentially affected if the enzyme isdistributed into two different subcellular compartments.The peroxisomal translocation signal in firefly and clickbeetle luciferases has been identified as the C-terminaltripeptide sequence, -Ser-Lys-Leu. Removal of this sequenceabolishes import into peroxisomes. To develop an optimalcytoplasmic form of the luciferase gene, Promega scientistsreplaced the existing C-terminal sequence with a newC-terminal sequence, -Ile-Ala-Val. Consistently, thismodified luciferase yielded about 4- to 5-fold greaterluminescence than the native enzyme when expressed inNIH/3T3 cells. Renilla and NanoLuc® luciferases do notcontain a targeting sequence and are not affected byperoxisomal targeting.Codon OptimizationAlthough redundancy in the genetic code allows aminoacids to be encoded by multiple codons, different organismsfavor some codons over others. The efficiency of proteintranslation in a non-native host cell can be increasedsubstantially by adjusting the codon usage frequency while

maintaining the same gene product. The native luciferasegenes cloned from beetles (firefly or click beetle), sea pansy(Renilla reniformis) and deep sea shrimp use codons thatare not optimal for expression in mammalian cells.Therefore, Promega scientists systematically altered thecodons to the preferred ones while removing inappropriateor unintended transcription regulatory sequences used inmammalian cells. As a result, a significant increase inluciferase expression levels was achieved, up to severalhundredfold in some cases (Figures 8.3 and 8.4).Cryptic Regulatory Sequence RemovalThe presence of cryptic regulatory sequences in a reportergene may adversely affect transcription, resulting inanomalous expression of the reporter gene. Removal ofthese sequences reduces the risk of anomalous expression.A cryptic regulatory sequence can be a transcriptionfactor-binding site or a promoter module (defined as atleast two transcription factor-binding sites separated by aspacer; Klingenhoff et al. 1999). Additionally, it is notuncommon for an enhancer element to elevate levels oftranscription in the absence of a promoter sequence orincrease basal levels of gene expression in the presence oftranscription regulatory sequences. These regulatorysequences can exhibit synergistic or antagonistic functions(Klingenhoff et al. 1999).These cryptic regulatory sequences in the luc gene wereremoved without changing the amino acid sequence tocreate the luc2 gene. In addition, sequences resemblingsplice sites, poly(A) addition sequences, Kozak sequences(translation start sites for mammalian cells), E. colipromoters or E. coli ribosome-binding sites were removedwherever possible. This process has led to a greatly reducednumber of cryptic regulatory sequences (Figure 8.5) andtherefore a reduced risk of anomalous transcription. Asimilar process was performed using Renilla luciferase toproduce the hRluc gene.Degradation Signal AdditionWhen performing reporter assays, the total accumulatedreporter protein within cells is measured. This accumulationoccurs over the intracellular lifetime of the reporter, whichis determined by both protein and mRNA stability. Iftranscription is changing during this lifetime, then theresulting accumulation of reporter will reflect a collectionof different transcriptional rates. The longer the lifetime ofthe reporter protein, the greater the collection of differenttranscriptional rates pooled into the reporter assay. Thispooling process has a "dampening effect" on the apparenttranscriptional dynamics, making changes in thetranscriptional rate more difficult to detect. This can beremedied by reducing the reporter lifetime, thus reducingthe pooling of different transcriptional rates into eachreporter measurement. The resulting improvement inreporter dynamics is applicable to both upregulation anddownregulation of gene expression.Ideally, the reporter lifetime would be reduced to zero,completely eliminating the pooling of differenttranscriptional rates in each assay measurement. Only the

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10,000,000

1,000,000

100,000

10,000

1,000CHO HeLa

Cell Line

Native (Rluc)Synthetic (hRluc)

Rela

tive

Ligh

t Uni

ts

3351

MC

07_1

A

Figure 8.3. The synthetic Renilla luciferase gene supports higher expression than the native gene in mammalian cells. CHO and HeLacells were transfected with the pGL3-Control Vector (Cat.# E1741; contains the SV40 enhancer/promoter) harboring either the synthetichRluc or native Rluc gene. Cells were harvested 24 hours after transfection and Renilla luciferase activity assayed using the Dual-Luciferase®

Reporter Assay System.

4731MA

luc2luc+

NIH/3T3

7.9

CHO

4.9

HEK 293

4.1

HeLa

11.8

1,000

10,000

100,000

1,000,000

10,000,000

Lum

ines

cenc

e(R

elat

ive

Ligh

t Uni

ts)

Figure 8.4. The firefly luc2 gene displays higher expression than the luc+ gene. The luc2 gene was cloned into the pGL3-Control Vector,replacing the luc+ gene. Thus both firefly luciferase genes were in the same pGL3-Control Vector backbone. The two vectors containing thefirefly 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 usingthe Dual-Luciferase® Reporter Assay System (Cat.# E1910). Firefly luciferase luminescence (in relative light units) was normalized to Renillaluciferase expression from the phRL-TK Vector. The increase in expression of luc2 compared to luc+, indicated as the fold increase, for eachcell line is listed above each pair of bars. A repeat of this experiment yielded similar results.

luc+POZ

Brn2

GBF3

GBF2

E4BP4COMP1

Avian C

Pbx1

Pbx1

Pbx1

Pax-3Pbx-1

HAND2

MOK-2

MyT1

Thing1

C2H2

COMP1

PXR/RXR/CAR

NMP4

NMP4P53

P53

NUR77/Nurr1/Nor-1

C2HC

GFI1

NKX3.1

TEF-1

RFX1

SRF

NGN1/3

ATF4AREB6

NF-KB

WHP

OBF1

MEIS 1

TALE

SOX-5

c-Myb

Clox

CDF-1

B-cell

AhRNMP4

Gfl-1B

NKX3.1

MOK-2

MOK-2 RF-7

NFY

MMC PBX/MEIS1

MEIS1

GFI1

MESI1MIBP-1/RFX1

v-Myb

v-Myb

v-Myb

PAX2/5/8

c-Myb

C-Myb

c-Myb

c-Myb

v-Myb

v-Myb

Pbx1/Meis1

RFX1

TATA

Brn3

CHOP

CCAAT

CCAAT

CCAAT

VIS1

CDE/CHR

Smad4

Se-CtRNA

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ST/TA

ST/TA

ST/TA

ST/TA

ST/TA

ST/TA

ST/TA

HNF1

SRF

SRF

GATA

MSX1/MSX-2

GBF1

GBF3

COMP1

E4BP4

Otx2

SRE

AP 2

Oct1,2

OBF1

Elk-1

Elk-1

Elk-1

FLI

FOXF2

MZF-1MAZ

ATFSRF

Sox-5

Tst-1/Oct-6CBF 3

HNF4

HNF1

HNF6MEF

PXR/CAR/RXR

Pax1

FOXF2

MIBP1

GLI-k

TCF/LEF-1

Pax-6

TALE

VIS1 HEN1

VIS1

GABP

COMP1

WHP

Pbx1/Meis1

AD-b

Pit1

CREBCREB

CREB

CREB

CREBCREB

CDE/CHR

RAR

PAR

PAR

PAR

Lmo2

SRF

Cut-like

Cut-like

Cut-like

Cut-like

Cut-like

OBF 1

Pbx-1

c-Ets-1

En-1

FAST-1/S MAD

VIS 1

Ikaros 3

POZ

Ikaros-3

VIS 1

FLI

E2F

E2

E2F

E2FE2F

E2F

E2FE2F

E2F

FAST-1/SMAD

MEF2HIF1

BPV

C-r

C-r

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BRN-23

POZ

TCF/LEF-1

Brn23

Pax-3

C-Ets-1

PAR

GLI-K

Lmo2

MyT1Mt

Tst-1/Oct-6

GSh-1

Albumin D-box

E4BP4

E4BP4

CDF-1C-Abl

MyT1

Tax/CREB

OBF1

MEF Lmo2

TCF/LEF-1

luc2

v-Myb

v-Myb

LBP1c/LSF

Bel-1

c-Myb

Baso

p53

CDF1

PAX9

FKHRL1

VDR/RXR

PAX9

p53

p53

NF-kB

4760

MA

Figure 8.5. Reduced number of consensus transcription factor-binding sites for the luc2 gene. The number of consensus transcriptionfactor-binding sites identified in the luc+ gene is greatly reduced in the luc2 gene.

transcription rate at the instant of the assay would berepresented by reporter protein accumulation within thecells. Unfortunately, a zero lifetime also would yield zeroaccumulation, and thus no reporter could be measured. Acompromise must be reached since, as reporter lifetimedecreases, so does the amount of reporter available fordetection. This is where the high sensitivity of luminescentassays is useful. Relative to other reporter technologies, the

intracellular stability of luciferase reporters may be greatlyreduced while still providing a signal well abovebackground. Thus, the high sensitivity of luciferase assayspermits greater dynamics of the luciferase reporters.

