Genetic reporter systems have contributed greatly to the study of eukaryotic gene
expression and regulation. Although reporter genes have played a significant role in
numerous applications, both in vitro and in vivo, they are most frequently used as
indicators of transcriptional activity in cells.
Typically, a reporter gene is joined to a promoter sequence in an expression vector
that is transferred into cells. Following transfer, cells are assayed for the presence
of the reporter by directly measuring the reporter protein itself or the enzymatic
activity of the reporter protein. An ideal reporter gene is one that is not endogenously
expressed in the cell of interest and is amenable to assays that are sensitive,
quantitative, rapid, easy, reproducible and safe.
Analysis of cis-acting transcriptional elements is a frequent application for
reporter genes. Reporter vectors allow functional identification and characterization of
promoter and enhancer elements because expression of the reporter protein correlates
with transcriptional activity of the reporter gene. For these types of studies, promoter
regions are cloned upstream or downstream of the gene. The promoter-gene fusion is
introduced into cultured cells by standard transfection methods or into a germ cell to
produce transgenic organisms. The use of reporter gene technology allows
characterization of promoter and enhancer elements that regulate cell, tissue and
development-defined gene expression.
Trans-acting factors can be assayed by co-transfer of the promoter-reporter gene
fusion DNA with another cloned DNA expressing a trans-acting protein or RNA of interest
or by activating the trans-acting factors through a treatment of the samples. The
protein could be a transcription factor that binds to the promoter region of interest
cloned upstream of the reporter gene. For example, when tat protein is expressed from
one vector in a transfected cell, the activity of different HIV-1 LTR sequences linked
to a reporter gene increases, and the activity increase is reflected in an increase of
reporter gene protein activity.
Reporter proteins can be assayed by detecting inherent characteristics, such as
enzymatic activity or spectrophotometric characteristics, or indirectly with
antibody-based assays. In general, enzymatic assays are quite sensitive due to the small
amount of reporter enzyme required to generate detectable levels of reaction products. A
potential limitation of enzymatic assays is high background if there is endogenous
enzymatic activity in the cell (e.g., β-galactosidase). Antibody-based assays are
generally less sensitive but will detect the reporter protein whether it is
enzymatically active or not.
Fundamentally, a reporter assay is a means to translate a biomolecular effect into
an observable parameter. While there are theoretically many strategies by which this can
be achieved, in practice the reporter assays capable of delivering the speed, accuracy
and sensitivity necessary for effective screening are based on photon production.
Photon production is realized primarily through fluorescence and
chemiluminescence. Both processes yield photons as a consequence of energy
transitions from excited-state molecular orbitals to lower energy orbitals. However,
they differ in how the excited-state orbitals are created. In chemiluminescence, the
excited states are the product of exothermic chemical reactions, whereas in
fluorescence the excited states are created by absorption of light.
This distinction of how the excited states are created greatly affects the
character of the photometric assay. For instance, fluorescence-based assays tend to
be much brighter, since the photons used to create the excited states can be pumped
into a sample at a very high rate. In chemiluminescence assays, the chemical
reactions required to generate excited states usually proceed at a much lower rate
and yield a lower rate of photon emission. The greater brightness of fluorescence
would appear to correlate with better assay sensitivity, but commonly this is not the
case. Assay sensitivity is determined by a statistical analysis of signal relative to
background or "noise", where the signal represents a sample measurement minus the
background measurement. Fluorescent assays tend to have much higher backgrounds,
leading to lower relative signals.
Fluorescent assays have higher backgrounds primarily because fluorometers must
discriminate between the very high influx of photons into the sample and the much
smaller emission of photons from the analytical fluorophores. This discrimination is
accomplished largely by optical filtration, since emitted photons have longer
wavelengths than excitation photons, and by geometry, since emitted photons typically
travel a different path than excitation photons. However, optical filters are not
perfect in their ability to differentiate between wavelengths, and photons can change
directions through scattering. Chemiluminescence has the advantage that, since
photons are not required to create the excited states, they do not constitute an
inherent background when measuring photon efflux from a sample. The resulting low
background permits precise measurement of very small changes in light.
Fluorescence assays also can be limited by interfering fluorophores within the
samples. This is especially problematic in biological samples, which can be replete
with a variety of heterocyclic compounds that fluoresce, typically in concentrations
much above the analytical fluorophores of interest. The problem is minimized in
simple samples, such as purified proteins, but for drug discovery, living cells are
increasingly used in high-throughput screening. Unfortunately, cells are enormously
complex in their chemical constitutions and can exhibit substantial inherent
fluorescence. Screening compound libraries also is inherently complex; although each
sample may contain only one or a few compounds, the data set from which the drug
leads are sifted is cumulated from many thousands of compounds. These compounds also
may present problems with fluorescence interference, since drug-like molecules
typically have heterocyclic structures.
For image analysis of microscopic structure, fluorescence is almost universally
preferred over chemiluminescence. Brightness counts because the optics required to
image cellular structures are relatively inefficient at light gathering. Thus the low
background inherent in chemiluminescence is of little advantage, since the signal is
usually far below the detection capabilities of imaging devices. Furthermore, imaging
is largely a matter of edge detection, which has different signal-to-noise
characteristics than simply detecting an analyte. Edge detection relies heavily on
signal strength and suffers less from uniform background noise.
In macroscopic measurements (such as in a plate well), which require accurate
quantification with high sensitivity, chemiluminescent assays often outperform
analogous fluorescence-based assays. Macroscopic measurements are the foundation for
most high-throughput screening, which relies heavily on the use of multiwell plates,
typically with 96, 384 or 1536 wells, to measure a single parameter in a large number
of samples as quickly as possible. Assays based on fluorescence or chemiluminescence
can provide high sample throughput. However, fluorescence is more likely to be
hindered by light contamination (from the excitation beam) or the chemical
compositions of samples and compound libraries. The use of chemiluminescence in
high-throughput screening has been limited largely by the lack of available assay
methods. Due to its long history, fluorescence has been more commonly used. But new
capabilities in chemiluminescence, particularly in bioluminescence, are now allowing
new bioluminescent techniques for high-throughput screening.
In nature, achieving efficient chemiluminescence is not a trivial matter, as
evidenced by the lack of this phenomenon in daily life. The large energy transitions
required for visible luminescence generally are disfavored over smaller ones that
dissipate energy as heat, normally through interactions with surrounding molecules,
especially water molecules in aqueous solutions. Because energy can be lost through
these interactions, chemiluminescence depends strongly on environmental conditions.
Thus, chemiluminescent assays often incorporate hydrophobic compounds such as
micelles to protect the excited state from water or rely on energy transfer to
fluorophores that are less sensitive to solvent quenching. Another difficulty with
chemiluminescence is efficient coupling of the reaction to the creation of
While chemiluminescence has relied on the ingenuity of chemists, bioluminescence,
a form of chemiluminescence, has instead relied on the processes of natural
evolution. Although most people are aware of bioluminescence primarily through the
nighttime displays of fireflies, there are many distinct classes of bioluminescence
derived through separate evolutionary histories. These classes are widely divergent
in their chemical properties, yet they all undergo similar chemical reactions, namely
the formation and destruction of a dioxetane structure. The classes are all based on
the interaction of the enzyme luciferase with a luminogenic substrate (Figure 8.1).
The luciferases that are used most widely in high-throughput screening are beetle
luciferases (including firefly luciferase), Renilla luciferase
and aequorin. Beetle luciferases are the most versatile of this group, and the number
of new applications is expanding rapidly. Click beetle luciferases, which also belong
to the beetle group, are becoming better known and offer a range of new luminescence
color options. Renilla luciferase is used primarily for reporter
gene applications, although its use also is expanding. Aequorin is used almost
exclusively to monitor intracellular calcium concentrations.
