SHORT TECHNICAL REPORTS Multicolor luciferase assay system: one-step monitoring of multiple gene expressions with a single substrate Yoshihiro Nakajima1, Takuma Kimura1, Kazunori Sugata1, Toshiteru Enomoto2, Atsushi Asakawa2, Hidehiro Kubota2, Masaaki Ikeda3, and Yoshihiro Ohmiya1 1National 2ATTO
Institute of Advanced Industrial Science and Technology (AIST), Osaka, Corporation, Tokyo, and 3Saitama Medical School, Saitama, Japan
BioTechniques 38:891-894(June 2005)
Reporter assays that use luciferase are widely employed for monitoring cellular events associated with gene expression. In general, firefly luciferase and Renilla luciferase are used for monitoring single gene expression. However, the expression of more than one gene cannot be monitored simultaneously by this system because one of the two reporting luciferases must be used as an internal control. We have developed a novel reporter assay system in which three luciferases that emit green, orange, and red light with a single substrate are used as reporter genes. The activities of the luciferases can be measured simultaneously and quantitatively with optical filters. This system enables us to simply and rapidly monitor multiple gene expressions in a one-step reaction.
INTRODUCTION Reporter assay systems are widely used for studying promoters, interactions between promoter and transcription factors, signal transduction, and other cellular activities, and are also applicable to drug screening both in vitro and in vivo (1–3). Of the reporter genes known to date, luciferases are the most frequently employed because their sensitivity and range of linear response are superior to those of other typical reporters, including β-galactosidase, chloramphenicol acetyltransferase, β-glucuronidase, and green fluorescent protein (4). Thus, luciferases are the most suitable reporter genes for the quantitative measurement of gene expression. In a typical in vitro luciferase reporter assay system, firefly luciferase is used for monitoring gene expression, whereas Renilla luciferase is used as an internal control [e.g., the DualLuciferase® Reporter Assay System; Promega, Madison, WI, USA (www. promega.com/tbs/tm040/tm040.html)]. Even with this current system, however, the expression of more than one gene cannot be monitored simultaneously because one of the two reporting luciferases must be used as an internal control to minimize experimental variability. To overcome the limitations Vol. 38, No. 6 (2005)
of the reporter assay system, additional luciferases and a novel measurement technique are required to monitor two or more gene expression levels at once. A reporter assay system for the simultaneous monitoring of multiple gene expressions and/or interactions is important to investigate cascades of transcriptional regulation that form the molecular basis of various biological functions. We have recently developed a novel reporter assay system, the tricolor reporter in vitro assay system, in which the expression of two genes can be monitored simultaneously by splitting the emissions from greenand red-emitting luciferases (5,6) with an optical filter (7). In this system, the green and red luciferase activities are simultaneously measured in the extracts of the transfected cells after the addition of firefly luciferin, and then Renilla luciferase activity is measured after adding Renilla luciferin to normalize the green and red luciferase activities. This reporter assay system is, however, slightly inconvenient because the Renilla luciferase activity must be measured separately from the green and red luciferase activities in another tube due to differences in their luciferin and assay chemistry. To improve this system, we have introduced an additional luciferase, the orange-emitting luciferase, which
emits orange light (8) in the presence of firefly luciferin, in place of Renilla luciferase. We have simultaneously successfully measured the activities of the green, orange, and red luciferases in a mixture by splitting their emissions with optical filters. We have confirmed that this system allows us to simultaneously simply and rapidly monitor the expression levels of three genes (two are test reporters and one is an internal control) by using a single luminescent substrate in one tube. MATERIALS AND METHODS Plasmids The sources of the luciferases used in this study were plasmids pB-RmL (9), pB-RolT226N (8), and pBl-PxRe (5). To make the luciferases suitable for high-level mammalian expression and to minimize inappropriate activation, we optimized the codons and deleted transcription factor binding sites within the cDNA without