NanoLuc® luciferase is more stable than beetle and Renillaluciferase reporter proteins. Due to protein stability,reporter response may lag behind the underlying

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transcriptional events by several hours. To reduce thecellular half-life of the reporter gene and improve reporterresponsiveness, Promega scientists developed destabilizedluciferase reporters by genetically fusing a proteindegradation sequence to the luciferase gene products (Liet al. 1998). After evaluating many degradation sequencesfor their effect on response rate and signal magnitude,Promega scientists chose two sequences: the PEST proteindegradation sequence and a second sequence composedof two protein degradation sequences (CL1 and PEST). Theluc2 gene with a PEST sequence (luc2P) and CL1 and PESTsequences (luc2CP), named the RapidResponse™ genes,respond more rapidly to stimuli but at the expense of signalintensity. The luc2P gene (approximately 1-hour proteinhalf-life; half-life varies by cell line) responds much morerapidly than luc2 (approximately 3-hour half-life), withmoderate signal intensity. The luc2CP gene (approximately0.4-hour half-life) responds more quickly and has lowestsignal intensity. The NanoLuc® gene product with a PESTsequence (NlucP) has a protein half-life of about 20 minutesbut still maintains a bright signal due to the enzyme'shigher specific activity. These destabilized reportersrespond faster and often display greatersignal-to-background compared to nondestabilizedcounterparts.Addition of a Secretion SignalNative Oplophorus luciferase catalyzes the luminescentoxidation of the substrate coelenterazine to produce bluelight. The enzyme is secreted by the shrimp as a defensemechanism against predation. In the laboratory, such asecreted reporter is useful when extracellular detection ispreferred—for example, to avoid cell lysis and maintaincell viability. To maximize the efficiency of protein secretionin mammalian cells, Promega scientists replaced the nativesecretion signal with a mammalian protein secretion signal,resulting in the secNluc gene. Secreted NanoLuc® luciferaseis ideal for nondestructive reporter gene assays in culturedcells due to the enzyme's thermostability, which allows astable enzyme to accumulate in the extracellular medium.

C. pGL4 Luciferase Reporter VectorsVectors used to deliver the reporter gene to host cells arecritical for overall reporter assay performance. Crypticregulatory sequences such as transcription factor-bindingsites and promoter modules within the vector backbonecan lead to high background and anomalous responses.This is a common issue for mammalian reporter vectors,including the pGL3 Luciferase Reporter Vectors. Promegascientists extended the successful "cleaning" strategydescribed above for reporter genes to the entire pGL3Vector backbone, removing cryptic regulatory sequenceswherever possible, while maintaining reporter functionality.Other modifications include a redesigned multiple cloningregion with a unique SfiI site to facilitate easy transfer ofthe DNA element of interest, removal of the f1 origin ofreplication and deletion of an intron. In addition, a syntheticpoly(A) signal/transcriptional pause site was placedupstream of either the multiple cloning region (in

promoterless vectors) or HSV-TK, CMV or SV40 promoter(in promoter-containing vectors). This extensive effortresulted in the totally redesigned and unique vectorbackbone of the pGL4 Vectors.The pGL4 family of luciferase vectors incorporates a varietyof features such as your choice of firefly or Renilla luciferase,Rapid Response™ versions for improved temporalresponse, mammalian selectable markers, basic vectorswithout promoters, promoter-containing control vectorsand predesigned vectors with your choice of severalresponse elements (Figure 8.6). These vectors encodeoptimized reporter genes that offer additional luminescencecolors, improved codon usage for mammalian expressionand fewer cryptic regulatory sequences such astranscription factor-binding sites that could affect proteinexpression in mammalian cells.

D. pNL VectorsNanoLuc® luciferase is available in a variety of vectors foruse in reporter gene assays of transcriptional control. ThesepNL vectors are based on the pGL4 vector backbone andthus offer many of the same advantages: Removal oftranscription factor-binding sites and other potentialregulatory elements to reduce the risk of anomalous results,easy sequence transfer from existing plasmids and a choiceof several promoter sequences. The family of vectors offera choice of NanoLuc® genes (unfused Nluc,PEST-destabilized NlucP and secreted secNluc). TheseNanoLuc® gene variations are codon-optimized and havehad many potential regulatory elements or otherundesirable features such as common restriction enzymesites removed.

III. Luciferase Reporter Assays and ProtocolsThe challenge when designing bioluminescent assays isharnessing this efficient light-emitting chemistry foranalytical methodologies. Most commonly this is done byholding the concentration of each reaction componentconstant, except for one component that is allowed to varyin relation to the biomolecular process of interest. Whenthe reaction is configured properly, the resultant light isdirectly proportional to the variable component, thuscoupling an observable parameter to the reaction outcome.In assays using luciferase, the variable component may bethe luciferase itself, its substrates or cofactors. Due to thevery low backgrounds in bioluminescence, the linear rangeof this proportionality can be enormous, typically extending104- to 108-fold over the concentration of the variablecomponent.The following section provides information about specificbioluminescent reporters and assays, including how tochoose the correct reporter genes to suit your researchneeds. Tables 8.1, 8.2, 8.3 and 8.4 include a summary ofavailable luciferase genes, assays and reagents.

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Selectable Marker

– None– Hygror

– Neor

– Puror

Luciferase Gene

– Firefly (luc2) • Rapid Response™ (–P, –CP)– Renilla (hRluc) • Rapid Response™ (–P, –CP)

UpstreamElement

– Multiple cloning region– Promoter/ response elements

4897

MA

Ampr

pGL4 Vectors

ori

SV40 latepoly(A) signal

SV40 earlyenhancer/promoter

Syntheticpoly(A)

Poly(A) block(for background

reduction)

Figure 8.6. The family of pGL4 Luciferase Reporter Vectors incorporates a variety of additional features, such as a choice of luciferasegenes, Rapid Response™ versions, a variety of mammalian selectable markers and vectors with or without promoters and responseelements.

A. Single-Reporter AssaysAssays based on a single reporter provide the quickest andleast expensive means to acquire gene expression data fromcells. However, because cells are inherently complex, theinformation gleaned from a single-reporter assay may beinsufficient to achieve detailed and accurate results. Thus,one of the first considerations when choosing a reportermethodology is deciding if the depth of information froma single reporter is sufficient or if you would benefit fromthe additional information that can be gleaned from asecond reporter (e.g., normalization of differences intransfection efficiency or cell number). If more informationis required, see the Dual-Reporter Assays section.When choosing a luciferase assay, a trade-off betweenluminescence intensity and duration is often necessarybecause bright reactions fade relatively quickly. Using afirefly or Renilla luciferase assay that yields maximumluminescence results in higher sensitivity, but using anassay with a longer signal half-life and a more stableluminescent signal is more convenient when performingassays in multiwell plates. Of the extended half-life fireflyluciferase assays, we recommend the ONE-Glo™ LuciferaseAssay System (Cat.# E6110) and ONE-Glo™ EX LuciferaseAssay System (Cat.# E8110). The ONE-Glo™ LuciferaseAssay System yields maximal luminescence intensity, hasa 45-minute half-life and is more tolerant of nonoptimalreaction conditions. The ONE-Glo™ EX Reagent has alonger signal half-life (approximately 2 hours), is morestable at 4°C or room temperature once reconstituted,exhibits less interference from phenol red in cell culturemedium and has less odor than other luciferase assayreagents.

The Steady-Glo® Luciferase Assay System provides evenlonger luminescence duration but with lower intensity. TheSteady-Glo® Reagent is added directly to the culturemedium for mammalian cells, so prior cell lysis is notnecessary. This allows you to grow cells in multiwell plates,

and then measure reporter expression in a single step.Renilla luciferase assays with different signal intensitiesand half-lives are also available.Sacrificing luminescence intensity for signal half-life is lessof a concern with NanoLuc® luciferase. The Nano-Glo®

Luciferase Assay System (Cat.# N1110) provides a simple,single-addition reagent that generates a glow-type signalwith a half-life of approximately 120 minutes in commonlyused tissue culture media. The reagent contains an integrallysis buffer, allowing direct use with cells expressingNanoLuc® luciferase, but is also compatible with culturemedium containing secreted luciferase.

Additional Resources for Single-Reporter AssaysTechnical Bulletins and Manuals

TM369 Nano-Glo® Luciferase Assay System TechnicalManual

TM292 ONE-Glo™ Luciferase Assay SystemTechnical Manual

TM432 ONE-Glo™ EX Luciferase Assay SystemTechnical Manual

TM052 Bright-Glo™ Luciferase Assay SystemTechnical Manual

TM051 Steady-Glo® Luciferase Assay SystemTechnical Manual

TB281 Luciferase Assay System Technical BulletinTM055 Renilla Luciferase Assay System Technical

ManualTM329 Renilla-Glo™ Luciferase Assay System

Technical ManualTM259 pGL4 Luciferase Reporter Vectors Technical

ManualCitationsEmonet , S.F. et al. (2009) Generation of recombinantlymphocytic choriomeningitis virus with trisegmented

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genomes stably expressing two additional genes of interest. Proc. Natl. Acad. Sci. USA 106, 3473–8.The lymphocytic choriomeningitis virus (LCMV) was usedas a model to create a trisegmented recombinant arenavirusin which viral genes were replaced by a gene of interest.One such engineered virus, r3LCMV CAT/FLuc, was usedin a pilot screen to identify anti-arenaviral compounds.Firefly luciferase (FLuc) activity was measured using theONE-Glo™ Luciferase Assay System.PubMed Number: 19208813Cai , Y. et al. (2014) DNA transposition by proteintransduction of the piggyBac transposase from lentiviralGag precursors. Nucleic Acids Res. 42, e28.Researchers were looking for alternative methods to usingtransposase vectors carried by lentiviruses to insert genesinto cellular DNA without the cytotoxicity that may occurif the transposase gene integrated into the genome. Theauthors generated lentiviral particles that carried thetransposase protein to deliver genes at an equal efficiencyas the conventional plasmid-based method. They clonedthe NanoLuc® luciferase reporter gene into agag-pol-integrase-defective packaging construct and clonedfirefly luciferase into the piggyBac transposon lentiviralvector. They also created Gag-pol constructs that expresseda hyperactive piggyBac transposase. Lentiviral particles(LPs) were generated by cotransfection of several plasmidsinto 293T cells. These NanoLuc® LPs were added to HeLacells with or without pseudotyping by Vesicular StomatitisVirus envelope glycoprotein, and after 48 hours,luminescence was measured using the Nano-Glo®