As luminous organisms through the eons were selected by the brightness of their
light, their luciferases have evolved both to maximize chemical couplings to generate
the excited states and to protect the excited states from water. In firefly
luciferase, the enzyme appears to exclude water by wrapping around the substrate, so
that the excited-state reaction products are completely secluded. The enzyme
structure shows two domains connected by a single polypeptide, which may act as a
hinge. It is likely that the substrates bind between the domains, causing them to
close like a lid on a box. The enzyme would act as an insulator between the
excited-state products and the environment around them. This strongly contrasts with
synthetic forms of chemiluminescence, where the excited states are exposed to the
solvent. In effect, a distinctive feature of bioluminescence is that the luciferase
serves to both generate and protect excited states.
Intracellular luciferase is typically quantified by adding a buffered solution
containing detergent to lyse the cells and luciferase substrates to initiate the
luminescent reaction. Luminescence will slowly decay due to side reactions, causing
irreversible inactivation of the enzyme. The nature of these side reactions is not
well understood, but they are probably due to the formation of damaging free
radicals. To maintain steady luminescence over an extended period of time, ranging
from minutes to hours, it is often necessary to inhibit the luminescent reaction to
various degrees. This reduces the rate of luminescence decay to the point where it
will not interfere over the time required to measure multiple samples. Even under
these conditions, as few as 10–20 moles of luciferase or
less per sample may be quantified. This corresponds to roughly 10 molecules per cell.
These assays are convenient for reporter gene applications because sample processing
is not necessary prior to reagent addition. Simply add the reagent, and read the
In some systems, a second reporter is used, expressed from a "control" vector, to
normalize results of the experimental reporter. For example, the second reporter can
control for variation between cell number or transfection efficiency. Typically, the
control reporter gene is driven by a constitutive promoter, and control vector is
cotransfected with the "experimental" vector. Different reporter genes are used for
the the control and experimental vectors so that the relative activities of the two
reporter products can be assayed individually. Control vectors also can be used to
optimize transfection methods. Gene-transfer efficiency can be assessed relatively in
cell lysates from different conditions by comparing reporter activity or assessed
absolutely by estimating the percentage of cells expressing the transferred gene by
in situ staining.
In general, bioluminescent reporters are preferred when experiments require high
sensitivity, accurate quantitation or rapid analysis of multiple samples.
Dual-reporter bioluminescence assays can be particularly useful for efficiently
Basic research into bioluminescence has led to practical applications,
particularly in molecular biology, where bioluminescent enzymes are widely used as
genetic reporters. Moreover, the value of these applications has grown considerably
over the past decade as the traditional use of reporter genes has broadened to cover
wide-ranging aspects of cell physiology.
The conventional use of reporter genes is largely to analyze and dissect the
function of cis-acting genetic elements such as promoters and enhancers (so-called
"promoter bashing"). In typical experiments, deletions or mutations are made in a
promoter region, and their effects on coupled expression of a reporter gene are
quantitated. However, the broader aspect of gene expression entails much more than
transcription alone, and reporter genes also can be used to study other cellular
Some examples of analytical methodologies that use luciferase include:
- Assays and biosensors that monitor cell-signaling pathways. For example, the
GloResponse™ Cell Lines facilitate rapid and convenient analysis of cell
signaling through the nuclear factor of activated T-cells (NFAT) pathway or
cyclic AMP (cAMP) response pathways via activation of a luciferase reporter
gene. The GloSensor™ biosensor is a genetically modified form of firefly
luciferase into which a cAMP-binding protein moiety has been inserted. cAMP
binding induces a conformational change, leading to increased light output.
- RNA interference to study how double-stranded RNA (dsRNA) suppresses
expression of a target protein by stimulating specific degradation of the
target mRNA. Luciferase reporters can be used to quantitatively evaluate
microRNA (miRNA) activity by inserting miRNA target sites downstream or 3′ of
the firefly luciferase gene.
- Identification of interacting pairs of proteins in vivo using a system known
as the two-hybrid system (Fields et al. 1989). The
interacting proteins of interest are brought together as fusion partners—one is
fused with a specific DNA-binding domain, and the other protein is fused with a
transcriptional activation domain. The physical interaction of the two fusion
partners is necessary for functional activation of a reporter gene driven by a
basal promoter and the DNA motif recognized by the DNA-binding protein. This
system was originally developed with yeast but also is used in mammalian
- Bioluminescence resonance energy transfer (BRET) to monitor protein-protein
interactions, where two fusion proteins are made, one using the bioluminescent
Renilla luciferase and another protein fused to a
fluorescent molecule. Interaction of the two fusion proteins results in energy
transfer from the bioluminescent molecule to the fluorescent molecule, with a
concomitant change from blue light to green light (Angers et
- Live-cell and in vivo imaging. Luciferase genes are commonly used as
light-emitting reporters in cellular and animal models. Visualization of
reporter expression using live-cell luciferase substrates allows
nondestructive, quantitative assays and repeat measures of the same samples
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Bioluminescence as a natural phenomenon is widely experienced with amazement at
the prospect of living organisms creating their own light. Luciferase genes have been
cloned from bacteria, beetles (e.g., firefly and click beetle),
Vargula and Gonyaulax (a dinoflagellate).
Of these, only luciferases from bacteria, beetles and Renilla
have found general use as indicators of gene expression. Bacterial luciferase,
although the first luciferase to be used as a reporter, is generally used to provide
autonomous luminescence in bacterial systems through expression of the lux operon.
Ordinarily, it is not useful for analysis in mammalian cells.
Firefly luciferase is by far the most commonly used bioluminescent reporter.
This monomeric enzyme of 61kDa catalyzes a two-step oxidation reaction to yield
light, usually in the green to yellow region, typically 550–570nm (Figure 8.1).
The first step is activation of the luciferyl carboxylate by ATP to yield a
reactive mixed anhydride. In the second step, this activated intermediate reacts
with oxygen to create a transient dioxetane that breaks down to the oxidized
products, oxyluciferin and CO2. Upon mixing with
substrates, firefly luciferase produces an initial burst of light that decays over
about 15 seconds to a low level of sustained luminescence. This kinetic profile
reflects the slow release of the enzymatic product, thus limiting catalytic
turnover after the initial reaction.
Various strategies have been tried to generate stable luminescence signal and
make the assay more convenient for routine laboratory use. The most successful of
these incorporates coenzyme A to yield maximal luminescence intensity that slowly
decays over several minutes. The mechanism of action for coenzyme A in the
luminescent reaction is unclear, although it probably stems from the evolutionary
ancestry of firefly luciferase. The amino acid sequence of firefly luciferase is
related to a diverse family of acyl-CoA synthetases. By analogy to the catalytic
mechanism of these related enzymes, formation of a thiol ester between CoA and
luciferin seems likely. An optimized assay containing coenzyme A generates
relatively stable luminescence in less than 0.3 seconds with linearity to enzyme
concentrations over a 100-millionfold range. The assay sensitivity allows
quantitation of fewer than 10–20 moles of enzyme.
The popularity of native firefly luciferase as a genetic reporter is due to the
sensitivity and convenience of the enzyme assay and tight coupling of protein
synthesis with enzyme activity. Firefly luciferase, which is encoded by the
luc gene, is a monomer that does not require any
post-translational modifications; it is available as a mature enzyme directly upon
translation of its mRNA. Catalytic competence is attained immediately after
release from the ribosome. Also, luciferase has a very short half-life in cells
(approximately 3 hours). Combined, these properties make luciferase an extremely
responsive reporter, far more so than other commonly used reporters.
Renilla luciferase is a 36kDa monomeric enzyme that catalyzes
the oxidation of coelenterazine to yield coelenteramide and blue light of 480nm
(Figure 8.1). The host organism, Renilla reniformis (sea
pansy), is a coelenterate that creates bright green flashes upon tactile
stimulation, apparently to ward off potential predators. The green light is
created through association of the luciferase with a green fluorescent protein and
represents a natural example of BRET.
