Professional Documents
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Yoshihiro Ohmiya
Biomedical Research Institute
National Institute of Advanced Industrial Science and Technology (AIST)
Tsukuba, 305-8566, Japan
y-ohmiya@aist.go.jp
Bioluminescence is a unique light source based on the luciferin-luciferase reaction, and their luciferases are good reporter enzymes
in the field of bioresearch. Indeed, bioluminescence reporters are widely used in various aspects of biological functions, such as
gene expression, post-translational modification, and protein-protein interaction in cell based assays (Wilson and Hastings, 1998;
Nakajima and Ohmiya, 2010). Advanced luciferase technologies also permit the quantitative visualization of gene expression at
single-cell resolution by imaging its luminescence in real-time using a highly sensitive charged-coupled device (CCD) camera.
Although fluorescence imaging techniques that use fluorescent proteins (e.g., green fluorescent protein (GFP) and its derivatives)
as probes have greatly contributed to the advancement of cell biology studies, bioluminescence imaging is emerging as a new and
sensitive approach for understanding cell physiology (Greer and Szalay, 2002; Roda et al., 2009).
Table 1 summarises the characteristic properties of bioluminescence systems based on commercially available luciferases from
luminescent beetles, sea pansy, copepods, and ostracods. Commercially available luciferins comprise only three types: firefly D-
luciferin, coelenterazine, and Cypridina luciferin, although dinoflagellate and Latia luciferins have been identified. In light-
emitting reactions, the emission maxima of firefly D-luciferin-type, coelenterazine-type, and Cypridina luciferin-type
bioluminescence are found at around 535–630 nm, 460–480 nm, and 460 nm, respectively. These values correspond to the
fluorescence emission maxima of their oxyluciferins. The mass of luciferases vary widely (20–62 kDa), and their structures, which
originate from phylogenetically distant systems, belong to different superfamilies. Cypridina, Gaussia, and Metridia luciferases
(CLuc, GLuc, and MetLuc, respectively) are secreted enzymes. The luciferin-luciferase reaction is triggered by adding luciferin,
although the bioluminescence in firefly, click beetle, and railroad worm needs ATP and magnesium ions as cofactors.
Table 1. Summary of Commercialized Luciferase Genes and Their Characteristic Properties. (see: Nakajima and Ohmiya, 2010)
Luciferase Reporter Assay
Cell based assay systems using reporter enzymes are used widely to study promoters, interactions between promoters and
transcription factors, signal transduction, and other cellular activities. Cell based assays are also applied to drug screening both in
vitro and in vivo. Of the reporter genes known to date, luciferases, enzymes that catalyze bioluminescence reactions, are used most
frequently because their sensitivity and linear response range are superior to those of typical reporters, including β-galactosidase,
chloramphenicol acetyltransferase, β-glucuronidase, and green fluorescent protein. Bioluminescence is a simple reaction that is
triggered by the addition of luciferin solution and some cofactors, and the equipment for measuring light intensity is simple
because it uses only a photomultiplier or a charge-coupled device (CCD) camera; thus, this assay can be applied to high-
throughput screening (HTS). Luciferases are the most suitable reporter enzymes for the quantitative measurement of gene
expression.
Figure 1 demonstrates the simple luciferase reporter assay (Bronstein et al., 1994). In general, the luciferase gene containing the
target promoter region of interest in the plasmid is transfected into target cells, and luciferase-expressing cells are lysed for an
appropriate period, e.g., 1-2 days. The amount of expressed luciferase protein can be estimated from the light intensity in vitro.
In the transient transfection luciferase assay, the luciferase-expressing cells are first lysed for an appropriate period. Luciferase
activity in the cell extracts, measured with a luminometer equipped with a photomultiplier tube, is the most frequently used
luciferase assay system because of its high sensitivity and broad linear response (up to 7-8 orders of magnitude) (Roda et al., 2009;
Silverman et al., 1998). This system is used widely for basic biological studies, including those of gene expression, post-
transcriptional modification, and protein-protein interactions, and for diagnostic and drug discovery applications, because it is
suited for HTS. However, we have to normalize the promoter activity using cell number or cellular internal enzyme activity, such
as β-galactosidase, β-glucuronidase, chloramphenicol acetyltransferase, thus minimizing inherent experimental variability that can
undermine experimental accuracy.