Luciferase Assay System. To analyze how well the fireflyluciferase gene was transferred, HaCaT and ARPE-19 cellswere incubated with increasing amounts of either wildtypeor mutated PBase/firefly luciferase transposon LPs. Afterten days, the transduced cells were assessed forluminescence using the ONE-Glo™ Luciferase AssaySystem. HEK293 cells, primary keratinocytes and normalhuman dermal fibroblasts also were incubated with eitherwildtype or mutated PBase/firefly luciferase transposonLPs, and after eight days, firefly luminescence wasmeasured.PubMed Number: 24089552Lin , P.F. et al. (2003) A small molecule HIV-1 inhibitor thattargets the HIV-1 envelope and inhibits CD4 receptorbinding. Proc. Natl. Acad. Sci. USA 100, 11013–8.To test the effect of BMS-378806, a new small-moleculeinhibitor of HIV-1, a cell fusion assay was developed. Targetcells that stably expressed CD4, CXCR4 or CCRS receptorsand carried a responsive luciferase plasmid were prepared.Effector cells were transiently transfected with the HIV coatprotein gp160 from various strains of virus and a plasmidto activate the response element controlling luciferaseexpression. If the cells fused, luciferase was synthesized.To measure cell fusion, effector cells were plated with targetcells at a ratio of 1:2 in 96-well plates, and then incubatedwith various concentrations of BMS-378806 for 12–24 hours.

Luciferase activity was measured using Steady-Glo®

Luciferase Assay System.PubMed Number: 12930892Promega PublicationsONE-Glo™ Luciferase Assay System: New substrate, betterreagent.The bioluminescence advantagepGL4 Vectors: A new generation of luciferase reportervectors.Bright-Glo™ and Steady-Glo® Luciferase Assay Systems:Reagents for academic and industrial applications.

B. Dual-Reporter AssaysThe most commonly used dual-reporter assays measureboth firefly and Renilla luciferase activities. These luciferasesuse different substrates and thus can be differentiated bytheir enzymatic specificities. Performing mostdual-luciferase assays involves adding two reagents to eachsample and measuring luminescence following eachaddition. Addition of the first reagent activates the fireflyluciferase reaction; addition of the second reagentextinguishes firefly luciferase activity and initiates thesecond luciferase reaction (Renilla luciferase). One suchassay is the Dual-Luciferase® Reporter Assay System (Cat.#E1910), which measures both luciferase activitiessequentially from a single sample. This system requires celllysis prior to performing the assay and requires the use ofreagent injectors with multiwell plates.

The Dual-Glo® Luciferase Assay System also measuresboth firefly and Renilla luciferase activities from a singlesample and provides longer luminescence duration (inother words, a longer luciferase signal half-life), which isuseful when performing reporter assays in multiwell plates.As with other reagents designed for use in multiwell plates,the Dual-Glo® Assay works directly in mammalian cellculture medium without prior cell lysis.

Another dual-luciferase assay option, the Nano-Glo®

Dual-Luciferase® Reporter Assay System (Cat.# N1610),involves firefly and NanoLuc® luciferases. This combinationof reporters and assay has several advantages, includinglower background levels due to better quenching of fireflyluciferase activity and higher sensitivity due to brighterNanoLuc® luminescence. The long signal half-lives(approximately 2 hours for firefly luciferase and NanoLuc®

luciferase) make this assay particularly useful forhigh-throughput screening. In addition, the lower fireflyluciferase background and brighter NanoLuc® signal allowsgreater flexibility in assay configuration. Either NanoLuc®

or firefly luciferase can be used as the experimentalreporter, with the other luciferase used as the normalizationcontrol, or both reporter genes can be used as theexperimental reporter, providing you with moreinformation from a single set of assays.

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In general, dual-reporter assays improve experimentalaccuracy and efficiency by: i) reducing variability that canobscure meaningful correlations; ii) normalizing interferingphenomena that may be inherent in the experimentalsystem; and iii) normalizing differences in transfectionefficiencies between samples. In addition, dual-reporterassays can reduce the number of nonrelevant hits (i.e.,"false" hits) due to direct interaction of compounds withthe reporter protein in high-throughput screenings. Theuse of co-incidence reporters—reporters that have dissimilarprofiles of compound interference—can help differentiatecompounds that modulate the biological pathway of interestfrom those that affect the stability or activity of the reporterenzyme.Reducing VariabilityBecause cells are complex micro-environments, significantvariability can occur between samples within an experimentand between experiments performed at different times.Challenges include maintaining uniform cell density andviability between samples and accomplishing reproducibletransfection of exogenous DNA. The use of multiwell platesintroduce variables such as edge effects, which are broughtabout by differences in heat distribution and humidityacross a plate. Dual-reporter assays can control for muchof this variability, leading to more accurate and meaningfulcomparisons between samples (Hawkins et al. 2002; Hannahet al. 1998; Wood, 1998; Faridi et al. 2003).Dual-Color AssaysIn some cases, researchers may prefer to activate bothluciferase assays simultaneously by adding a single reagent.This reduces total assay volume and liquid-handlingrequirements. Promega scientists have developed clickbeetle luciferases, which are related to firefly luciferase andcan be differentiated by the color of luminescence: red andgreen. The sequences and structures of these Chroma-Luc™luciferases are nearly identical, with only a few amino acidsubstitutions necessary to create the different colors. Thisstructural similarity means that both the control andexperimental reporters are likely to respond similarly tobiochemical changes within the cell, resulting in moreaccurate normalization to the control reporter. For situationswhere genetic recombination between the two luciferasegenes is a concern, Promega scientists have developed twogenes encoding the green-emitting reporter: one that isnearly identical to the luciferase emitting red luminescence,and one that is maximally divergent from it. TheChroma-Luc™ genes encoding both the red- andgreen-emitting luciferases are codon-optimized formammalian cells.The Chroma-Glo™ Luciferase Assay System measuresChroma-Luc™ activities in multiwell plates. TheChroma-Glo™ Reagent formulation supports optimalreaction kinetics for both reporters simultaneously andworks directly in culture medium. Because colordifferentiation is required for the Chroma-Glo™ Assay, aluminometer capable of using colored optical filters isrequired. Since the light is transmitted through optical

filters, sensitivity relative to other assay methods is reduced.Both red- and green-emitting Chroma-Luc™ luciferaseactivities are detectable using optical filters when therelative concentrations differ by up to 100-fold. This is lessthan dual-luciferase assays that use chemical differentiation,where the relative concentrations may differ by more than1,000-fold.

Additional Resources for Dual-Reporter and Dual-ColorAssaysTechnical Bulletins and Manuals

TM040 Dual-Luciferase® Reporter Assay SystemTechnical Manual

TM426 Nano-Glo® Dual-Luciferase® Reporter AssaySystem Technical Manual

TM058 Dual-Glo® Luciferase Assay System TechnicalManual

TM062 Chroma-Glo™ Luciferase Assay SystemTechnical Manual

TM259 pGL4 Luciferase Reporter Vectors TechnicalManual

Online ToolsDual-Luciferase® Reporter Assay System videoA more powerful dual luciferase assay chalk talk.Nano-Glo® Dual-Luciferase® Reporter Assay Systemmanual protocol video.Nano-Glo® Dual-Luciferase® Reporter Assay Systeminjectors protocol video.CitationsGuo, R. et al. (2013) Novel microRNA reporter uncoversrepression of Let-7 by GSK-3β. PLos ONE 8, e66330.Members of the let-7 microRNA family are thought to actas tumor suppressors. The authors of this paper describea sensitive luciferase-based reporter assay for detectinglet-7 miRNA activity in cells. The reporter construct wasbased on the pmirGLO Vector, which contains fireflyluciferase as the reporter gene and Renilla luciferase as aninternal control. The authors inserted let-7 miRNA targetsites at the 3′ end of the firefly luciferase gene. Interactionof let-7 miRNA with these target sequences resulted inreduced luciferase activity. The authors used the reporterconstruct to screen a kinase inhibitor library for compoundsthat repressed let-7 activity in ovarian cancer cells andidentified GSK-3β as a potential target for therapeutics.PubMed Number: 23840442Mateo, M. et al. (2011) VP24 is a molecular determinant ofEbola virus virulence in guinea pigs. J. Infect. Dis. 204,1011–20.The authors used the Dual-Glo® Luciferase Assay Systemto measure a pISG54 promoter-driven firefly luciferasegene and pRL-TK plasmid constitutively expressing Renillaluciferase, and either a plasmid expressing thecorresponding variants of Ebola virus (EBOV) structuralprotein VP24 construct (phCMV-EBOV-VP24) or emptyphCMV in HEK 293T and GPC-16 transfected cells. Cells