Although Renilla and Aequorea are
both luminous coelenterates based on coelenterazine oxidation and both have a
green fluorescent protein, their respective luciferases are structurally
unrelated. In particular, Renilla luciferase does not require
calcium in the luminescent reaction. As a reporter molecule,
Renilla luciferase, which is encoded by the
Rluc gene, provides many of the same benefits as firefly
luciferase. Historically, the presence of nonenzymatic luminescence, termed
autoluminescence, reduced assay sensitivity; however, improvements in assay
chemistry have nearly eliminated this problem. In addition, the simplicity of the
Renilla luciferase chemistry and, more recently,
improvements to the luciferase substrate have enabled quantitation of
Renilla luciferase from living cells, in situ or in vivo.
Click Beetle Luciferase
Click beetle and firefly luciferase belong to the same beetle luciferase
family. Hence, the size and enzymatic mechanism of click beetle luciferase are
similar to those of firefly luciferase. What makes the click beetle unique is the
variety of luminescence colors they emit. Genes cloned from the ventral light
organ of a luminous click beetle, Pyrophorus plagiophthalamus
encode four luciferases capable of emitting luminescence ranging from green to
orange (544–593nm). The Chroma-Luc™ luciferases were developed from these
naturally occurring luciferase genes to generate luminescence colors as different
as possible: a red luciferase (611nm) and two green luciferases (544nm each).
These luciferase genes were codon-optimized for mammalian cells and are nearly
identical to one another, with a maximum of 8 amino acids difference between any
two of these genes. The two green luciferase genes generate very similar
luciferase proteins; however, one is maximally similar to (~98%) the DNA sequence
for the red luciferase, while the other is divergent (~68%) . Therefore
experimental and control reporter genes and proteins within an experiment can be
almost identical. Under circumstances where genetic recombination is a concern,
the divergent luciferase gene pair may be useful.
An ideal genetic reporter should: i) express uniformly and optimally in the host
cells; ii) only generate responses to effectors that the assay intends to monitor
(avoid anomalous expression); and iii) have a low intrinsic stability to quickly
reflect transcriptional dynamics. Despite their biology and enzymology, native
luciferases are not necessarily optimal as genetic reporters. In the past decade,
Promega scientists have made significant improvements in expression, reducing the
risk of anomalous expression and destabilizing these reporters. The key strategies to
achieve these improvements are described here.
Peroxisomal Targeting Site Removal
Normally, in the firefly light organ, luciferase is located in specialized
peroxisomes of photocytic cells. When the enzyme is expressed in foreign hosts, a
conserved translocation signal causes luciferase to accumulate in peroxisomes and
glyoxysomes. With moderate to high levels of expression, the peroxisomes typically
become saturated with luciferase and much of the reporter is found in the
cytoplasm. Localization to peroxisomes, however, might interfere with normal
cellular physiology in two ways. First, large amounts of a foreign protein in the
peroxisomes could impair their normal function. Second, many other peroxisomal
proteins use the same translocation signals, so saturation with luciferase implies
competition for the import of other peroxisomal proteins. Peroxisomal and
glyoxysomal localization of luciferase also may interfere with the performance of
the genetic reporter. For instance, luciferase accumulation in the cell might be
differentially affected if the enzyme is distributed into two different
The stability of luciferase in peroxisomes is not known but could be different
than its stability in the cytosol. If so, luciferase expression could be affected
by changes in the distribution of luciferase between peroxisomes and the cytosol.
Measurements of in vivo luminescence also might be affected, since the
availability of ATP, O2 and luciferin within peroxisomes is
The peroxisomal translocation signal in firefly and click beetle luciferases
has been identified as the C-terminal tripeptide sequence, -Ser-Lys-Leu. Removal
of this sequence abolishes import into peroxisomes. However, the relative specific
activity of this modified luciferase has not been determined. To develop an
optimal cytoplasmic form of the luciferase gene, Promega scientists followed two
strategies: i) design a new C-terminal tripeptide sequence based on available data
to minimize peroxisomal import, -Gly-Lys-Thr; and ii) apply random mutagenesis to
the C-terminal region and select brightly luminescent colonies of E.
coli transformed with the mutagenized luciferase genes. From
sequence data of these selected mutants, we chose a clone with the sequence
-Ile-Ala-Val. Consistently, both modified luciferases yielded about 4- to 5-fold
greater luminescence than the native enzyme when expressed in NIH/3T3 cells. We
chose the luciferase containing an -Ile-Ala-Val sequence for the cytoplasmic form
because it usually yielded slightly greater luminescence than the luciferase with
-Gly-Lys-Thr. Renilla luciferase does not contain a targeting
sequence and is not affected by peroxisomal targeting.
Although redundancy in the genetic code allows amino acids to be encoded by
multiple codons, different organisms favor some codons over others. The efficiency
of protein translation in a non-native host cell can be increased substantially by
adjusting the codon usage frequency while maintaining the same gene product. The
native luciferase genes cloned from beetles (firefly or click beetle) or sea pansy
(Renilla reniformis) use codons that are not optimal for
expression in mammalian cells. Therefore, we systematically altered the codons to
the preferred ones while removing inappropriate or unintended transcription
regulatory sequences used in mammalian cells. As a result, a significant increase
in luciferase expression levels was achieved, up to several hundredfold in some
cases (Figures 8.2 and 8.3).
Figure 8.3. The firefly luc2 gene displays higher expression
than the luc+ gene.
The luc2 gene was cloned into the pGL3-Control
Vector (Cat.# E1741), replacing the
luc+ gene. Thus both firefly luciferase genes
were in the same pGL3-Control Vector backbone. The two vectors containing
the firefly luciferase genes were co-transfected into NIH/3T3, CHO, HEK
293 and HeLa cells using the phRL-TK Vector as a transfection control.
Twenty-four hours post-transfection the cells were lysed with Passive
Lysis 5X Buffer (Cat.# E1941), and
luminescence was measured using the
Dual-Luciferase® Reporter Assay System
(Cat.# E1910). Relative light units
were normalized to Renilla luciferase expression
from the phRL-TK Vector. The fold increase in expression is listed above
each pair of bars. A repeat of this experiment yielded similar
Cryptic Regulatory Sequence Removal
Anomalous expression is defined as departure from normal or expected levels of
expression. The presence of cryptic regulatory sequences in the reporter gene may
adversely affect transcription, resulting in anomalous expression of the reporter
gene. Removal of these sequences reduces the risk of anomalous expression. A
cryptic regulatory sequence can be a transcription factor-binding site and/or a
promoter module (defined as two transcription factor-binding sites separated by a
spacer; Klingenhoff et al. 1999). Transcription
factor-binding sites located downstream from a promoter are believed to affect
promoter activity. Additionally, it is not uncommon for an enhancer element to
exert activity, resulting in elevated levels of transcription in the absence of a
promoter sequence or increased basal levels of gene expression in the presence of
transcription regulatory sequences. Promoter modules can exhibit synergistic or
antagonistic functions (Klingenhoff et al. 1999).
We removed these cryptic regulatory sequences in the luc
genes without changing the amino acid sequence to create the
luc2 gene. In addition, sequences resembling splice sites,
poly(A) addition sequences, Kozak sequences (translation start sites for mammalian
cells), E. coli promoters or E. coli
ribosome-binding sites also were removed wherever possible. This process has led
to a greatly reduced number of cryptic regulatory sequences (Figure 8.4) and
therefore a reduced risk of anomalous transcription.
Degradation Signal Addition
When performing reporter assays, measurements are made on the total
accumulated reporter protein within cells. This accumulation occurs over the
intracellular lifetime of the reporter, which is determined by both protein and
mRNA stability. If transcription is changing during this lifetime, then the
resulting accumulation of reporter will reflect a collection of different
transcriptional rates. The longer the lifetime of the reporter protein, the
greater the collection of different transcriptional rates pooled into the reporter
assay. This pooling process has a "dampening effect" on the apparent
transcriptional dynamics, making changes in the transcriptional rate more
difficult to detect. This can be remedied by reducing the reporter lifetime, thus
reducing the pooling of different transcriptional rates into each reporter
measurement. The resulting improvement in reporter dynamics is applicable to both
upregulation and downregulation of gene expression.