Figure 1. Principle of a simple luciferase reporter assay. The reporter plasmid vector consists of the target promoter sequence and
a luciferase gene sequence. After transfection of the plasmid into target cells, the promoter region regulates the expression of
luciferase gene in living cells. The expressed luciferase protein catalyzes a reaction with luciferin to produce light. In the transient
transfection luciferase assay, luciferase-expressing cells are lysed for an appropriate period. The amount of expressed luciferase
protein can be estimated from the light intensity, which indicates the promoter activity in living cells. In this case, the promoter
activity is normalized by cell numbers or cellular enzymatic activity. (see: Bronstein et al., 1994)
Today, there are many examples of the gene expression reporter assays that use a protein or gene of GLuc and CLuc in
mammalian cells (Tannous et al., 2005; Nishide et al., 2006; Yamagishi et al., 2006). Figure 3 shows the principle of the dual
secreted luciferase reporter assay. In general, for reporter assays, the promoter activities of the target and the control must be
determined, because the reproducibility of each assay is the most important consideration. A dual-luciferase reporter assay system
using CLuc and GLuc is suitable for the HTS of multiple gene expression, because nonsecreted luciferase reporter assays require
sample lysis or special equipment, such as a filtered luminometer (Wu et al., 2007). The activities of the two secreted luciferases
can be measured separately and accurately. The dual-reporter assay system using CLuc and GLuc is suitable for the HTS of gene
transcription in living cells. However, the dual-reporter assay system can be used only in eukaryotes, because the active form of
CLuc is not expressed in Escherichia coli, although GLuc can be expressed as an active form in E. coli.
Figure 3. Principle of a dual secreted luciferase reporter assay. The two reporter plasmid vectors consist of the target promoter
sequence, the CLuc gene sequence and the constitutive promoter sequence, with the GLuc gene sequence serving as the control.
After transfection of the two plasmids into target cells, the promoter region regulates the expression of the luciferase genes in
living cells. Both expressed CLuc and GLuc secrete into the medium. CLuc protein catalyzes a reaction with cypridinid luciferin to
produce blue light. GLuc protein catalyzes a reaction with coelenterazine to produce blue light. For the two-tube dual secreted
luciferase assay, two partial aliquots of luciferases secreted cell medium are collected for an appropriate period. The amounts of
expressed luciferase proteins in each tube can be estimated from the light intensity separately. The light intensities indicate the
target promoter and control promoter activities in living cells. In this case, the target promoter activity is normalized by control
promoter activity. (see: Wu et al., 2007)
Because all of these luciferases emit light with firefly D-luciferin, their individual activities can be detected in a one-step reaction
in a single sample. Figure 5A shows a schematic of the measurement of the three luciferase activities in a mixture using >560-nm
and >600-nm long-pass filters (O56 and R60) (Nakajima et al., 2005). First, the total relative light units (F0) were measured in the
absence of the filters. Then, the F1 value that passed through the O56 filter (striped area) was measured, and finally, the F2 value
that passed through the R60 filter (striped area) was measured. Each luciferase activity was calculated using the simultaneous
equation (Figure 5B) by substituting F0, F1, and F2 values: where G, O, and R are the activities of the green-, orange-, and red-
emitting luciferases, respectively; κGO56, κOO56, and κRO56 are the transmission coefficients of the green-, orange-, and red-
emitting luciferases of the O56 filter, respectively; and κGR60, κOR60, and κRR60 are the transmission coefficients of the green-,
orange-, and red-emitting luciferases of the R60 filter, respectively. Using this method, we estimated that the linear response range
of the system exceeds two orders of magnitude (Figure 5B), although the low-threshold light intensities requires one order of
magnitude higher intensity than those estimated in the dual-color luciferase assay system.
Figure 5. Schematic diagram of the measurement of luciferase activities by splitting emissions using optical filters. (A)
Bioluminescence spectra of green-emitting (green dotted line), orange-emitting (orange dotted line) and red-emitting (red dotted
line) luciferases, and the transmission spectra of >560 nm (O56, black dotted line) and >600 nm (R60, gray dotted line) long-pass
filters. First, the total relative light units (F0) were measured in the absence of the filters. Then, the F1 value that passed through
the O56 filter (striped area) was measured, and finally, the F2 value that passed through the R60 filter (striped area) was measured.
Each luciferase activity was calculated using the Equations (Figure 5B) by substituting F0, F1 and F2 values. G, O and R are the
activities of the green-, orange- and red-emitting luciferases, respectively; kGO56, kOO56 and kRO56 are the transmission
coefficients of the green-, orange- and red-emitting luciferases of the O56 filter, respectively; and kGR60, kOR60 and kRR60 are
the transmission coefficients of the green-, orange- and red-emitting luciferases of the R60 filter, respectively. (B) Quantitative
relationship among green (green circle), orange (orange circles), and red (red circles) luciferase activities in a mixture. (see:
Nakajima et al., 2005)
In Figure 6, after transfection of reporter gene vector, the luciferase-expressing cells in a culture plate is measured with a real time
monitoring luminometer equipped with a photomultiplier tube. The noninvasive multicolor luciferase assay was also used in in
vitro and in vivo studies of post-translational stabilization, signaling pathways, and protein-protein interactions by using a CCD
camera to capture the luciferase emissions (Noguchi, 2008, 2010).
