Dual Luminescence-Based Reporter Gene Assay for ... - BioTechniques

Dual Luminescence-Based Reporter Gene Assay for Luciferase and β-Galactosidase Chris S. Martin, Patricia A. Wight1, Anna Dobretsova1 and Irena Bronstein Tropix, Inc., Bedford, MA and 1University of Arkansas for Medical Sciences, Little Rock, AR, USA BioTechniques 21:520-524 (September 1996)

ABSTRACT A unique combined luminescence assay for firefly (Photinus pyralis) luciferase and β-galactosidase (β-gal) reporter gene products is described. Luciferase and β-gal activities are determined with the same aliquot of cell lysate prepared from cells contransfected with both reporter genes, thereby reducing manual labor and increasing experimental accuracy. With the Dual-Light assay system, luciferase activity is measured first with an enhanced luciferase assay, followed by quantitation of β-gal with Galacton-Plus chemiluminescent substrate and Sapphire-II enhancer. Highly sensitive detection of luciferase (2 fg) and β-gal (8 fg) is achieved with a dynamic range over seven orders of magnitude of enzyme concentration. Comparative analysis of both independent and combined (Dual-Light) detection methods for cells contransfected with luciferase and β-gal reporter genes is also described.

INTRODUCTION The use of reporter genes to study the regulation of gene expression is common practice. In these studies, the effect of a particular DNA sequence or trans-acting factor on gene transcription is evaluated by quantitation of the expressed reporter protein. Widely used reporter proteins (1,14) include the enzymes chloramphenicol acetyltransferase (CAT) (11), β-galactosidase (β-gal) (1,14), firefly (Photinus pyralis) luciferase (2), β-glucuronidase (GUS) (5,10) and secreted placental alkaline phosphatase (SEAP) (5,9), as well as human growth hormone (1), aequorin (13) and green fluorescent protein (6). Because of inherent amplification, reporter enzymes provide the most sensitive detection of gene products through enzymatic substrate turnover. The adaptation of the luciferase/luciferin system for reporter gene analysis and the use of luminescent substrates for β-gal (4) and GUS (5) have 520 BioTechniques

enabled detection of femtogram quantities of these reporter proteins. The nonradioactive luminescence assays are 1000fold more sensitive and less labor-intensive compared to standard CAT assays. Bioluminescent luciferase reporter gene assays have gained in popularity due to their simplicity and high sensitivity. The use of commercially available kits has simplified assay protocols to the addition of one or two reagents to an aliquot of cell extract, followed by measurement of the luminescence signal. While colorimetric and fluorescence assays for β-gal have been available for some time, sensitivity equal to or greater than that achieved with luciferase assays was not possible until the introduction of chemiluminescent 1,2-dioxetane substrates (12). By using Galacton or GalactonPlus 1,2-dioxetane substrates (Tropix, Bedford, MA, USA) and a light-emission accelerator containing a polymeric enhancer, sensitive detection of β-gal in the femtogram range is achieved (5). Thus, both luciferase and β-gal activities from cells cotransfected with the two reporter genes are determined in a single instrument capable of measuring luminescence. Until now, luciferase and β-gal activities have been measured separately by using multiple aliquots of cell extract. In this article, we describe the development of a combined luminescence-based reporter gene assay for luciferase and β-gal. With the Dual-Light Reporter Gene Assay System (Tropix), a single extract sample is assayed sequentially for both enzyme activities. Luciferase reporter enzyme activity is quantitated first with an enhanced luciferase reaction, and then β-gal reporter enzyme activity is determined with Galacton-Plus chemiluminescent β-gal substrate. The Dual-Light assay simplifies the determination of luciferase and β-gal activities from cotransfected cells and increases precision between the two measurements. This assay system is particularly useful in experiments where one of the reporter genes is used to normalize transfection efficiencies. Vol. 21, No. 3 (1996)