Ideally, the reporter lifetime would be reduced to zero, completely eliminating
the pooling of different transcriptional rates in each assay measurement. Only the
transcription rate at the instant of the assay would be represented by reporter
protein accumulation within the cells. Unfortunately, a zero lifetime also would
yield zero accumulation, and thus no reporter could be measured. A compromise must
be reached since, as lifetime decreases, so does the amount of reporter available
for detection. This is where the high sensitivity of luminescent assays is useful.
Relative to other reporter technologies, the intracellular stability of luciferase
reporters may be greatly reduced without losing measurable signal. Thus, the high
sensitivity of luciferase assays permits greater dynamics of the luciferase
The speed by which a genetic reporter can respond to changes in transcriptional
rate correlates to reporter stability within cells. Highly stable reporters
accumulate to greater levels in cells, but their concentrations change slowly
relative to changes in transcription. Conversely, lower stability yields less
accumulation but a much faster rate of response. To provide reporters that meet
different experimental needs, the Rapid Response™ Reporter family of luciferase
genes were developed with different intracellular stabilities.
Beetle and Renilla luciferase reporters have an intrinsic
protein half-life of ~3 hours. However, reporter response may still lag behind the
underlying transcriptional events by several hours. To further improve reporter
performance, Promega scientists developed destabilized luciferase reporters by
genetically fusing a protein degradation sequence to the luciferase gene products
(Li et al. 1998). After evaluating many degradation sequences
for their effect on response rate and signal magnitude, we chose two sequences:
the PEST protein degradation sequence and a second sequence composed of two
protein degradation sequences (CL1 and PEST). Due to an increased rate of
degradation, these destabilized reporters respond faster and often display a
greater magnitude of response to rapid transcriptional events and are therefore
called the Rapid Response™ Reporters.
Vector Backbone Design
Vectors used to deliver the reporter gene to host cells are critical for
overall reporter assay performance. Cryptic regulatory sequences such as
transcription factor-binding sites and/or promoter modules within the vector
backbone can lead to high background and anomalous responses. This is a common
issue for mammalian reporter vectors, including the pGL3 Luciferase Reporter
Vectors. Promega scientists extended the successful "cleaning" strategy for
reporter genes to the entire pGL3 Vector backbone, removing cryptic regulatory
sequences wherever possible, while maintaining functionality. Other modifications
include a redesigned multiple cloning region to facilitate easy transfer of the
DNA element of interest, removal of the f1 origin of replication and deletion of
an intronic sequence. In addition, a synthetic poly(A) signal/transcriptional
pause site was placed upstream of the multiple cloning region (in promoterless
vectors) or the HSV-TK, CMV or SV40 promoter (in promoter-containing vectors).
This extensive effort resulted in the totally redesigned and unique vector
backbone of the pGL4 Vectors.
By manipulating luciferase genes Promega scientists have developed a series of
optimized reporter genes featuring additional luminescence colors and improved codon
usage, while deleting cryptic regulatory sequences such as transcription
factor-binding sites that could decrease protein expression in mammalian cells. The
pGL4 family of luciferase vectors incorporates a variety of features such as your
choice of firefly or Renilla luciferase, Rapid Response™
versions, mammalian-selectable markers, basic vectors without promoters,
promoter-containing control vectors and predesigned vectors with your choice of
several response elements (Figure 8.5).
Advantages of the pGL4 Vectors include:
- Improved sensitivity and biological relevance due to:
- Increased reporter gene expression: Codon optimization of synthetic
genes for mammalian expression
- Reduced background and risk of expression artifacts: Removal of
cryptic DNA regulatory elements and transcription factor-binding sites
- Improved temporal response: Rapid Response™ technology using
destabilized luciferase genes
- Additional advantages include:
- Flexible detection options: Choice of reporter genes
- Easy transition from transient to stable cells: Choice of mammalian
- Easy transfer from one vector to another: Common multiple cloning site
and a unique SfiI transfer scheme
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The challenge when designing bioluminescent assays is harnessing this efficient
light-emitting chemistry for analytical methodologies. Most commonly this is done by
holding the reaction component concentrations constant, except for one component that is
allowed to vary in relation to the biomolecular process of interest. When the reaction
is configured properly, the resultant light is directly proportional to the variable
component, thus coupling an observable parameter to the reaction outcome. In assays
using luciferase, the variable component may be the luciferase itself, its substrates or
cofactors. Because of very low backgrounds in bioluminescence, the linear range of this
proportionality can be enormous, typically extending 104- to
108-fold over the concentration of the variable component.
The following section provides information about specific bioluminescent reporters
and assays, including how to choose the correct reporter genes to suit your research
needs. Tables 8.2 and 8.3 show available luciferase genes, assays and reagents.
Assays based on a single reporter provide the quickest and least expensive means
to acquire gene expression data from cells. However, because cells are inherently
complex, the quantity of information gleaned from a single-reporter assay may be
insufficient to achieve detailed and accurate results. Thus, one of the first
considerations when choosing a reporter methodology is deciding whether the speed and
depth of information from a single reporter is sufficient or whether a greater
density of information is desired. If a greater density of information is required,
see the Dual-Reporter Assays section.
Using an assay reagent that produces stable luminescence is more convenient when
performing assays in multiwell plates. Unfortunately, because bright reactions fade
relatively quickly, a trade-off is necessary between luminescence intensity and
duration. The Bright-Glo™ Luciferase Assay System yields maximal firefly luciferase
luminescence intensity and sufficient signal duration for analysis in a multiwell
plate. The Steady-Glo® Luciferase Assay System provides
even greater luminescence duration but with lower intensity. Both reagents work
directly in culture medium for mammalian cells, so prior cell lysis is not necessary.
This allows you to grow cells in multiwell plates, then measure expression in a
Additional Resources for Single-Reporter Assays
Technical Bulletins and Manuals
Luciferase Assay System Technical Bulletin
Renilla Luciferase Assay System Technical
Renilla-Glo™ Luciferase Assay System Technical
Bright-Glo™ Luciferase Assay System Technical Manual
Steady-Glo® Luciferase Assay System Technical
ONE-Glo™ Luciferase Assay System Technical Bulletin
pGL4 Luciferase Reporter Vectors Technical Manual
Ashfield , T. et al.
(2004) Convergent evolution of disease resistance gene specificity in two
flowering plant families. Plant Cell 16
Leaves of Glycine max (soybean) were
co-transfected by particle bombardment with various combinations of
vectors encoding plant disease-resistance genes and a luciferase reporter
construct containing the constitutive 35S promoter of cauliflower mosaic
virus. Leaf disks from the transfected areas were frozen in liquid
nitrogen, ground and resuspended in 240μl of Cell Culture Lysis Reagent.
The lysates were assayed for luciferase activity with the Luciferase
de Haan, C.A.M. et al.
(2004) Cleavage inhibition of the murine coronavirus spike protein by a
furin-like enzyme affects cell-cell but not virus-cell fusion. J. Virol. 78
The Renilla Luciferase Assay System was used to
analyze mouse hepatitis coronavirus strain A59 (MHV-A59) entry into
cells. A mouse hepatitis coronavirus construct expressing
Renilla luciferase was used to infect LR7 cells
in the presence or absence of a furin protease inhibitor.
Lin , P.F. et al.
(2003) A small molecule HIV-1 inhibitor that targets the HIV-1 envelope and
inhibits CD4 receptor binding. Proc. Natl. Acad. Sci. USA 100
To test the effect of BMS-378806, a new small molecule inhibitor of
HIV-1, a cell fusion assay was developed. Target cells that stably
expressed CD4, CXCR4 or CCRS receptors and carried a responsive
luciferase plasmid were prepared. Effector cells were transiently
transfected with the HIV coat protein gp160 from various strains of virus
and a plasmid to activate the response element controlling luciferase
expression. If the cells fused, luciferase was synthesized. To measure
cell fusion, effector cells (1 × 104
cells/well) were plated with target cells at a ratio of 1:2 in 96-well
plates, then incubated with various concentrations of BMS-378806 for
12–24 hours. Luciferase activity was determined using
Steady-Glo® Luciferase Assay System.