Figure 6. Principle of a real time luciferase reporter assay. The reporter plasmid vector consists of the target promoter sequence
and the luciferase gene sequence. After transfection of the plasmid into target cells, the promoter region regulates the expression of
the luciferase gene in living cells. For the real time luciferase assay, firefly luciferin in the medium enters into the living luciferase
expressed cells, and is oxidized by luciferase immediately, resulting in the production of light. The amount of expressed luciferase
protein can be estimated from the light intensity. The light intensity indicates the promoter activity in living cells. In this case, the
promoter activity is measured by a real-time monitoring luminometer. (see: Noguchi et al., 2008)
Calcium Ion and ATP Monitoring Using Photoprotein and Beetle Luciferase
A dual-color system for simultaneously monitoring of intracellular Ca 2+ and ATP levels through a combination of a Ca2+-sensitive
aequorin and ATP-sensitive beetle red luciferase (PxRE) is shown in Figure 7. Aequorin is composed of two distinct units, the
apoprotein apoaequorin and the prosthetic group hydroperoxycoelenterazine. When calcium ions bind the calcium binding sites
(EF hand motifs), the protein undergoes a conformational change and converts coelenterazine into excited coelenteramide and
CO2. As the excited coelenteramide relaxes to the ground state, blue light (λmax = 465 nm) is emitted. PxRe luciferase catalyzes
the oxidation of firefly D-luciferin by using ATP in the presence of O2 and Mg2+, and then forms oxyluciferin in an electronically
excited state. As the excited oxyluciferin relaxes to the ground state, red light (λmax = 630 nm) is emitted. Using this system,
Kwon et al. (2010) detect the dynamic changes simultaneously in both intracellular Ca 2+ and ATP levels during chondrogenesis.
Figure 7. Principle of calcium ion and ATP monitoring using photoprotein and beetle luciferase. This is a dual-color system for
simultaneously monitoring the intracellular Ca2+ and ATP levels through a combination of a Ca2+-sensitive aequorin and ATP-
sensitive PxRe. Aequorin is composed of two distinct units, the apoprotein apoaequorin and the prosthetic group coelenterazine, a
luciferin. When Ca2+ occupies such sites, the protein undergoes a conformational change and converts coelenterazine into excited
coelenteramide and CO2. As the excited coelenteramide relaxes to the ground state, blue light (λmax = 465 nm) is emitted. PxRe
luciferase catalyzes the oxidation of D-luciferin by using ATP in the presence of O2 and Mg2+, and then forms oxyluciferin in an
electronically excited state. As the excited oxyluciferin relaxes to the ground state, red light (λmax = 630 nm) is emitted. (see:
Kwon et al., 2010)
Bioluminescence Imaging
Among known optical imaging techniques, molecular imaging using either fluorescent or bioluminescent reporters is one of the
most sensitive methods, and uses the most cost-effective and simplest procedure. Fluorescence imaging using a fluorescent dye
molecule or a genetically encoded fluorescent protein and its derivatives has contributed immensely to the advancement of cell
biology, and provides a powerful tool to image an extensive array of samples, ranging from single molecules to whole organisms
(Giepmans et al., 2006; Ghasemi et al., 2009; Day and Davidson, 2009; DiPilato and Zhang, 2010). However, because fluorescent
reporters require exogenous illumination to emit light, this technique is not appropriate for long-term and quantitative imaging for
the following reasons: (1) the fluorescent reporter is bleached by repetitive illumination; (2) repetitive exogenous light illumination
causes phototoxic damage to cells; and (3) exogenous illumination perturbs the physiology in light-sensitive tissue such as the
retina. In contrast, bioluminescence imaging using luciferase reporters does not need exogenous light illumination, and the
luminescence reaction is quantitative, and has an extremely low background. This imaging method is particularly useful for
longitudinal and quantitative imaging.
Figure 9 shows the time-lapse imaging of the nucleocytoplasmic shuttling of ELuc-fused importin-α (mRch1) (Imamoto et al.,
1996; Goldfarbet al., 2004), a shuttling nuclear import receptor for nuclear localization signal-containing protein. The
luminescence signal was detected initially in the cytosol; after 24 min, the signal increased gradually in the nucleus, and this was
accompanied by a decrease of the signal in the cytosol. Conversely, the signal in the nucleus decreased gradually after 52 min of
measurement with a concomitant increase in cytosolic signal for up to 80 min of measurement. An accumulation of the signal in
the nucleus was observed again from 84 to 128 min of measurement (Nakajima et al., 2010).