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were stimulated with interferon 24 hours post-transfection,harvested 16 hours later and assayed for dual-luciferaseactivity using the Dual-Glo® Luciferase Assay System. Dataindicated that mutations in the V24 protein were associatedwith EBOV virulence but that this virulence was not linkedto the interferon-antagonist function of V24 protein.PubMed Number: 21987737Elbashir, S.M. et al. (2001) Duplexes of 21-nucleotide RNAsmediate RNA interference in cultured mammalian cells. Nature 411, 494–8.In this landmark paper decribing RNA interference inmammalian cells, firefly and Renilla luciferase geneproducts were targeted for degradation. NIH/3T3, HEK293,HeLa S3, COS-7 and S2 cells were transfected with 1μg ofpGL2-Control or pGL3-Control Vector, 0.1μg pRL-TKVector and 0.21μg siRNA duplex targeting either firefly orRenilla luciferase. The Dual-Luciferase® Reporter AssaySystem was used 20 hours post-transfection to monitorluciferase expression. Transfection with 21bp dsRNAcaused specific degradation of a targeted sequence. Thiswas the first demonstration of the RNAi effect inmammalian cells.PubMed Number: 11373684Promega PublicationsNIH Chemical Genomics Center: Small-molecule screeningfor investigating fundamental biological questions.Deciphering the pGL4 Vector code.pGL4 Vectors: A new generation of luciferase reportervectors.Increased Renilla luciferase sensitivity in theDual-Luciferase® Reporter Assay System.Introducing Chroma-Luc™ technology.Dual-Glo™ Luciferase Assay System: Convenient dualreporter measurements in 96- and 384-well plates.

C. Live-Cell SubstratesResearchers strive to monitor cellular activities with as littleimpact on the cell as possible. The endpoint of anexperiment, however, sometimes requires completedisruption of cells so that the environment surroundingthe reporter enzyme can be carefully controlled. Recently,Promega scientists developed a variety of live-cellsubstrates to monitor Renilla and firefly luciferase activitywithout disrupting cells.Renilla luciferase requires only oxygen and coelenterazineto generate luminescence, providing a simple luciferasesystem to measure luminescence from living cells.Unfortunately, coelenterazine is unstable in aqueoussolutions, so it can be difficult and inconvenient to measureRenilla luciferase. EnduRen™ and ViviRen™ Live CellSubstrates overcome this difficulty and easily generateluminescence from live cells expressing Renilla luciferase.Because luminescence is generated from living cells, thesesubstrates are ideal for multiplexing with assays thatdetermine viable cell number.

An alternative to live-cell substrates is a secreted form ofreporter protein, which can be quantified by measuringreporter activity in the cell culture medium. NanoLuc®

luciferase is ideally suited for both standard (lytic) andsecretion-based (nonlytic) reporter gene applications.Promega scientists have fused NanoLuc® luciferase to anN-terminal secretion signal, resulting in a secreted form(secNluc) that does not require cell lysis prior to or duringthe reporter assay.

Additional Resources for Live-Cell SubstratesTechnical Bulletins and Manuals

TM244 EnduRen™ Live Cell Substrate TechnicalManual

TM064 ViviRen™ Live Cell Substrate TechnicalManual

CitationsDragulescu-Andrasi, A. et al. (2011) Bioluminescenceresonance energy transfer (BRET) imaging ofprotein-protein interactions within deep tissues of livingsubjects. Proc. Natl. Acad. Sci. USA 108, 12060–5.The authors constructed red light-emitting reporter systemsbased on bioluminescence resonance energy transfer (BRET)for ratiometric imaging of protein:protein interactions incell culture and deep tissues of small animals. These BRETsystems consist of Renilla luciferase (RLuc) variants as BRETdonors, combined with two red fluorescent proteins asBRET acceptors. They used the EnduRen™ Live CellSubstrate to produce a red-shift emission maxima optimalfor deep-tissue imaging. To demonstrate this capability,the authors imaged HT-1080 cells accumulating in the lungsof nude mice. The cells expressed BRET fusion proteins inthe context of rapamycin-induced association ofFK506-binding protein 12 (FKBP12) and FKBP12rapamycin-binding domain. Mice were injected withluciferase substrate at 1.5 hours after cell injection toproduce a bioluminescence image. Their data suggest thatthe BRET systems could be used for drug screening andtarget validation applications.PubMed Number: 21730157Promega PublicationsIn vivo evaluation of regulatory sequences by analysis ofluciferase expression.Measuring Renilla luminescence in living cells.Perform multiplexed cell-based assays on automatedplatforms.Bioluminescence imaging of live trout for virus detectionusing EnduRen™ Live Cell Substrate.

D. Bioluminescent Reporters to Normalize for Changes in CellPhysiologyEvents associated with cell physiology can affect reportergene expression. Of particular concern is the effect ofcytotoxicity, which can mimic genetic downregulationwhen using a single-reporter assay. Reporter assays thatcan be multiplexed with a cell viability or cytotoxicity assay

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allow independent monitoring of both reporter expressionand cell viability to avoid data misinterpretation (Farfan etal. 2004). The use of multiplexed assays allows correlationof events within cells, such as the coupling of targetsuppression by RNAi, to the consequences on cellularphysiology (Hirose et al. 2002).Luminescent cell viability or cytotoxicity assays, such asthe CellTiter-Glo® Luminescent Cell Viability Assay andCytoTox-Glo™ Cytotoxicity Assay, use a stabilized fireflyluciferase to generate a luminescent signal that is indicativeof cell health. Because these assays contain firefly luciferase,they cannot be directly combined with a firefly luciferasereporter assay. However, the assays can be readilycombined with nondestructive luciferase assays. Forexample, expression of Renilla luciferase may be quantitatedby adding EnduRen™ or ViviRen™ Live Cell Substrate tothe culture medium and measuring luminescence. Whenthe reporter measurements are completed, theCellTiter-Glo® or CytoTox-Glo™ Reagent is added to thesample to inactivate Renilla luciferase and initiateluminescence, which is indicative of cell viability.Alternatively, secNluc can used as the reporter, and activitycan be measured from an aliquot of culture medium usingthe Nano-Glo® Luciferase Assay System.Fluorescent cell viability and cytotoxicity assays are alsoavailable to monitor cell health and normalizesingle-reporter assay results. For example, theCellTiter-Fluor™ Cell Viability Assay is a nonlytic,fluorescence assay that measures the relative number ofviable cells in a population. The CellTiter-Fluor™ Substrateenters intact cells, where it is cleaved by a live-cell proteasethat is restricted to intact cells to generate a fluorescentsignal proportional to the number of living cells. Thenumber of nonviable cells does not affect fluorescencebecause the live-cell protease becomes inactive upon lossof membrane integrity and leakage into the culturemedium. Similarly, the CytoTox-Fluor™ Cytotoxicity Assayuses a fluorogenic peptide substrate to measure the relativenumber of dead cells in cell populations by quantifying adistinct protease activity associated with cytotoxicity. Thesetwo assays are combined in the MultiTox-Fluor MultiplexCytotoxicity Assay. Finally, the CellTox™ GreenCytotoxicity Assay uses a proprietary dye that is excludedfrom viable cells but preferentially binds to DNA from deadcells. Upon DNA binding, fluorescence of the dye issubstantially enhanced, and the resulting fluorescence isproportional to the level of cytotoxicity. These assays arewell suited for multiplexing with homogeneous luciferaseassay reagents such as Bright-Glo™, Steady-Glo® andONE-Glo™ Luciferase Assay Systems (Zakowicz et al. 2008)as well as the Renilla-Glo® Luciferase Assay System.

E. Bioluminescent Reporters to Monitor RNA InterferenceRNA interference (RNAi) is a phenomenon by whichdouble-stranded RNA complementary to a target mRNAcan specifically inactivate a gene by stimulating degradationof the target mRNA. As such, RNAi has emerged as apowerful tool to analyze gene function. Since its report in

C. elegans (Fire et al. 1998), RNAi has been reported in avariety of organisms, including zebrafish, planaria, hydra,fungi, Drosophila and plant and mammalian systems. Thesephenomena have been collectively termed RNA silencingand appear to use a common set of proteins and shortRNAs. These processes are mechanistically similar but notidentical.Bioluminescent reporters have been harnessed to studyRNAi. For example, the pmirGLO Dual-Luciferase miRNATarget Expression Vector quantitatively evaluatesmicroRNA (miRNA) activity through insertion of miRNAtarget sites downstream or 3´ of the firefly luciferase gene.Reduced firefly luciferase expression indicates binding ofendogenous or introduced miRNAs to the cloned miRNAtarget sequence. This vector also encodes Renilla luciferase(hRluc-neo) as a control reporter for normalization andselection. For more information about the RNAi processand technologies that can be used to design, synthesize andevaluate short interfering RNAs (siRNAs) and small hairpinRNAs (shRNAs), refer to the RNA Interference chapter.