ONE-Glo™ Luciferase Assay System: New substrate, better reagent.
The bioluminescence advantage
pGL4 Vectors: A new generation of luciferase reporter vectors.
Bright-Glo™ and Steady-Glo®: Reagents for
academic and industrial applications.
The most commonly used dual-reporter assays measure both firefly and
Renilla luciferase activities. These luciferases use
different substrates and thus can be differentiated by their enzymatic specificities.
The method involves adding two reagents to each sample and measuring luminescence
following each addition. Addition of the first reagent activates the firefly
luciferase reaction; addition of the second reagent extinguishes firefly luciferase
and initiates the Renilla luciferase reaction. The
Dual-Luciferase® Reporter Assay System requires cell
lysis prior to performing the assay and requires the use of reagent injectors with
multiwell plates. The Dual-Glo™ Reagent is optimized for multiwell plates, providing
longer luminescence duration (in other words, a longer luciferase signal half-life).
As with other reagents designed for use in multiwell plates, the Dual-Glo™ Assay
works directly in mammalian cell culture medium without prior cell lysis.
In general dual-reporter assays improve experimental accuracy and efficiency by:
i) reducing variability that can obscure meaningful correlations; ii) normalizing
interfering phenomena that may be inherent in the experimental system; and iii)
normalizing differences in transfection efficiencies between samples.
Because cells are complex micro-environments, significant variability can occur
between samples within an experiment and between experiments performed at
different times. Challenges include maintaining uniform cell density and viability
between samples and accomplishing reproducible transfection of exogenous DNA.
Multiwell plates introduce variables such as edge effects, brought about by
differences in heat distribution and humidity across a plate. Dual-reporter assays
can control for much of this variability, leading to more accurate and meaningful
comparisons between samples (Hawkins et al. 2002; Hannah
et al. 1998; Wood, 1998; Faridi et
In some cases, researchers may prefer to activate both luciferase assays
simultaneously by adding a single reagent. This reduces total assay volume and
liquid-handling requirements. The light emission of the two luciferases can be
differentiated by the color of luminescence. Promega scientists have developed
click beetle luciferases, which are related to firefly luciferase, to yield red
and green luminescence. The structures of these luciferases are nearly identical,
with only a few amino acid substitutions necessary to create the different colors.
This structural similarity means that both the control and experimental reporters
are likely to respond similarly to biochemical changes within the cell, resulting
in even more accurate normalization to the control reporter.
The genes encoding these reporters, the Chroma-Luc™ genes, are codon-optimized
for mammalian cells. Promega scientists developed two genes encoding the
green-emitting reporter: one which is nearly identical to the luciferase emitting
red luminescence, and one that is maximally divergent from it. These genes encode
reporter proteins that have nearly identical sequences. The divergent gene may be
useful in situations where genetic recombination is a concern.
The Chroma-Glo™ Luciferase Assay System measures Chroma-Luc™ activity in
multiwell plates. The Chroma-Glo™ Reagent formulation supports optimal reaction
kinetics for both reporters simultaneously, and it works directly in culture
medium. Because color differentiation is required for the Chroma-Glo™ Assay, a
luminometer capable of using colored optical filters is required. Since the light
is transmitted through optical filters, sensitivity relative to other assay
methods is reduced. Both red- and green-emitting Chroma-Luc™ luciferase activities
are detectable using optical filters when the relative concentrations differ by up
to 100-fold. This is less than dual-luciferase assays that use chemical
differentiation, where the relative concentrations may differ by over 1,000-fold.
Distinguishing among the Dual Assays
Dual-reporter and dual-color assays allow you to measure expression of two
different reporter genes or, when using a luciferase-based cell viability assay,
one reporter gene and cell health. In all cases, the assays allow both
measurements to be made sequentially from each sample. Most dual assays are
optimized for use in multiwell plates.
Additional Resources for Dual-Reporter and Dual-Color Assays
Technical Bulletins and Manuals
Dual-Luciferase® Reporter Assay System
Dual-Glo™ Luciferase Assay System Technical Manual
Chroma-Glo™ Luciferase Assay System Technical Manual
pGL4 Luciferase Reporter Vectors Technical Manual
Elbashir, S.M. et al.
(2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured
mammalian cells. Nature 411
In this landmark paper decribing RNA interference in mammalian cells,
firefly and Renilla luciferase gene products were
targeted for degradation. NIH/3T3, HEK293, HeLa S3, COS-7 and S2 cells
were transfected with 1μg of pGL2-Control or pGL3-Control Vector, 0.1μg
pRL-TK Vector and 0.21μg siRNA duplex targeting either firefly or
Renilla luciferase. The
Dual-Luciferase® Reporter Assay System was
used 20 hours post-transfection to monitor luciferase expression.
Transfection with 21bp dsRNA caused specific degradation of a targeted
sequence. This was the first demonstration of the RNAi effect in
Yamaguchi, K. et al.
(2004) Identification of nonsteroidal anti-inflammatory drug-activated gene
(NAG-1) as a novel downstream target of phosphatidylinositol
3-kinase/AKT/GSK-3 pathway. J. Biol. Chem. 279
The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 was used
to identify nonsteroidal anti-inflammatory drug-activated gene (NAG-1) as
a novel downstream target of the PI3K pathway. For these experiments,
HCT-116 cells were treated with 50μM LY294002, and NAG-1 protein
expression was assessed by Western blotting. Gene upregulation during
LY294002 treatment was measured using a luciferase reporter construct
containing the NAG-1 promoter, the pRL-null Vector as a transfection
control and the Dual-Luciferase® Reporter
Verge, V. et al.
(2004) Localization of a promoter in the putative internal ribosome entry site
of Saccharomyces cerevisiae
TIF4631 gene. RNA 10
Researchers cloned the Photinus and
Renilla luciferase ORFs into the pSP64 Poly(A)
Vector to create a dual-reporter vector named SP6P. A similar vector,
SP6R.4G(-508/-3).P, was created in which a 5′ untranslated region from
the Saccharomyces TIF4631 gene was cloned between
the two reporter genes. These two vectors were used to transform yeast
strains. The resultant transformants were lysed using Passive Lysis
Buffer and a modified lysis procedure. Lysates were analyzed for
luciferase activities with the
Dual-Luciferase® Reporter Assay System and a
TD20/20 luminometer. The researchers also cloned and sequenced the 5′
untranslated region of TIF4631 using a RACE-PCR technique followed by
cloning the PCR amplimers.
Chen, Q.Y. et al.
(2004) Human CD1D gene has TATA boxless dual promoters: An SP1-binding element
determines the function of the proximal promoter. J. Immunol. 172
The authors demonstrated that the human CD1D gene has distal and
proximal TATA boxless promoter sequences. Distal and proximal promoters
to CD1D were cloned into the pGL3-Basic Vector to create reporter
constructs. One construct contained the entire 4,986 base pair region,
including the distal and proximal CD1D promoter. Transient transfections
were performed using 5 x 105 Jurkat cells in
24-well plates, 0.8μg of pGL3-Basic Vector with the insert of interest
and 30ng of pRL-CMV Vector as a transfection normalization control. The
Dual-Glo™ Luciferase Assay System was used to assay luciferase
NIH Chemical Genomics Center: Small-molecule screening for investigating
fundamental biological questions.
Deciphering the pGL4 Vector code.
pGL4 Vectors: A new generation of luciferase reporter vectors.
Increased Renilla luciferase sensitivity in the
Dual-Luciferase® Reporter Assay
Introducing Chroma-Luc™ technology.
Dual-Glo™ Luciferase Assay System: Convenient dual reporter measurements
in 96- and 384-well plates.