changing the deduced amino acid sequences (Y. Nakajima, K. Sugata, and Y. Ohmiya, unpublished data). The resulting plasmids were SYN384 (green luciferase), SNY385 (orange luciferase), and SYN325 (red luciferase). Each luciferase coding region was excised with NcoI and XbaI, and the fragment was ligated into the NcoI/XbaI site of expression vector pGV-C2 (Toyo Ink, Tokyo, Japan) from which the firefly luciferase had been removed, resulting in pSV40-mGR, pSV40-mOR, and pSV40-mRED, respectively. The Bp/915-mOR reporter plasmid carrying the 5′ flanking region (-816/+99) of murine Bmal1 (mBmal1) was constructed by replacing the NcoI/XbaI fragment of the Bp/915-Luc reporter vector (10) with orange luciferase from SYN385. To construct a red reporter plasmid carrying Rev-Erb and retinoic acid receptor-related orphan receptor (ROR) response element (RORE), the region (-13/+69) in the mBmal1 promoter was PCR-amplified with the Bp/915-Luc vector as previously described (7), and the product was ligated into the SmaI site immediately upstream of the simian virus 40 (SV40) promoter of pSV40-mRED, resulting in pRORE-mRED. cDNA coding for the full open reading frame of murine BioTechniques 891
SHORT TECHNICAL REPORTS
Expression of Luciferases in Silkworm To make the expression plasmids for silkworm, luciferases were excised from pB-RmL, pB-RolT226N, and pBl-PxRe and subcloned into the pYNG expression vector (Katakura Industries, Saitama, Japan) in which luciferases were expressed under the control of a polyhedrin promoter. Recombinant luciferases were produced by a silkworm-baculovirus expression system (Katakura Industries). Infected silkworms were homogenized with 20 mM Tris-HCl, pH 8.0, supplemented with 10% (w/v) glycerol, 150 mM NaCl, 1 mM phenylmethanesulfonyl fluoride (PMSF), and 1 mM phenylthiourea. The resultant supernatant was precipitated with 4%–10% (w/v) polyethylene glycol. Measurement of Bioluminescence Spectra Bioluminescence was measured with an AB1850 spectrophotometer (Atto, Tokyo, Japan). Measurement was carried out by injecting 15 µL of PicaGene as a substrate into 15 µL of the transfected cell extract, and bioluminescence spectra were collected 892 BioTechniques
[][
F0 1 F1 = κGO56 F2 = κGR60
1 κOO56 κOR60
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1 G κRO56 O κRR60 R [Eq. 1] where G, O, and R are the green, orange, and red luciferase activities, respectively; F0 is the total relative light units (RLU) measured in the absence of the optical filter; F1 and F2 are the RLU that pass through the O56 and R60 filters, respectively; κGO56, κOO56, and κRO56 are the transmission coefficients of the green, orange, and red luciferases of the O56 filter, respectively; and κGR60, κOR60, and κRR60 are the transmission coefficients of the green, orange, and red luciferases of the R60 filter, respectively. RESULTS AND DISCUSSION To construct the simultaneous monitoring system, we chose the following three luciferases as reporter genes because all of these luciferases emit light with a common substrate (firefly luciferin), and their bioluminescence spectra are constant even if the intracellular pH is changed (5,8): the green-emitting luciferase (green luciferase) of Rhagophthalmus ohbai (9), the orange-emitting luciferase (orange luciferase) that is a point mutant of the green luciferase (T226N; Reference 8), and the red-emitting luciferase (red luciferase) of Phrixothrix hirtus (5). Figure 1A shows the
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Forty-five microliters of cell lysate were mixed with 100 µL PicaGene that had been adapted at 30°C, and the mixture was incubated for 5 min at 30°C. Then, the green, orange, and red luciferase activities were measured for 60 s at 1-s intervals in the absence or presence of 560- and 600-nm long pass filters (O56 and R60 filters, respectively; Hoya, Tokyo, Japan) using an AB2250 luminometer (Atto). Each activity was calculated using the simultaneous equation
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Murine fibroblast NIH/3T3 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; ICN Biochemicals, Aurora, OH, USA) at 37°C. Cells were seeded in 24-well plates at a density of 5 × 104 cells per well, 1 day before transfection. Transfection was carried out using Lipofectamine™ Plus (Invitrogen) according to the manufacturer’s instructions. Two days after transfection, the cells were washed once with 300 μL cold phosphate-buffered saline (PBS) and disrupted in 300 μL of PicaGene™ Dual lysis buffer (Toyo Ink) supplemented with 10% (w/v) glycerol for 10 min at 25°C, and then the lysate was kept on ice.