F. Bioluminescent Reporters in Cell-Signaling AssaysLuciferase reporter assays are widely used to investigatecellular signaling pathways and as high-throughputscreening tools for drug discovery (Brasier et al. 1992,Zhuang et al. 2006). Synthetic constructs with clonedregulatory elements directing reporter gene expression canbe used to monitor signal transduction and identify thesignaling pathways involved. By linking luciferaseexpression to specific response elements (REs) within thereporter construct, transfecting cells with this construct,subjecting the transfected cells to a particular treatment,and then measuring reporter activity, researchers candetermine what REs are used, and thus, what signalingpathways are involved. The use of inhibitors and siRNAscan be used to confirm what factors are involved in thisresponse.To speed this type of research, Promega scientists havedesigned several convenient pGL4 Vectors with your choiceof a number of response elements and regulatory sequencesto take advantage of the benefits of the pGL4 Vectorbackbone and luc2P gene. See Table 8.1. Many of thesevectors encode the hygromycin-resistance gene to allowselection of stably transfected cell lines. Alternatively,Promega offers cell lines that are already stably transfectedwith pGL4-based vectors with specific response elements.See the Luciferase Reporter Cell Lines section.Bioluminescent reporters also enable characterization ofnuclear receptors, a class of ligand-regulated transcriptionfactors that sense the presence of steroids and othermolecules inside the cell. Nuclear receptors typically residein the cytoplasm and are often complexed with associatedregulatory proteins. Ligand binding triggers translocationinto the nucleus, where the receptors bind specific responseelements via the DNA-binding domain, leading toupregulation of the adjacent gene. Bioluminescent reporterscan be harnessed to identify and characterize nuclearreceptor agonists, antagonists, co-repressors and

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co-activators using a universal receptor assay, which issimilar in many ways to the two-hybrid assay. In atwo-hybrid assay, two proteins that are thought to interactare expressed as fusion proteins, one fused with theDNA-binding domain (DBD) of the yeast GAL4transcription factor and the other fused to the VP16activation domain. Protein:protein interaction brings thetwo domains together to yield expression of a reporter genedownstream of tandem GAL4-binding sites and a minimalpromoter. The universal nuclear reporter assay can bethought of as a "one-hybrid" assay, where theligand-binding domain (LBD) of a nuclear receptor replacesthe bait and prey proteins and VP16 activation domain(Figure 8.7).To perform the universal nuclear receptor assay, simplycotransfect the cell line of interest with a construct encodingthe LBD-GAL4 DBD fusion protein and a suitable reportervector with multiple copies of the GAL4 upstreamactivation sequence (UAS) upstream of the promoter andreporter gene. Two to three days post-transfection, treatcells with the test compounds of interest, then measureluciferase activity. This approach allows you to convertany cell line into a nuclear receptor-responsive cell line,which you can use to identify receptor agonists, antagonists,co-activators and co-repressors. You can even performmutagenesis on the ligand-binding domain to determinethe effect in your responsive cell line without interferencefrom the endogenous receptor. An example of a suitablereporter construct is thepGL4.35[luc2P/9XGAL4UAS/Hygro] Vector (Cat.# E1370),which contains nine copies of the GAL4 UAS immediatelyupstream of a minimal promoter driving expression ofluc2P reporter gene. For added convenience, Promega offersHEK293 cells that are stably transfected with thepGL4.35[luc2P/9XGAL4UAS/Hygro] Vector: theGloResponse™ 9XGAL4UAS-luc2P HEK293 cells. For moreinformation, see the Luciferase Reporter Cell Lines section.Promega offers a number of additional reagents to simplifyuniversal nuclear receptor assays. The pBIND-ERα Vector(Cat.# E1390) contains the yeast Gal4 DBD and an estrogenreceptor ligand-binding domain (ER-LBD) gene fusion, andthe pBIND-GR Vector (Cat.# E1581) contains the yeast Gal4DBD and glucocorticoid receptor ligand-binding domain(GR-LBD) gene fusion. Promega also offers the pFN26A(BIND) hRluc-neo Flexi® Vector (Cat.# E1380), which allowsexpression of a fusion protein comprised of the GAL4 DBD,a linker segment and an in-frame protein-coding sequenceunder the control of the human cytomegalovirus (CMV)immediate early promoter. You simply clone the DNAfragment encoding the ligand-binding domain of yourreceptor into SgfI and PmeI sites at the 5′ and 3′ ends of alethal barnase gene, which acts as a positive selection forsuccessful ligation of the insert. Each BIND vector containsa Renilla luciferase/neomycin resistance co-reporter fornormalization of transfection efficiency or construction ofa double-stable cell line without the need for additionalcloning.

Bioluminescent reporters also are useful for studying Gprotein-coupled receptors (GPCRs), which regulate awide-range of biological functions and are one of the mostimportant target classes for drug discovery (Klabunde etal. 2002). The firefly luciferase-based GloSensor™ cAMPAssay provides a sensitive and easy-to-use format tointerrogate overexpressed or endogenous GPCRs that signalvia changes in intracellular cAMP concentration. The assayuses genetically encoded biosensor variants comprised ofcAMP-binding domains fused to mutant forms of Photinuspyralis luciferase. cAMP binding induces conformationalchanges that promote large increases in light output.Following pre-equilibration with the GloSensor cAMPReagent, cells transiently or stably expressing the biosensorvariant can be used to assay GPCR function using a nonlyticassay format, enabling kinetic measurements of cAMPaccumulation or turnover in living cells. Moreover, theassay offers a broad dynamic range, with up to 500-foldchanges in light output. Extreme sensitivity allows detectionof Gi-coupled receptor activation or inverse agonist activityin the absence of artificial stimulation by compounds suchas forskolin. For more information, visit the GloSensor™Technology page.Similar luminescent biosensors exist to detect site-specificprotease activity. To detect the protease, an oligonucleotideencoding the protease recognition sequence is cloned intothe pGloSensor™-10F Linear Vector (Cat.# G9461), and theGloSensor™ protein containing the protease site of interestis synthesized in a cell-free protein expression system andsubsequently used as a protease substrate. Cleavage of theprotease recognition sequence leads to activation of theGloSensor™ protein and light emission. The level ofluminescence correlates to protease activity. Luminescentbiosensors to detect cGMP and caspase-3/7 activity in vivoare also available.

Additional Resources for Bioluminescent Reporters inCell-Signaling AssaysTechnical Bulletins and Manuals

9PIE847 pGL4.29[luc2P/CRE/Hygro] Vector ProductInformation

9PIE848 pGL4.30[luc2P/NFAT-RE/Hygro] VectorProduct Information

9PIC935 pGL4.31[luc2P/GAL4UAS/Hygro] VectorProduct Information

9PIE849 pGL4.32[luc2P/NF-κB-RE/Hygro] VectorProduct Information

9PIE134 pGL4.33[luc2P/SRE/Hygro] Vector ProductInformation

9PIE135 pGL4.34[luc2P/SRF-RE/Hygro] VectorProduct Information

9PIE137 pGL4.35[luc2P/9XGAL4UAS/Hygro] VectorProduct Information

9PIE136 pGL4.36[luc2P/MMTV/Hygro] VectorProduct Information

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9052

MA

pG5lucVector GAL4 GAL4 GAL4 GAL4 GAL4

X Y

luc+TATA

pG5lucVector

VP16

GAL4

GAL4UAS TATAGAL4

UASGAL4UAS

GAL4UAS

GAL4UAS luc+

GAL4

Nuclear ReceptorLigand-BindingDomain

A. Two-Hybrid Assay

B. Universal Nuclear Receptor Assay

Figure 8.7. The two-hybrid assay and universal nuclear receptor assay. Panel A. The traditional two-hybrid assay. The pG5luc Vectorcontains five GAL4 upstream activator sequences (UAS) upstream of a minimal TATA box, which in turn is upstream of the firefly luciferasegene. Interaction between the two test proteins, expressed as GAL4-X and VP16-Y fusion proteins, results in an increase in luciferasetranscription and expression. Panel B. The universal nuclear receptor assay. The ligand-binding domain of the nuclear receptor replacesthe bait and prey proteins and VP16 activation domain. Within the cell, binding of the appropriate ligand to the nuclear receptor-GAL4fusion protein releases any co-repressors bound to the ligand-binding domain. Co-activators help recruit the transcription machinery tothe luciferase reporter gene, resulting in luciferase expression and an increase in luminescence.

Promega PublicationsNovel pGL4 reporter vector panel for profiling cellularstress and chemical toxicity.

G. Luciferase Reporter Cell LinesThe GloResponse™ Cell Lines contain optimized luciferasereporter technology integrated into a cell line. These cellsuse the destabilized and optimized luc2P gene for greatersensitivity and shorter induction times compared to nativereporter enzymes. The GloResponse™ NFAT-RE-luc2PHEK293 Cell Line, NFκB-RE-luc2P HEK293 Cell Line andCRE-luc2P HEK293 Cell Line allow rapid and convenientanalysis of cell signaling through the NFAT, NF-κB orcAMP response pathways, respectively, via activation of aluciferase reporter gene. Non-native activators of thesepathways, including GPCRs, can be studied after theappropriate proteins are introduced by transfection.GPCR signaling pathways can be categorized into threeclasses based on the G protein α-subunit involved: Gs, Gi/oand Gq. The GloResponse™ CRE-luc2P HEK293 Cell Linecan be used to study and configure screening assays forGs- and Gi/o-coupled GPCRs, which signal through cAMPand the cAMP reponse element (CRE). For Gq-coupledGPCRs, which signal through calcium ions and NFAT-RE,use the GloResponse™ NFAT-RE-luc2P HEK293 Cell Line.GPCR assays that use the GloResponse™ Cell Lines areamenable to high-throughput screening. These assaystypically have greater response dynamics (fold of induction)than other assay formats and generate high-quality dataas indicated by the high Z′ values.