Researchers strive to monitor cellular activities with as little impact on the
cell as possible. The endpoint of an experiment, however, sometimes requires complete
disruption of cells so that the environment surrounding the reporter enzyme can be
carefully controlled. Recently, Promega scientists developed a variety of live-cell
substrates to monitor Renilla and firefly luciferase activity
without disrupting cells.
Renilla luciferase requires only oxygen and coelenterazine to
generate luminescence, providing a simple luciferase system to measure luminescence
from living cells. Unfortunately, coelenterazine is unstable in aqueous solutions, so
it has been difficult and inconvenient to measure Renilla
luciferase. EnduRen™ and ViviRen™ Live Cell Substrates overcome this difficulty and
easily generate luminescence from live cells expressing Renilla
luciferase. Because luminescence is generated from living cells, these substrates are
ideal for multiplexing with assays that determine cell number.
Promega also offers a number of live-cell firefly luciferase substrates, including
VivoGlo™ Luciferin, the potassium salt of D-luciferin; VivoGlo™ Caspase-3/7 Substrate
(Z-DEVD-Aminoluciferin, Sodium Salt), a firefly luciferase prosubstrate containing
the DEVD tetrapeptide sequence recognized by caspase-3 and -7; and VivoGlo™
Luciferin-β-Galactoside Substrate (6-O-β-galactopyranosyl-luciferin), a substrate for
the reporter enzyme β-galactosidase. These substrates are useful for imaging firefly
luciferase in live cells and organisms.
Normalizing Interfering Phenomena
When correlations between experimental conditions and reporter gene expression
are examined, other events associated with cell physiology can affect reporter
gene expression. Of particular concern is the effect of cytotoxicity, which can
mimic genetic downregulation when using a single-reporter assay. Reporter assays
that can be multiplexed with a cell viability assay allow independent monitoring
of both reporter expression and cell viability to avoid data misinterpretation
(Farfan et al. 2004). The use of multiplexed assays allows
correlation of events within cells, such as the coupling of target suppression by
RNAi to the consequences on cellular physiology (Hirose et
The CellTiter-Glo® Luminescent Cell Viability Assay
provides a rapid and sensitive cell viability assay based on luminescent detection
of cellular ATP. Because the CellTiter-Glo® Assay uses
a stabilized firefly luciferase, it cannot be directly combined with a firefly
luciferase reporter assay. However, the assay can be readily combined with
nondestructive Renilla luciferase assays.
Expression of Renilla luciferase may be quantitated or
continuously monitored by adding EnduRen™ Substrate to the culture medium and
measuring luminescence. When reporter measurements are completed, the
CellTiter-Glo® Reagent is added to the sample to
inactivate Renilla luciferase and initiate ATP-dependent
luminescence, which is indicative of cell viability. Because the
CellTiter-Glo® Assay is an endpoint assay, further
sample monitoring after measuring cell viability is not possible.
Fluorescent cell viability assays also are available to monitor cell health and
normalize single-reporter assay results to live cell number. For example, the
CellTiter-Fluor™ Cell Viability Assay is a nonlytic, fluorescence assay that
measures the relative number of viable cells in a population. The CellTiter-Fluor™
Substrate enters intact cells, where it is cleaved by a live-cell protease that is
restricted to intact cells to generate a fluorescent signal proportional to the
number of living cells. The number of nonviable cells does not affect fluorescence
because the live-cell protease becomes inactive upon loss of membrane integrity
and leakage into the culture medium. This assay is well-suited for multiplexing
with homogeneous luciferase assay reagents such as Bright-Glo™, Steady-Glo™ and
ONE-Glo™ Luciferase Assay Systems (Zakowicz, H. et al. 2008)
because it exhibits no intrinsic color quenching, which can limit luminescent
assay sensitivity. Viability assays using older resazurin-based substrates can
quench up to 82% of luminescence due to color intensity of the dye.
Additional Resources for Live-Cell Substrates
Technical Bulletins and Manuals
EnduRen™ Live Cell Substrate Technical Manual
ViviRen™ Live Cell Substrate Technical Manual
Dinh, D.T. et al.
(2005) Helix I of Beta-arrestin is involved in postendocytic trafficking but is
not required for membrane translocation, receptor binding and
internalization. Mol. Pharmacol. 67
Type 1 angiotensin II receptor-Renilla luciferase
(AT1R-Rluc), and β-arrestin1 and β-arrestin2 GFP fusion constructs were
transfected into COS-7 cells. The COS-7 cell cultures were activated with
100μM angiotensin II in the presence of 60μM EnduRen™ Live Cell
Substrate, and BRET fluorescence readings were taken at 475nm and 515nm
over a 1-hour period. The authors also described analysis of helix I
mutants of β-arrestin1 and β-arrestin2 in similar β-arrestin GFP BRET
studies. Data were displayed as a ratio of fluorescence readings with
both constructs compared to fluorescence from the AT1R-Rluc construct
Measuring Renilla luminescence in living
Bioluminescence imaging of live trout for virus detection using EnduRen™
Live Cell Substrate.
Bioluminescent reporters have been harnessed to study RNA interference (RNAi), a
phenomenon by which double-stranded RNA complementary to a target mRNA can
specifically inactivate a gene by stimulating degradation of the target mRNA. As
such, RNAi has emerged as a powerful tool to analyze gene function. Since its report
in C. elegans (Fire et al. 1998), RNAi has
been reported in a variety of organisms, including zebrafish, planaria, hydra, fungi,
Drosophila and plant and mammalian systems. These phenomena
have been collectively termed RNA silencing and appear to use a common set of
proteins and short RNAs. These processes are mechanistically similar but not
identical. For more information about the RNAi process and technologies and products
that can be used to design, synthesize and evaluate siRNAs and shRNAs, refer to the
RNA Interference chapter.
Luciferase reporter assays are widely used to investigate cellular signaling
pathways and as high-throughput screening tools for drug discovery (Brasier
et al. 1992, Zhuang et al. 2006).
Synthetic constructs with cloned regulatory elements directing reporter gene
expression can be used to monitor signal transduction and identify the signaling
pathways involved. By linking luciferase expression to specific response elements
(REs) within the reporter construct, transfecting cells with this construct,
subjecting the transfected cells to a particular treatment, then measuring reporter
activity, researchers can determine what REs are used, and thus, what signaling
pathways are involved. The use of inhibitors and short interfering RNAs (siRNAs) can
be used to confirm what factors are involved in this response.
To speed this type of research, Promega scientists have designed several
convenient pGL4 Vectors with your choice of a number of response elements and
regulatory sequences to take advantage of the benefits of the pGL4 Vector backbone
and luc2P gene. See Tables 8.1 and 8.2. Many of these vectors
encode the hygromycin-resistance gene to allow selection of stably transfected cell
lines. Alternatively, Promega offers cell lines that are already stably transfected
with pGL4-based vectors with specific response elements. See Section III.E,
Luciferase Reporter Cell Lines.
|Table 8.1. Response Element pGL4 Vectors.
||cAMP response element
||NFAT response element
||GAL4 upstream activating sequence
||Varies (requires binding and activation by
||Nuclear Factor κB response element
||serum response element
||Serum response factor response element
||GAL4 upstream activating sequence
||Varies (requires binding and activation by
||Murine mammary tumor virus long terminal repeat
||Several nuclear receptors, including androgen receptor
and glucocorticoid receptor
Bioluminescent reporters also enable characterization of nuclear receptors, a
class of ligand-regulated transcription factors that sense the presence of steroids
and other molecules inside the cell. Nuclear receptors typically reside in the
cytoplasm and are often complexed with associated regulatory proteins. Ligand binding
triggers translocation into the nucleus, where the receptors bind specific response
elements via the DNA-binding domain, leading to upregulation of the adjacent gene.