Figure 9. Time-lapse bioluminescence imaging of the nucleocytoplasmic shuttling. The reporter plasmid vector consists of the
constitutive promoter sequence and an importin-α fused luciferase gene sequence. After transfection of the plasmid into target
cells, the promoter region regulates the expression of luciferase genes in living cells. For the time-lapse bioluminescence imaging
experiment, firefly luciferin that is added to the medium enters the importin-α luciferase expressed cells, where it is catalyzed by
the expressed firefly luciferase to generate light. The light signal indicates the nucleocytoplasmic shuttling of importin-α in living
cells. In this case, the bioluminescence imaging is measured by special equipment using a CCD photon imaging system. (see:
Nakajima et al., 2010)
Another approach for in vivo bioluminescence imaging is based on a luciferase protein probe. The antibody-fused luciferase
visualizes disease-specific antigens on cell surfaces in a living body after the conjugation of luciferase and antibody. The purified
probe is injected into mice. Twenty-four hours after the administration of the antibody fused luciferase probe, luciferin was
injected, and the bioluminescence images were obtained using a CCD photon imaging system (Figure 11).
For this technique, Wu et al. (2009) developed a far-red luminescence imaging technology for the visualization of disease specific
antigens on cell surfaces in a living body. First, they conjugated a far-red fluorescent indocyanine derivative to biotinylated
Cypridina luciferase (CLuc). This conjugate produced a bimodal spectrum that has long-wavelength bioluminescence emission in
the far-red region, as a result of bioluminescence resonance energy transfer. To generate a far-red luminescent probe with targeting
and imaging capabilities for tumors, they then linked this conjugate to an anti-human Dlk-1 monoclonal antibody via the biotin-
avidin interaction. This far-red luminescent probe is a convenient analytical tool for the evaluation of monoclonal antibody
localization in a living body.
Figure 11. In vivo bioluminescence imaging using an antibody-fused luciferase probe. Cypridina luciferase is expressed in a
suitable expression system, and is purified by chromatography. An antibody fused Cypridina luciferase consists of Cypridina
luciferase and antibody protein for an antigen on the cell membrane via, e.g., biotin-avidin conjugation. The targeted antigen
expressed cell line was implanted into the back of mice. Tumor growth was monitored until it reached an acceptable size. For in
vivo imaging, the antibody fused Cypridina luciferase was injected into mice intravenously. To obtain a bioluminescence image,
the mice were given injections of Cypridina luciferin at appropriate times. Bioluminescence imaging was performed by special
equipment using an intensified CCD camera. (see: Wu et al., 2009)
After incubating the sample for 30-60 min with the anti-cancer antibody-luciferase conjugate, the sample is directly visualized by
the luciferin-luciferase reaction. After incubation and washing, a cypridinid luciferin solution is added to the section under
darkened conditions; images of the bioluminescence from the tumor cells are quickly acquired using a cooled CCD camera system
operated at a high sensitivity range. The bioluminescence images were strong, and corresponded to those obtained by
immunohistochemical staining.
Figure 12. Ex vivo bioluminescence imaging using an antibody-fused luciferase probe. (A) Procedure of immunohistochemical
visualization for serial paraffin sections using an indirect peroxidase labeling is as follows. The immunohistochemical staining was
carried out using a monoclonal antibody. After washing with PBS, the section was incubated with the secondary peroxidase
labeled antibody. Finally, the immunohistochemical staining used diaminobenzidine. (B) Cypridina luciferase is expressed in a
suitable expression system, and is purified by chromatography. An antibody fused Cypridina luciferase consists of antibody
protein for antigen and Cypridina luciferase via, e.g., biotin-avidin conjugation. The procedure for immunohistochemical
visualization for serial paraffin sections using a direct luciferase labeling is as follows; the immunohistochemical staining was
carried out using the antibody fused Cypridina luciferase. After washing with PBS, the section is imaged using the Cypridina
luciferin-luciferase reaction. Bioluminescence imaging was performed with special equipment using an intensified CCD camera.
(see: Wu et al., 2013)
Conclusion
In the post genome era, we must clarify quantitatively and spatially the complex phenomena of biological systems in real time.
Bioluminescence based on the diversity of luminescent molecules has great potential as an assay tool, because the light is produced
by enzyme reactions inherent within the physiological systems of living organisms. Such assays can be used to analyze the
complex phenomena in biological systems, which can be detected as light signal outputs that correspond to specific biological
events. As described in this review, bioluminescence has many applications and bioluminescence assays are expected to become
essential research tools. Finally, it is essential to consider the underlying principle and the characteristics of bioluminescent
reactions, and to select a suitable bioluminescence system depending on the assay purpose.
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