The GloResponse™ Cell Lines were generated by clonalselection of HEK293 cells stably transfected withpGL4-based vectors carrying specific response elementsfor the pathway of interest. These cell lines incorporateimprovements developed for the pGL4 Vectors forenhanced performance and reduced background reporterexpression. The destabilized luc2P luciferase reporterimproves responsiveness to transcriptional dynamics andis codon-optimized for enhanced expression in mammaliancells. The result is cell lines with very high reporterinduction levels when the pathway of interest is activated.

Additional Resources for Luciferase Reporter Cell LinesTechnical Bulletins and Manuals

TB362 GloResponse™ CRE-luc2P HEK293 Cell LineTechnical Bulletin

TB363 GloResponse™ NFAT-RE-luc2P HEK293Cell Line Technical Bulletin

TB380 GloResponse™ NFκB-RE-luc2P HEK293 CellLine Technical Bulletin

H. Bioluminescent Reporters to Study Protein DynamicsThe rate of protein turnover is tightly regulated for manysignaling proteins. Protein stabilization and subsequentaccumulation occur in response to changing cellularconditions, resulting in activation of downstreamtranscriptional events. Promega offers reporter vectors tostudy the rate of protein turnover of two key signalingproteins involved in response to cellular stress:hypoxia-inducible factor-1A (HIF1A) and nuclear factorerythroid 2-related factor-2 (NRF2). The pNLF1-HIF1a

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[CMV/neo] Vector (Cat.# N1381) enables simplequantification of intracellular HIF1A protein levels to studythe dynamics of this signaling protein in mediating cellularresponse to hypoxia. The vector encodes NanoLuc®

luciferase fused to the C-terminus of the HIF1A proteinunder control of the CMV promoter. By using a constitutivepromoter such as CMV to drive expression of theHIF1A-NanoLuc® fusion protein, changes in light outputcorrelate to dynamic changes in protein levels. Similarly,the pNLF1-NRF2 [CMV/neo] Vector (Cat.# N1391) enablessimple quantification of intracellular NRF2 protein levelsto study the dynamics of this signaling protein in mediatingcellular response to oxidative stress. The vector encodesNanoLuc® luciferase fused to the C-terminus of the NRF2protein under the control of the CMV promoter and issupplied with the pKEAP1 [CMV/Hygro] Vector for properregulation of intracellular NRF2 levels.

Additional Resources for Bioluminescent Reporters toStudy Protein DynamicsTechnical Bulletins and Manuals

9PIN138 pNLF1-HIF1a [CMV/neo] Vector ProductInformation

9PIN139 pNLF1-NRF2 [CMV/neo] Vector ProductInformation

Promega PublicationsDetection of HIF1α expression at physiological levels usingNanoLuc® Luciferase and the GloMax® Discover SystemMeasuring intracellular protein lifetime dynamics usingNanoLuc® luciferase.

I. Promega Reporter Vectors and AssaysThere are many factors to consider when choosing areporter vector and assay. The tables in this section showthe various features of reporter vectors, including thereporter gene, presence of a multiple cloning region, genepromoter, protein degradation sequences and mammalianselectable marker (Tables 8.1, 8.2 and 8.3), as well as thefeatures of Promega reporter assays (Table 8.4). These tableswill help you choose a reporter vector or assay. For themost up-to-date selection of reporter vectors and assays,visit the Promega web site. For a step-by-step guide to helpyou choose the best luciferase reporter vector for yourstudies, use the Luciferase Reporter Vector Selector.

IV. Getting the Most Out of Your Genetic Reporter AssaysA. Introduction

When performed properly, experiments using geneticreporters can yield tremendous amounts of information.However, there are several important considerations whendesigning and performing these types of experiments toensure that the data are sound.

B. Choice of Reporter Gene and AssayThe ideal reporter gene is one that is not endogenouslyexpressed in the cell of interest and is amenable to assaysthat are sensitive, quantitative, rapid, easy, accurate,

reproducible and safe. One of the first considerations whenperforming genetic reporter asays is choosing a reportergene. The characteristics and advantages of commonluminescent reporters are discussed earlier; see theLuciferase Genes and Vectors section.Also important when choosing a reporter methodology isdeciding if a single reporter is sufficient or if the greaterinformation density of a dual assay is preferred. The useof two reporters can improve experimental outcomes bytwo means: 1) normalizing interfering phenomena that maybe inherent in biological systems such as cytotoxicity; and2) reducing random variability due to factors such asdifferences in cell density and viability, differences intransfection efficiency and variability due to "edge effects"brought about through differences in heat capacity andhumidity across a multiwell plate. For more information,see the Luciferase Reporter Assays and Protocols section.Another important consideration is reporter assaycharacteristics such as signal half-life, signal intensity, lyticor nonlytic protocol, and compatibility with multiwellplates in a high-throughput format. Assays based on asingle reporter provide the quickest and least expensivemeans to acquire data. However, because cells areinherently complex, the quantity of data available from asingle reporter may be insufficient to achieve reliableresults. A discussion of the most common reporter assaysis provided in the Luciferase Genes and Vectors section.

C. Reporter Construct DesignA common question when designing reporter vectors forpromoter dissection is “What sequences should I clone intomy vector?”. Unfortunately, there is no one correct answer.The necessary sequences depend on the biological questionyou are trying to answer and the vector into which you arecloning. An advantage of transgenic reporter assays is thatyou can control which elements are examined. You mightinclude the entire proximal promoter (including ~1kbupstream of the promoter), a specific promoter subsectionor as little as a single response element. When generatinga transcriptional fusion or using a vector with notranscription start site, you might include the +1transcription start site. The reporter might include the 5′UTR if you want to understand how this sequence mayaffect promoter activity, but keep in mind that UTRsequences also can affect post-transcriptional regulation.The reporter might include an intron and all or part of oneor both flanking exons to study RNA splicing orcharacterize regulatory elements contained within theintron (but if the exons include coding sequence, be sureto clone the reporter gene in frame). The reporter mightcontain the 3′ UTR to focus on post-transcriptionalregulation through miRNA effects. You might clone anycombination of sequences from your gene of interest tolook at the integration of regulatory pathways; reporterassays allow this flexibility and refined experimentaldesign.

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D. ControlsThe proper controls are an important part of reporterassays. The most important is a control using untransfectedcells to define the background signal in the assay (fromluminometer noise or reagent background). Generally,background luminescence is inconsequential, and thesignal:background ratio is quite high in luciferase assays.Additional controls may include the parent vector used toprepare the reporter vector (minus any inserts) and apositive control vector. Measuring luciferase activity fromthe parent vector allows you to discount any reporterresponse due to the vector backbone, not the insert;experimental results can be expressed as the ratio ofexperimental vector response to parent vector response.The same function is generally provided by the secondreporter in a dual-reporter assay when using matchedvectors (i.e., pGL4 firefly and Renilla vectors).A reporter vector with a relatively strong promoter canserve as a positive control for luciferase expression anddetection in the cell line of interest and can be used tonormalize for interfering phenomena. However, strongpromoters, such as cytomegalovirus (CMV) and SV40promoters, can easily interfere with transcriptional activityof weaker promoters by sequestering transcription factorsand are more likely to be regulated by experimentaltreatments due to the high number of transcriptionfactor-binding sites. In general, we recommend avoidingpromoters with the highest activities in your cells. Vectorswith weaker promoters often are a better choice, and evenvectors without a promoter yield sufficient luciferaseactivity for normalization purposes in most cells and areless likely to be regulated by the treatment.

E. Transfection ParametersTransfection is necessary to introduce the reporter vectorinto a cell. Transient transfection is the most commonmethod, but stable transfection should be considered if youare performing the same reporter assay frequently. Bothapproaches have advantages and disadvantages. Transienttransfection allows you to vary the reporter vectors andvector ratios, but cells must be transfected for each set ofexperiments and will lose the reporter vector over time.Transfection efficiency can be low for primary cells andsome cell lines and can vary considerably. Often a secondreporter is cotransfected into cells to normalize fordifferences in transfection efficiency. These differences canbe assessed relatively in cell lysates by comparing reporteractivity or assessed absolutely by estimating the percentageof cells expressing the transferred gene by in situ staining.A second reporter also can help to determine if a responsewas due to cell toxicity and not a promoter-specific event.Because transfection can be stressful, cells are often allowedto recover prior to experimental treatment. Stabletransfection eliminates variability in transfection efficiencyand the stress of transfection but requires additional timeand effort to select stably transfected cells.

The optimal transfection reagent and conditions dependon the cell line used and must be determined empirically.Prior to performing an experiment, we recommend usinga control reporter vector with a strong promoter in the cellline of interest and varying the transfection parameters,such as vector ratio, amount of DNA and amount oftransfection reagent, to determine the optimal transfectionconditions. Efficient transfection can be critical when usingless sensitive reporters such as chloramphenicolacetyltransferase (CAT) or β-galactosidase but is less of aconcern with sensitive reporters such as luciferase. For adetailed discussion of transfection, see the Transfectionchapter of the Protocols and Applications Guide.Two important factors when transfecting cells prior to adual-reporter assay are the reporter vector ratio and relativepromoter strengths in the cell line of interest. The optimalratio often is related to promoter strength and must bedetermined empirically. It is important, especially if youmust use a reporter vector with a strong promoter, totransfect cells with several different ratios of reportervectors. For assays using pGL4 Vectors with firefly andRenilla luciferases, we recommend a 20:1 ratio as a goodstarting point, but the ratio could be as high as 1,000:1,especially if the promoter of one vector is dramaticallystronger than that of the other. The ratio also might behigher if NanoLuc® luciferase is used as one of the reporterproteins due to the enzyme's bright signal. For vectors withpromoters of equal strengths, the ratio might be closer to1:1. When performing vector titrations, be sure to transfectall cells with a constant amount of DNA to minimizedifferences in transfection efficiency due to differing DNAamounts. The ideal ratio will provide moderate butconsistent detection of the experimental luciferase signalthat is not influenced by the amount of control luciferasevector present. Regardless of the vector ratio, be sure touse enough control plasmid to provide a signal that is belowthe saturation point of the luminometer and at least threestandard deviations above background levels.