Measurement of Luciferase Activities
bioluminescence spectra of the luciferases expressed in NIH/3T3 cells, and the transmission spectra of 560- (O56) and 600-nm (R60) long pass filters (which collect emissions at >540 and >575 nm, respectively) for splitting emissions. The luciferases emitted green (λmax = 550 nm), orange (λmax = 580 nm), and red (λmax = 630 nm) light, similar to those measured in Escherichia coli extract (5,8). Figure 1B shows the bioluminescence spectrum
Relative intensity
Cell Culture and Transfection
for 1 min with 1 mm slit width. All spectra were corrected for equipment photosensitivity and normalized.
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RORα4 (mRORα4) was amplified by reverse transcription PCR as previously described (7) and cloned into pCR3.1 (Invitrogen, Carlsbad, CA, USA).
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E F1
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1 1 1 F0 F1 = �GO56 �OO56 �RO56 �GR60 �OR60 �RR60 F2
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Figure 1. Measurement of luciferase activities by splitting emissions using long pass filters. (A) Bioluminescence spectra of green (green line), orange (orange line), and red (red line) luciferases, and the transmission spectra of 560(O56, black solid line) and 600-nm (R60, black dotted line) long pass filters. Expression plasmids (400 ng) pSV40-mGR, pSV40-mOR, and pSV40-mRED were transfected independently into NIH/3T3 cells, and the cells were harvested and disrupted in PicaGene Dual lysis buffer after 48 h. Spectra were measured as described in the Materials and Methods section. The photograph (inset) shows the bioluminescence of silkworm expressing green (left), orange (middle), and red (right) luciferases, after mixing luciferin (PicaGene). The exposure was for 1 min using Superia Venus 1600 film (Fujifilm, Tokyo, Japan). (B) Bioluminescence spectrum of NIH/3T3 cells expressing green, orange, and red luciferases (black line), in which the cell lysates with the same bioluminescence intensity were mixed, and the transmission spectra of the O56 (orange line) and R60 (red line) filters. (C–E) Measurement of luciferase activities using long pass filters. The simultaneous equation for calculating each luciferase activity is shown at the bottom of the figure. Vol. 38, No. 6 (2005)
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Figure 2. Quantitative relationship among green (green circle), orange (orange circles), and red (red circles) luciferase activities in a mixture. Fifty microliters each of the luciferases that were expressed in silkworm and diluted with PicaGene Dual lysis buffer at the indicated volume ratio were mixed. The respective luciferase activities were measured with an AB2250 luminometer after injecting 100 µL PicaGene. RLU, relative light unit. Vol. 38, No. 6 (2005)
added and showed good linearity (r2 = 0.997 for orange luciferase; r2 = 0.995 for red luciferase), whereas the activity of the green luciferase was constant. However, we note that this relationship does not hold linearity when the respective luciferase activities are below 1 × 104 RLU/20 s (data not shown). The transmission coefficients of the luciferases, the raw (F0, F1, and F2) data and the calculated data are indicated in the supplementary tables available online at www.BioTechniques.com. The results indicate that our novel assay system can accurately measure three luciferase activities simultaneously, and the equation for calculating the activities of the green, orange, and red luciferases by splitting emissions is valid. The dynamic range of this system spans at least three orders of magnitude, suggesting that this linearity is sufficient for the simultaneous monitoring of any gene expressions because, to our knowledge, the usual changes in transcriptional activities upon interacting with transcription factors occur within this range. To further verify this system, we analyzed the effect of the transcription factor RORα4, an orphan nuclear receptor (11), on clock gene transcription. Recently, we and others have reported that RORα4 positively regulates the transcription of the clock