Bioluminescent reporters can be harnessed to identify and characterize nuclear
receptor agonists, antagonists, co-repressors and co-activators using a universal
receptor assay, which is similar in many ways to the two-hybrid assay. In a two-hybrid assay, two proteins that are thought to
interact are expressed as fusion proteins, one fused with the DNA-binding domain
(DBD) of the yeast GAL4 transcription factor and the other fused to the VP16
activation domain. Protein:protein interactions bring the two domains together to
yield expression of a reporter gene downstream of tandem GAL4-binding sites and a
minimal promoter. The universal nuclear reporter assay can be thought of as a
"one-hybrid" assay, where the ligand-binding domain (LBD) of a nuclear receptor
replaces the bait and prey proteins and VP16 activation domain (Figure 8.6).
To perform the universal nuclear receptor assay, simply cotransfect the cell line
of interest with a construct encoding the LBD-GAL4 DBD fusion protein and a suitable
reporter vector with multiple copies of the GAL4 UAS upstream of the promoter and
reporter gene. Two to three days posttransfection, treat cells with the test
compounds of interest, then measure luciferase activity. This approach allows you to
convert any cell line into a nuclear receptor-responsive cell line, which you can use
to identify receptor agonists, antagonist, co-activators and co-repressors. You can
even perform mutagenesis on the ligand-binding domain to determine the effect in your
responsive cell line without interference from the endogenous receptor. An example of
a suitable reporter construct is the
(Cat.# E1370), which contains nine copies of the
GAL4 UAS immediately upstream of a minimal promoter driving expression of
luc2P reporter gene. For added convenience, Promega offers
HEK293 cells that are stable transfected with the
the GloResponse™ 9XGAL4UAS-luc2P HEK293
cells. For more information, see the Luciferase Reporter Cell Lines section below.
Promega offers a number of additional reagents to simplify universal nuclear
receptor assays. The pBIND-ERα Vector (Cat.# E1390)
contains the yeast Gal4 DBD and an estrogen receptor-ligand binding domain (ER-LBD)
gene fusion, and the pBIND-GR Vector (Cat.# E1581)
contains the yeast Gal4 DBD and glucocorticoid receptor-ligand binding domain
(GR-LBD) gene fusion. Promega also offers the pFN26A (BIND)
hRluc-neo Flexi® Vector
(Cat.# E1380), which allows expression of a fusion
protein comprised of the GAL4 DBD, a linker segment and an in-frame protein-coding
sequence under the control of the human cytomegalovirus (CMV) immediate early
promoter. You simply clone the DNA fragment encoding the ligand-binding domain of
your receptor into SgfI and PmeI sites at the 5′ and 3′ ends of a lethal barnase
gene, which acts as a positive selection for successful ligation of the insert. Each
BIND vector contains a Renilla luciferase/neomycin resistance
co-reporter for normalization of transfection efficiency or construction of a
double-stable cell line without the need for additional cloning.
Bioluminescent reporters also are useful for studying G-protein coupled receptors
(GPCRs), which regulate a wide-range of biological functions and are one of the most
important target classes for drug discovery (Klabunde et al.
2002). The firefly luciferase-based GloSensor™ cAMP assay provides a sensitive and
easy-to-use format to interrogate overexpressed or endogenous GPCRs that signal via
changes in intracellular cAMP concentration. The assay uses genetically encoded
biosensor variants comprised of cAMP-binding domains fused to mutant forms of
Photinus pyralis luciferase. cAMP binding induces
conformational changes that promote large increases in light output. Following
pre-equilibration with a luciferase substrate, cells transiently or stably expressing
the biosensor variant can be used to assay GPCR function using a nonlytic assay
format, enabling kinetic measurements of cAMP accumulation or turnover in living
cells. Moreover, the assay offers a broad dynamic range, with up to 500-fold changes
in light output. Extreme sensitivity allows detection of
Gi-coupled receptor activation or inverse agonist activity in
the absence of artificial stimulation by compounds such as forskolin. For more
information, visit: www.promega.com/glosensor/
The GloResponse™ Cell Lines contain optimized, state-of-the-art luciferase
reporter technology integrated into a cell line. These cells use the destabilized and
optimized luc2P gene, allowing greater sensitivity and shorter
induction times than native reporter enzymes. The GloResponse™
NFAT-RE-luc2P HEK293 Cell Line,
NFκB-RE-luc2P HEK293 Cell Line and
CRE-luc2P HEK293 Cell Line allow rapid and convenient analysis
of cell signaling through the NFAT, NF-κB or cAMP response pathways via activation of
a luciferase reporter gene. Non-native activators of these pathways, including GPCRs,
can be studied after the appropriate proteins are introduced by transfection.
GPCR signaling pathways can be categorized into three classes based on the G
protein α-subunit involved: Gs, Gi/o
and Gq. The GloResponse™ CRE-luc2P HEK293
Cell Line can be used to study and configure screening assays for
Gs- and Gi/o-coupled GPCRs, which
signal through cAMP and the cAMP reponse element (CRE). For
Gq-coupled GPCRs, which signal through calcium ions and
NFAT-RE, the GloResponse™ NFAT-RE-luc2P HEK293 Cell Line should
be used. GPCR assays that use the GloResponse™ Cell Lines are amenable to
high-throughput screening. These assays typically have greater response dynamics
(fold of induction) than other assay formats and generate high-quality data as
indicated by the high Z′ values.
The GloResponse™ Cell Lines were generated by clonal selection of HEK293 cells
stably transfected with pGL4-based vectors carrying specific response elements for
the pathway of interest. These cell lines incorporate improvements developed for the
pGL4 Vectors for enhanced performance. The destabilized luc2P
luciferase reporter improves responsiveness to transcriptional dynamics and is
codon-optimized for enhanced expression in mammalian cells. The pGL4 vector backbone
was engineered to reduce background reporter expression. The result is cell lines
with very high reporter induction levels when the pathway of interest is activated.
Additional Resources for Luciferase Reporter Cell Lines
Technical Bulletins and Manuals
GloResponse™ CRE-luc2P HEK293 Cell Line Technical
GloResponse™ NFAT-RE-luc2P HEK293 Cell Line
GloResponse™ NFκB-RE-luc2P HEK293 Cell Line
The tables in this section show the various features of reporter vectors,
including the reporter gene, presence of a multiple cloning region, gene promoter,
protein degradation sequences and mammalian selectable marker (Tables 8.2 and 8.3),
as well as the features of Promega reporter assays (Table 8.3). These tables and the
following tools will help you choose a pGL4 Vector or reporter assay.
For a step-by-step guide to help you choose the best pGL4 Vector for your studies,
use the pGL4 Vector Selector. To go to
the tool, click on the link, select the Solution Finder tab, then choose "pGL4 Vector
The Introduction to Reporter Gene Assays animation demonstrates the basic
design of a reporter assay using the Dual-Luciferase®
Reporter Assay System to study promoter structure, gene regulation and signaling
|Table 8.2. pGL4 Luciferase Reporter Vectors.
||Multiple Cloning Region
||Protein Degradation Sequence
||Mammalian Selectable Marker
||serum response element
||murine mammary tumor virus long terminal repeat
|Table 8.3. Other Luciferase Reporter Vectors.
||Multiple Cloning Region
||Protein Degradation Sequence
||Mammalian Selectable Marker
|Table 8.4. Luciferase Reporter Assays.
||Single-Sample or Plate Assay
|Luciferase Assay System
luc, luc+, luc2
||Single or Plate2
|Steady-Glo® Luciferase Assay
luc, luc+, luc2
|Bright-Glo™ Luciferase Assay System
luc, luc+, luc2
|ONE-Glo™ Luciferase Assay System
luc, luc+, luc2
||Long (≥45 minutes)
Renilla Luciferase Assay System
||Single or Plate2
|Renilla-Glo™ Luciferase Assay System
||Long (≥60 minutes)
|Dual-Glo™ Luciferase Assay System
luc+, luc2, Rluc, hRluc
luc+, luc2, Rluc, hRluc
||Single (Cat.# E1910)
|Chroma-Glo™ Luciferase Assay System
CBRluc, CBG99luc, CBG68luc
|EnduRen™ Live Cell Substrate
|ViviRen™ Live Cell Substrate
1Do not use this product or reagent with automated reagent injectors
available on certain luminometers.