F. Assay TimingThe times between plating and transfection, transfectionand experimental treatment, and treatment and reporterassay need to be consistent within a set of experiments tominimize variability and improve assay precision andaccuracy. When plating cells prior to transfection, take intoaccount the growth rate of the cells so that cells reachproper confluency at the time of transfection. If necessary,allow time for cells to recover after transfection and thereporter to reach steady state levels of expression. Duringinitial assay optimization, perform a time course todetermine the time of peak reporter expression. The optimaltime between treatment and reporter assay depends on anumber of factors, including the kinetics of your system,longevity of the change you are monitoring (i.e., the assaywindow) and stability of the reporter protein.For early-responding genes, we recommend a reporter witha short protein half-life such as the Rapid Response™luciferase genes, which encode protein degradation

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sequences (PEST; Li et al. 1998; or CL1; Gilon et al. 1998) todestabilize the reporter protein. The Rapid Response™luciferase genes (luc2P and luc2CP) respond more quicklyand with greater magnitude to changes in transcriptionalactivity than their more stable counterpart (luc2). Adestabilized version of NanoLuc® luciferase (NlucP) is alsoavailable in certain pNL Vectors (see Table 8.2). The onsetof the response is more tightly coupled to the inductionevent. The use of more stable luciferase versions will resultin a later and longer response with a wider assay window,so assay timing is not as critical. In these cases, reporterassays are commonly performed 12–24 hours aftertreatment.

G. Cells and Cell Culture ConditionsCells and cell culture conditions used to dissect a promotercan affect assay design and results. Three types of cells arecommonly used: 1) fibroblasts, which are easy to maintainand amenable to most reporter assays but may not expressnecessary co-factors or be as biologically relevant as othercells; 2) cancer cells, which may be more relevant and areeasy to use; and 3) primary cells, which may be the mostbiologically relevant but often are difficult to obtain,maintain and transfect. Cell cultures should not beconfluent during the experiment, since confluent cells canexhibit differences in metabolism, gene expression andphysiological response compared to preconfluent cellcultures. Likewise, cells at higher passage numbers maynot behave in the same way as cells at lower passagenumbers. If necessary, grow and freeze a large quantity ofcells at a lower passage number to ensure that yourexperiments are performed with cells at a similar passagenumber. Cells should be healthy and, ideally,easy-to-transfect. When repeating an experiment, be sureto replicate cell culture and transfection conditions asclosely as possible to ensure consistency. Be aware thatcomponents of cell culture media can affect the intensityand duration of the luminescent signal of a luciferase assay.For example, phenol red can decrease relative luminescence.

H. Special Considerations for High-Throughput AssaysDue to their sensitivity and broad linearity, luminescentreporter genes are particularly well suited to a wide rangeof high-throughput applications, including genetic reporterassays and luciferase-based assays to measure diversetargets such as kinases, cytochrome p450s, proteases,apoptosis, cell viability and cytotoxicity. We recommendusing the optimized luciferase genes described earlier inthis chapter—luc2, hRluc and NanoLuc® luciferase—forhigh-throughput screening (HTS) due to the substantialincrease in expression efficiency. The resulting increase inassay sensitivity is particularly important in HTS, wherethe trend is toward miniaturization to reduce consumptionof costly screening compounds by using high-densitymultiwell plates (e.g., 1,536- or 3,456-well format).NanoLuc® luciferase has the added benefits of higherspecific activity for a brighter signal and enhanced thermalstability for fewer false hits in primary screens.

The destabilized versions of these luciferases (luc2P, luc2CP,hRlucP, hRlucCP and NlucP) enhance reporter responsedynamics, reduce assay time and minimize secondaryeffects that may arise from the prolonged incubation ofcells with screening compounds.The reporter gene assay is also an important considerationfor HTS. Many newer luciferase assays were developedwith HTS applications in mind. They often have a longersignal half-life (≥2 hours) so that the assay reagent can beadded to all wells of a set of plates and then luminescencemeasured without a noticeable drop in luminescenceintensity from beginning to end. The longer signal half-lifeeliminates the need for a luminometer with reagent injector.These newer assays often are more tolerant of inhibitorsand nonoptimal reaction conditions. For this reason, thereagent can be added directly to cells in growth medium,and there is no need to remove the medium, wash cells orperform a separate cell lysis step. This higher tolerance forinhibitors also reduces the risk of false screening hits dueto luciferase inhibition by compound libraries used incontemporary drug discovery to identify agonists andantagonists of pharmacological targets.The risk of false screening hits often can be decreasedfurther by multiplexing your reporter gene assays with cellviability or cytotoxicity assays to control for artifacts thatmay arise due to changes in cell health and by usingco-incidence reporters that have dissimilar profiles ofcompound interference to help differentiate compoundsthat modulate the biological pathway of interest from thosethat affect the stability or activity of the reporter protein.An example of a co-incidence reporter system involvesstoichiometric expression of firefly luciferase andPEST-destabilized NanoLuc® luciferase from the samepromoter using ribosome skipping mediated by the P2Apeptide. Luciferase activities are measured sequentiallyfrom the same sample using the Nano-Glo®

Dual-Luciferase® Reporter Assay System, a homogeneouslytic assay performed in an “add-read-add-read” format.For more information, view the Reducing False Hits in HTSUsing a Coincidence Reporter System presentation.To improve data quality and maximize the amount of dataobtained from a single sample during high-throughputscreenings, researchers often multiplex assays. Multiplexingrequires that the assay chemistries are compatible and thatthe separate assay signals can be distinguished usingdifferent detection modalities such as fluorescence andluminescence. For noncompatible assays, measurementsmust be done sequentially or a portion of the sample mustbe physically removed to a separate plate. One easy wayto separate a sample is to use a secreted reporter, whichaccumulates in the culture medium and can be measuredin a nondestructive, nonlytic way by using an aliquot ofculture medium to perform the assay. NanoLuc® luciferaseis readily available in a secreted form.

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V. ReferencesAngers, S. et al. (2000) Detection of β2-adrenergic receptordimerization in living cells using bioluminescence resonanceenergy transfer (BRET). Proc. Natl. Acad. Sci. USA 97, 3684–9.

Brasier, A.R. and Ron, D. (1992) Luciferase reporter gene assay inmammalian cells. Methods Enzymol. 216, 386–97.

Farfan, A. et al. (2004) Multiplexing homogeneous cell-based assaysCell Notes 10, 15–8.

Faridi, J. et al. (2003) Expression of constitutively active Akt-3 inMCF-7 breast cancer cells reverses the estrogen and tamoxifenresponsivity of these cells in vivo. Clin. Can. Res. 9, 2933–9.

Fields, S. et al. (1989) A novel genetic system to detectprotein-protein interactions. Nature 340, 245–6.

Fire, A. et al. (1998) Potent and specific genetic interference bydouble-stranded RNA in Caenorhabditis elegans. Nature 391, 806–11.

Gilon, T., Chomsky, O. and Kulka, R.G. (1998) Degradation signalsfor ubiquitin system proteolysis in Saccharomyces cerevisiae. EMBOJ. 17, 2759–66.

Hall, M.P. et al. (2012) Engineered luciferase reporter from a deepsea shrimp utilizing a novel imidazopyrazinone substrate. ACSChem. Biol. 7, 1848–57.

Hannah, R. et al. (1998) Rapid luciferase reporter assay systemsfor high-throughput studies. Promega Notes 65, 9–14.

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.

Hirose, F. et al. (2002) Drosophila Mi-2 negatively regulates dDREFby inhibiting its DNA-binding activity. Mol. Cell. Biol. 22, 5182–93.

Klabunde, T. and Hessler G. (2002) Drug design strategies fortargeting G-protein-coupled receptors. Chembiochem. 4, 928–44.

Klingenhoff, A. et al. (1999) Functional promoter modules can bedetected by formal models independent of overall nucleotidesequence similarity. Bioinformatics 15, 180–6.

Li, X. et al. (1998) Generation of destabilized green fluorescentprotein as a transcription reporter. J. Biol. Chem. 273, 34970–5.

Robers, M. et al. (2014) Measuring intracellular protein lifetimedynamics using NanoLuc® luciferase. PubHub.

Wood, K.V. (1998) The chemistry of bioluminescent reporter assays.Promega Notes 65, 14–20.

Zakowicz, H. et al. (2008) Measuring cell health and viabilitysequentially by same-well multiplexing using the GloMax ® -MultiDetection System. Promega Notes 99, 25–8.

Zhuang, F. and Liu, Y.H. (2006) Usefulness of the luciferase reportersystem to test the efficacy of siRNA. Methods Mol. Biol. 342, 181–7.