gene Bmal1 through RORE in the promoter region of Bmal1 (7,12,13). Furthermore, we have analyzed in detail the role of RORα4 in the transactivation of Bmal1 by means of a tricolor reporter in vitro assay system, in which the effect of RORα4 on the transcription from RORE and the Bmal1 promoter was directly compared using green, red, and Renilla luciferases (7). In this context, we repeated the experiment using green, orange, and red luciferases, in which the green luciferase was used as an internal control in place of Renilla luciferase. Reporter plasmids pRORE-mRED and Bp/915-mOR were co-transfected with various amounts of RORα4 expression plasmid and pSV40-mGR plasmid as an internal control into NIH/3T3 cells. As shown in Figure 3, RORα4 produced substantial increases in the transcriptional activities of both RORE
(red bars) and the Bmal1 promoter (orange bars) in a dose-dependent manner. The dose dependence of the RORα4-induced transcription of RORE was identical to that of the Bmal1 promoter; however, the response of the Bmal1 promoter to RORα4 was much greater than that of RORE, and this may be due to the presence of additional enhancer element(s) responding to RORα4 other than RORE in the 5′ flanking region of the Bmal1 promoter. The dose dependence of the transactivations shown in the present study is almost identical with that measured previously (7). We note, however, that the induction ratios of the RORE and Bmal1 promoter by RORα4 are relatively lower than those of our previous study, which is probably caused by the differences in the experimental conditions, such as promoter used and/or the amount of reporter constructs used. We therefore assume that the inconsistent results are due not to the difference in sensitivity between the two reporter assay systems but to differences in the experimental conditions. Taken together, the results
6 Relative Activity (Fold)
of NIH/3T3 cells expressing the green, orange, and red luciferases, in which cell lysates with the same bioluminescence intensity were mixed, as well as the transmission spectra of the O56 and R60 long pass filters. The splitting of the emissions using long pass filters is advantageous in that the emission loss is less than that when interference filters that collect a narrow range of emissions are used, thereby improving the signal-to-noise ratio. Figure 1, C–E, shows the measurement of the green, orange, and red luciferase activities in a mixture using long pass filters. First, total RLU (F0) was measured in the absence of the filters (Figure 1C). Then, the F1 value that passed through the O56 filter (shadowed area) was measured (Figure 1D), and finally, the F2 value that passed through the R60 filter (shadowed area) was measured (Figure 1E). Each value was substituted into the simultaneous equation (bottom), and the respective activities were calculated. To examine whether the respective activities of the three luciferases in a mixture can be measured simultaneously, the activities in mixtures with various volume ratios were measured (Figure 2), where the amount of the green luciferase added was fixed to give approximately half the maximum activities of the orange and red luciferases. The measured activities of the orange and red luciferases were found to increase in proportion to the amount
RORE SV40
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Figure 3. Simultaneous monitoring of mRORα4-dose-dependent induction of RORE-mediated (red bars) and mBmal1 promoter fragment-driven (orange bars) transcription. Reporter plasmids pRORE-mRED (200 ng) and Bp915-mOR (100 ng) and 4 ng pSV40-mGR were co-transfected without (0) or with the indicated amounts (ng) of expression plasmids carrying mRORα4 into NIH/3T3 cells. The amount of DNA added per well was kept constant (379 ng) by adding pBluescript® SK(–) (Stratagene, La Jolla, CA, USA). All values are shown as multiples (mean ± sd; n = 6) of the control (lacking the expression plasmid). The diagram of the reporter plasmids shows the location of elements. Key: RORE, Rev-Erb/ ROR response element in the mBmal1 promoter; mBmal1 915, 915-bp fragment of the mBmal1 promoter region; SV40, simian virus 40 promoter; Orange, orange luciferase; Red, red luciferase; Green, green luciferase. BioTechniques 893