2Use with plate assays only when luminometer has a reagent
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When performed properly, experiments using genetic reporters can yield tremendous
amounts of information. However, there are several important considerations when
designing and performing these types of experiments to ensure that the data are sound.
A common question when designing reporter vectors for promoter dissection is “What
sequences should I clone into my vector?”. Unfortunately, there is no one correct
answer. The necessary sequences depend on the biological question you are trying to
answer and the vector into which you are cloning. An advantage of transgenic reporter
assays is that you can control which elements are examined. You might include the
entire proximal promoter (including ~1kb upstream of the promoter), a specific
promoter subsection or as little as a single response element. When generating a
transcriptional fusion or using a vector with no transcription start site, you might
include the +1 transcription start site. The reporter might include the 5′ UTR if you
want to understand how this sequence may affect promoter activity, but keep in mind
that UTR sequences also can affect post-transcriptional regulation. The reporter
might include an intron and all or part of one or both flanking exons to study RNA
splicing or characterize regulatory elements contained within the intron (but if the
exons include coding sequence, be sure to clone the reporter gene in frame). The
reporter might contain the 3′ UTR to focus on only post-transcriptional regulation
through miRNA effects. You might clone any combination of sequences from your gene of
interest to look at the integration of regulatory pathways; reporter assays allow
this flexibility and refined experimental design.
The proper controls are an important part of reporter assays. The most important
is a control using untransfected cells to define the background signal in the assay
(from luminometer noise or reagent background). Generally, background luminescence is
inconsequential, and the signal:background ratio is quite high in luciferase assays.
Additional controls may include the parent vector used to prepare the reporter vector
(minus any inserts) and a positive control vector. Measuring luciferase activity from
the parent vector allows you to discount any reporter response due to the vector
backbone, not the insert; experimental results can be expressed as the ratio of
experimental vector response to parent vector response. The same function is
generally provided by the second reporter in a dual-reporter assay when using matched
vectors (i.e., pGL4 firefly and Renilla vectors). A reporter
vector with a relatively strong promoter can serve as a positive control for
luciferase expression and detection in the cell line of interest.
Transfection is necessary to introduce the reporter vector into a cell. Transient
transfection is the most common method, but stable transfection should be considered
if you are performing the same reporter assay frequently. Both approaches have
advantages and disadvantages. Transient transfection allows you to vary the reporter
vectors and vector ratios, but cells must be transfected for each set of experiments
and will lose the reporter vector over time. Transfection efficiency can be low for
primary cells and some cell lines and can vary considerably. Often a second reporter
must be cotransfected into cells to normalize for differences in transfection
efficiency; this reporter also can help to determine if a response was due to cell
toxicity and not a promoter-specific event. Because transfection is stressful, cells
must be allowed to recover prior to experimental treatment. Stable transfection
eliminates variability in transfection effiency and the stress of transfection but
requires additional time and effort to select stably transfected cells.
The optimal transfection reagent and conditions depend on the cell line used and
often must be determined empirically. Efficient transfection can be critical when
using less sensitive reporters such as chloramphenicol acetyltransferase (CAT) or
β-galactosidase) but is less of a concern with sensitive reporters such as
luciferase. For a detailed discussion of transfection, see the Transfection chapter of the Protocols and Applications
Two important factors when transfecting cell prior to dual-reporter assays are the
reporter vector ratio and relative promoter strengths in the cell line of interest.
The optimal ratio often is related to promoter strength and must be determined
empirically. Strong promoters, such as cytomegalovirus (CMV) and SV40 promoters, can
easily interfere with transcriptional activity of weaker promoters by sequestering
transcription factors and are more likely to be regulated by experimental treatments
due to the high number of transcription factor-binding sites. In general, we
recommend avoiding promoters with the highest activities in your cells. Vectors with
weaker promoters often are a better choice,and even vectors without a promoter yield
sufficient luciferase activity for normalization purposes in most cells and are less
likely to be regulated by the treatment.
It is important, especially if you must use a reporter vector with a strong
promoter, to transfect cells with several different ratios of reporter vectors. For
assays using pGL4 Vectors with firefly and Renilla luciferases,
we recommend a 20:1 ratio as a good starting point, but the ratio could be as high as
200:1 when the promoter of one vector is dramatically stronger than that of the
other. For vectors with promoters of equal strengths, the ratio might be 1:1. When
performing vector titrations, be sure to transfect all cells with a constant amount
of DNA to minimize differences in transfection efficiency due to differing DNA
amounts. The ideal ratio will provide moderate but consistently detectable
Renilla luciferase signal that is not influenced by the
amount of firefly luciferase vector present, and the firefly and
Renilla luciferase signals will be at least 3 standard
deviations above background levels, below the saturation point of the luminometer and
within 4 orders of magnitude of each other.
The times between plating and transfection, transfection and experimental
treatment, and treatment and reporter assay need to be consistent within a set of
experiments to minimize variability and improve assay precision and accuracy. When
plating cells prior to transfection, take into account the growth rate of the cells
so that cells reach proper confluency at the time of transfection. After transfection
allow time for cells to recover and the reporter to reach steady state levels of
expression. During initial assay optimization, perform a time course to determine the
time of peak reporter expression. The optimal time between treatment and reporter
assay depends on a number of factors, including the kinetics of your system,
longevity of the change you are monitoring (i.e., the assay window) and stability of
the reporter protein.
For early-responding genes, we recommend a reporter with a short protein half-life
such as those found in the Rapid Response™ luciferase genes, which encode protein
degradation sequences (PEST; Li et al. 1998 or CL1; Gilon
et al. 1998) to destabilize the reporter protein. The Rapid
Response™ luciferase genes (luc2P and
luc2CP) respond more quickly and with greater magnitude to
changes in transcriptional activity than their more stable counterparts
(luc and luc2). The onset of the
response is more tightly coupled to the induction event, and the assay window is
narrower. The use of more stable luciferase versions will result in a later and
longer response with a wider assay window, so assay timing is not as critical. In
these cases, reporter assays are commonly performed 12–24 hours after treatment.
There are many factors to consider when choosing a reporter assay, including the
reporter gene used, assay format (individual tubes or multiwell plates), luminometer
capabilities, and the need to maintain cell viability. A summary of luciferase
reporter assay features is provided in Table 8.4.
Cells and cell culture conditions used to dissect a promoter can affect assay
design and results. Three types of cells are commonly used: 1) fibroblasts, which are
easy to maintain and amenable to most reporter assays but may not express necessary
co-factors or be as biologically relevant as other cells; 2) cancer cells, which may
be more relevant and are easy to use; and 3) primary cells, which may be the most
biologically relevant but often are difficult to obtain, maintain and transfect. Cell
cultures should not be confluent during the experiment, since confluent cells can
exhibit differences in metabolism, gene expression and physiological response
compared to preconfluent cell cultures. Likewise, cells at higher passage numbers may
not behave in the sameway as cells at lower passage numbers. If necessary, grow and
freeze a large quantity of cells at a lower passage number to ensure that your
experiments are performed with cells at the same passage number. Cells should be
healthy and, ideally, easy-to-transfect. The cell culture medium should replaced
several days before and after transfection. When repeating an experiment, be sure to
replicate cell culture and transfection conditions as closely as possible to ensure
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(1999) Functional promoter modules can be detected by formal models independent of
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(1998) Generation of destabilized green fluorescent protein as a transcription
J. Biol. Chem.
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(2008) Measuring cell health and viability sequentially by same-well multiplexing
using the GloMax
-Multi Detection System.
- Zhuang, F. and Liu, Y.H. (2006) Usefulness of the luciferase reporter system to test the efficacy of siRNA.
Methods Mol. Biol.
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