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VI. Vector Tables

Table 8.1. pGL4 Luciferase Reporter Vectors.Mammalian

SelectableMarkerGene Promoter

ProteinDegradation

Sequence

MultipleCloningRegionReporter GeneVector

NoNoNoYesluc2pGL4.10NoNohPESTYesluc2PpGL4.11NoNoCL1-hPESTYesluc2CPpGL4.12NoSV40NoNoluc2pGL4.13

HygroNoNoYesluc2pGL4.14HygroNohPESTYesluc2PpGL4.15HygroNoCL1-hPESTYesluc2CPpGL4.16

NeoNoNoYesluc2pGL4.17NeoNohPESTYesluc2PpGL4.18NeoNoCL1-hPESTYesluc2CPpGL4.19

PuroNoNoYesluc2pGL4.20PuroNohPESTYesluc2PpGL4.21PuroNoCL1-hPESTYesluc2CPpGL4.22

NominPNoYesluc2pGL4.23NominPhPESTYesluc2PpGL4.24NominPCL1-hPESTYesluc2CPpGL4.25

HygrominPNoYesluc2pGL4.26HygrominPhPESTYesluc2PpGL4.27HygrominPCL1-hPESTYesluc2CPpGL4.28HygrominP + CREhPESTNoluc2PpGL4.29HygrominP + NFAT

REhPESTNoluc2PpGL4.30

Hygroadenovirusmajor late +GAL4 UAS

hPESTNoluc2PpGL4.31

HygrominP + NF-kBRE

hPESTNoluc2PpGL4.32

Hygroserum responseelement

hPESTNoluc2PpGL4.33

HygroSRF REhPESTNoluc2PpGL4.34HygroGAL4 UAShPESTNoluc2PpGL4.35Hygromurine

mammaryhPESTNoluc2PpGL4.36

tumor viruslong terminal

repeatHygrominP +

antioxidant REhPESTNoluc2PpGL4.37

HygrominP + p53 REhPESTNoluc2PpGL4.38HygrominP + ATF6

REhPESTNoluc2PpGL4.39

HygrominP + metalRE

hPESTNoluc2PpGL4.40

HygrominP + heatshock RE

hPESTNoluc2PpGL4.41

HygrominP + hypoxiaRE

hPESTNoluc2PpGL4.42

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MammalianSelectable

MarkerGene Promoter

ProteinDegradation

Sequence

MultipleCloningRegionReporter GeneVector

HygrominP +xenobiotic RE

hPESTNoluc2PpGL4.43

HygrominP + AP1 REhPESTNoluc2PpGL4.44HygrominP +

interferonstimulated RE

hPESTNoluc2PpGL4.45

HygrominP +Sis-inducibleelement RE

hPESTNoluc2PpGL4.47

HygrominP +SMAD3/SMAD4

hPESTNoluc2PpGL4.48

binding elementRE

HygrominP +TCF-LEF RE

hPESTNoluc2PpGL4.49

HygroCMVNoNoluc2pGL4.50NeoCMVNoNoluc2pGL4.51

HygrominP + STAT5RE

hPESTNoluc2PpGL4.52

Nophosphoglyceratekinase (PGK)

NoNoluc2pGL4.53

Nothymidinekinase (TK)

NoNoluc2pGL4.54

NoNoNoYeshRlucpGL4.70NoNohPESTYeshRlucPpGL4.71NoNoCL1-hPESTYeshRlucCPpGL4.72NoSV40NoNohRlucpGL4.73NoHSV-TKNoNohRlucpGL4.74NoCMVNoNohRlucpGL4.75

HygroNoNoYeshRlucpGL4.76HygroNohPESTYeshRlucPpGL4.77HygroNoNoYeshRlucCPpGL4.78

NeoNoNoYeshRlucpGL4.79NeoNohPESTYeshRlucPpGL4.80NeoNoCL1-hPESTYeshRlucCPpGL4.81

PuroNoNoYeshRlucpGL4.82PuroNohPESTYeshRlucPpGL4.83PuroNoCL1-hPESTYeshRlucCPpGL4.84

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Table 8.2. pNL Reporter Vectors.Mammalian

SelectableMarker

GenePromoter

ProteinDegradation

Sequence

MultipleCloningRegion

ReporterGeneVector

NoNoNoYesNlucpNL1.1Nluc]NoCMVNoNoNlucpNL1.1.CMV[Nluc/CMV]NoNohPESTYesNlucPpNL1.2[NlucP]NoNoNoYessecNlucpNL1.3secNluc]NoCMVNoNosecNlucpNL1.3.CMV

[secNluc/CMV]HygroNoNoYesNlucpNL2.1[Nluc/Hygro]HygroNohPESTYesNlucPpNL2.2[NlucP/Hygro]HygroNoNoYessecNlucpNL2.3[secNluc/Hygro]

NominPNoYesNlucpNL3.1[Nluc/minP]NoCMVhPESTNoNlucPpNL3.2.CMV

HygrominPhPESTNoNlucPpNL3.2.NF-κB-RE[NlucP/NF-κB-RE/Hygro]

NominPhPESTYesNlucPpNL3.2[NlucP/minP]NominPNoYessecNlucpNL3.3[secNluc/minP]

HygroNoYes for NlucP, Nofor luc2

Yesluc2, NlucPpNLCoI1[luc2-P2A-NlucP/Hyg]

HygrominPYes for NlucP, Nofor luc2

Yesluc2, NlucPpNLCoI2[luc2-P2A-Nluc/minP/Hyg]

HygroCMVYes for NlucP, Nofor luc2

Noluc2, NlucPpNLCoI3[luc2-P2A-NlucP/CMV/Hyg]

HygroPGKYes for NlucP, Nofor luc2

Noluc2, NlucPpNLCoI4[luc2-P2A-NlucP/PGK/Hyg]

Table 8.3. Other Luciferase Reporter Vectors.Mammalian

SelectableMarker

GenePromoter

ProteinDegradation

Sequence

MultipleCloningRegionReporter GeneVector

NoNoNoYesluc+pGL3-BasicNoSV40NoYesluc+pGL3-ControlNoNoNoYesluc+pGL3-EnhancerNoSV40NoYesluc+pGL3-PromoterNoNoNoYesCBRlucpCBR-BasicNoNoNoNoCBRlucpCBR-ControlNoNoNoYesCBG68lucpCBG68-BasicNoNoNoNoCBG68lucpCBG68-ControlNoNoNoYesCBG99lucpCBG99-BasicNoNoNoNoCBG99lucpCBG99-Control

CellTiter-Glo, Dual-Glo, Dual-Luciferase, Flexi, GloMax, Nano-Glo,NanoLuc, Renilla-Glo and Steady-Glo are registered trademarks of PromegaCorporation. Bright-Glo, CellTiter-Fluor, CellTox, Chroma-Glo, Chroma-Luc,CytoTox-Fluor, CytoTox-Glo, EnduRen, GloResponse, GloSensor, ONE-Glo,Rapid Response and ViviRen are trademarks of Promega Corporation.Products may be covered by pending or issued patents or may have certainlimitations. Please visit our Web site for more information.All prices and specifications are subject to change without prior notice.Product claims are subject to change. Please contact Promega TechnicalServices or access the Promega online catalog for the most up-to-dateinformation on Promega products.© 2004–2015 Promega Corporation. All Rights Reserved.

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Table 8.4. Luciferase Reporter Assays.

Live-CellAssay

SignalStability

Single-Sampleor PlateAssayGene AssayedAssay System

Single ReporterNoShort (<0.5h)Single or

Plate1luc, luc+, luc2Luciferase Assay System

NoLong (>0.5h)Plate2luc, luc+, luc2Steady-Glo® LuciferaseAssay System

NoLong (>0.5h)Plate2luc, luc+, luc2Bright-Glo™ LuciferaseAssay System

NoLong (≥45minutes)

Plate2luc, luc+, luc2ONE-Glo™ Luciferase AssaySystem

NoLong (≥2hours)

Plate2luc, luc+, luc2ONE-Glo™ EX LuciferaseAssay System

NoShort (<0.5h)Single orPlate1

Rluc, hRlucRenilla Luciferase AssaySystem

NoLong (≥60minutes)

Plate2Rluc, hRlucRenilla-Glo® LuciferaseAssay System

Yes3Long (≥2.0h)Single orPlate2

Nluc, secNlucNano-Glo® Luciferase AssaySystemDual Reporter

NoLong (>0.5h)Plate2luc+, luc2, Rluc, hRlucDual-Glo® Luciferase AssaySystem

NoShort (<0.5h)Single (Cat.#E1910)

luc+, luc2, Rluc, hRlucDual-Luciferase® ReporterAssay System

NoShort (<0.5h)Plate1 (Cat.#E1980)

NoLong (≥2h)Plate2Nluc, luc, luc+, luc2Nano-Glo® Dual-Luciferase®Reporter Assay System

NoLong (>0.5h)Plate2CBRluc, CBG99luc, CBG68lucChroma-Glo™ LuciferaseAssay SystemLive-Cell

YesLong (>0.5h)Plate1Rluc, hRlucEnduRen™ Live CellSubstrate

YesShort (<0.5h)PlateRluc, hRlucViviRen™ Live CellSubstrate

1Use with plates only when the luminometer has a reagent injector.2We do not recommend the use of this product with automated reagent injectors.3Use for live-cell assays by quantifying the secreted form of NanoLuc® luciferase in culture medium.

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