SHORT TECHNICAL REPORTS shown in Figure 3 suggest again that our new reporter assay system using green, orange, and red luciferases can monitor simultaneously respective transcriptional activities with a single luciferin and that the simultaneous equation for estimating the three luciferase activities by splitting emissions is appropriate. In conclusion, this system has enabled us to directly compare two or more transcriptional activities and/or interactions with transcription factors in the same cell population in a onestep reaction with a single luciferin. In addition, this system can easily be applied to the high-throughput monitoring of gene expression levels, including the real-time monitoring of genes showing rhythmic, constitutive, or acute expression profiles in culture cells or tissues or in vivo. ACKNOWLEDGMENTS
We thank Drs. K. Honma and S. Honma of Hokkaido University for helpful suggestions and discussions. We also thank A. Oka and N. Ueda of AIST for excellent technical assistance. This study was supported by a NEDO grant (Dynamic Biology Project) from the Ministry of Economy, Trade and Industry of Japan, and in part by a Grant-in-Aid for the Support of Young Researchers (grant no. 13073-2125-14) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. COMPETING INTERESTS STATEMENT
The authors declare no competing interests. REFERENCES 1.Bronstein, I., J. Fortin, P.E. Stanley, G.S. Stewart, and L.J. Kricka. 1994. Chemiluminescent and bioluminescent reporter gene assays. Anal. Biochem. 219:169-181. 2.Wilson, T. and J.W. Hastings. 1998. Bioluminescence. Annu. Rev. Cell Dev. Biol. 14:197-230. 3.Greer, L.F. and A.A. Szalay. 2002. Imaging of light emission from the expression of luciferases in living cells and organisms: a review. Luminescence 17:43-74. 4.Naylor, L.-H. 1999. Reporter gene technology: the future looks bright. Biochem. Pharmacol. 58:749-757. 894 BioTechniques
5.Viviani, V.R., E.J. Bechara, and Y. Ohmiya. 1999. Cloning, sequence analysis, and expression of active Phrixothrix railroad-worms luciferases: relationship between bioluminescence spectra and primary structures. Biochemistry 38:8271-8279. 6.Nakajima, Y., T. Kimura, C. Suzuki, and Y. Ohmiya. 2004. Improved expression of novel red- and green-emitting luciferases of Phrixothrix railroad worms in mammalian cells. Biosci. Biotechnol. Biochem. 68:948-951. 7.Nakajima, Y., M. Ikeda, T. Kimura, S. Honma, Y. Ohmiya, and K. Honma. 2004. Bidirectional role of orphan nuclear receptor RORα in clock gene transcriptions demonstrated by a novel reporter assay system. FEBS Lett. 565:122-126. 8.Viviani, V.R., A. Uchida, N. Suenaga, M. Ryufuku, and Y. Ohmiya. 2001. Thr226 is a key residue for bioluminescence spectra determination in beetle luciferases. Biochem. Biophys. Res. Commun. 280:1286-1291. 9.Ohmiya, Y., M. Sumiya, V.R. Viviani, and N. Ohba. 2000. Comparative aspects of a luciferase molecule from Japanese luminous beetle, Rhagophthalmus ohbai. Sci. Rept. Yokosuka City Mus. 47:31-38. 10.Yu, W., M. Nomura, and M. Ikeda. 2002. Interactivating feedback loops within the mammalian clock: BMAL1 is negatively autoregulated and upregulated by CRY1, CRY2, and PER2. Biochem. Biophys. Res. Commun. 290:933-941. 11.Jetten, A.M., S. Kurebayashi, and E. Ueda. 2001. The ROR nuclear orphan receptor subfamily: critical regulators of multiple biological processes. Prog. Nucleic Acid Res. Mol. Biol. 69:205-247. 12.Sato, T.K., S. Panda, L.J. Miraglia, T.M. Reyes, R.D. Rudic, P. McNamara, K.A. Naik, G.A. FitzGerald, et al. 2004. A functional genomics strategy reveals Rora as a component of the mammalian circadian clock. Neuron 43:527-537. 13.Akashi, M. and T. Takumi. 2005. The orphan nuclear receptor RORα regulates circadian transcription of the mammalian core-clock Bmal1. Nat. Struct. Mol. Biol. 12:441-448.
Received 7 December 2004; accepted 2 February 2005. Address correspondence to Yoshihiro Ohmiya, Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Midorigaoka, Ikeda, Osaka 563-8577, Japan. e-mail:
[email protected]
Vol. 38, No. 6 (2005)