MATERIALS AND METHODS Luminescence Assays Purified E. coli β-gal (G-5635) was obtained from Sigma Chemical (St. Louis, MO, USA). Purified firefly (P. pyralis) luciferase was obtained from Analytical Luminescence Laboratory (San Diego, CA, USA). Lysis solution, Buffer A, Buffer B and Accelerator-II are components of the Dual-Light Reporter Gene Assay System. The Dual-Light assay was performed in triplicate at room temperature. For experiments utilizing purified enzyme, dilutions were performed in Lysis Solution (0.1 M potassium phosphate, pH 7.8, 0.2% Triton X-100) containing 0.5 mM dithiothreitol (DTT) and 1 mg/mL bovine serum albumin (BSA) Fraction V (A-3059; Sigma Chemical). A 10-µL aliquot of diluted enzyme was added per well to an opaque white microplate (Microlite 2; Dynatech Laboratories, Chantilly, VA, USA). Subsequently, 25 µL of Buffer A were added to each well, and the plate was placed in a Dynatech ML2250 luminometer. The relative light units (RLU) obtained with luciferase were measured for 5 s beginning 2 s after the injection of 100 µL of Buffer B. RLUs for β-gal were determined 30 min after the addition of Buffer B and were initiated by the injection of 100 µL of Accelerator-II. Luminescence signal intensity was measured for 5 s following a 2s delay. For experiments using cell lysates, Dual-Light assays were performed as described above with 10 µL of 1/10 diluted lysate, except for the following modifications. Accelerator-II was added 60 min after the addition of Buffer B, and luminescence was measured using a Monolight 2010 luminometer (Analytical Luminescence Laboratory). Independent assessment of reporter gene expression was performed as per manufacturers’ instructions utilizing the Luciferase Assay System (Promega, Madison, WI, USA) for luciferase activity and the Galacto-Light Kit (Tropix) for β-gal activity, with 20 µL or 10 µL of 1/10 diluted cell lysate, respectively. Luminescence was measured in the Monolight 2010 luminometer, following a 2-s delay, for either 10 s with luciferase assays or 5 s with βgal assays. Plasmid Construction Construction of poLUC, a promoterless vector with multiple unique restriction sites upstream of the luciferase coding

sequence, has been described previously (2). A 1.5-kb BamHI fragment encompassing the murine myelin proteolipid protein (PLP) gene promoter was cloned into the unique BamHI site upstream of the luciferase coding sequence in poLUC. This construct was denoted PLP-1393LUC and contains PLP DNA from positions 1393 to +88, relative to the start site of transcription. Plasmid PLP-2400LUC contains PLP sequence from positions 2400 (approximate) to +88, relative to the start site of transcription. PLP-2400LUC was constructed from plasmids PLP-1393LUC and PLP(+)Z. Plasmid PLP(+)Z (15) contains approximately 2.4 kb of 5′-flanking PLP DNA immediately upstream of the cap site with an ApaI site at the 5′ end. The unique ApaI site in PLP(+)Z was converted to a SalI site by digestion of PLP(+)Z with ApaI, removal of the 3′ overhang with T4 DNA polymerase and subsequent ligation of SalI linkers (5′-GGTCGACC-3′; Promega). Thereafter, a SalI (approximate PLP position -2400) to NcoI (PLP position -435) fragment was exchanged with the corresponding SalI (multiple cloning site) to NcoI (PLP position -435) fragment in PLP-1393LUC, resulting in plasmid PLP-2400LUC. This construct is capable of driving the expression of luciferase, under the control of the PLP promoter, in glial cells. Transfection The transfection efficiency for every transfection assay was monitored by the transient expression of β-gal. β-Gal was encoded by the plasmid RSVlacZ (7), which contains the lacZ gene under the control of the Rous sarcoma viral long terminal repeat (LTR) promoter. N20.1 glial cells (14) were transiently transfected with equimolar amounts of a given luciferase construct as well as RSVlacZ. Cells were grown in Ham’s F-12/ Dulbecco’s modified Eagle medium (DMEM) (Irvine Scientific, Santa Ana, CA, USA) supplemented with 10% fetal bovine serum (Intergen, Purchase, NY, USA) in an atmosphere of 5% CO2 at 34°C. Twenty-four hours before transfection, the cells were plated at a density of 6.57 × 105 per 60-mm dish. The DNA to be transfected was prepared as follows. First, either 16.7 or 10.9 µg of PLP-2400LUC or poLUC, respectively, were combined with 5 µg of RSVlacZ. Then Bluescript SK(+) vector DNA (Stratagene, La Jolla, CA, USA) was added to bring the total quantity of DNA to 28.8 µg, or it was used as the sole source of DNA for mocktransfected cells. Finally, 11.5 µg of the DNA mixture were added per dish directly to the growth medium (5 mL), in duplicate, according to the methods described by Chen and

Figure 1. Dual-Light assay procedure. Vol. 21, No. 3 (1996)

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Table 1. Comparison of Transfection Results Obtained When a Given Cell Lysate Was Assayed for Luciferase and β-Gal Activities Either Independently or with Dual-Light

Luciferase (Promega)

β-Gal (Galacto-Light)

Fold Over poLUC

Luciferase (Dual-Light)

β-Gal (Dual-Light)

Fold Over poLUC

PLP-2400LUC RSVlacZ

21 826

37 913

3.02

4387

111 791

3.05

PLP-2400LUC RSVlacZ

24 118

37 807

3.35

4928

110 462

3.48

poLUC RSVlacZ

6811

35 293

1.00

1470

108 636

0.98

poLUC RSVlacZ Bluescript SK(+)

7451

38 608

1.00

1610

115 539

1.02

140

262

NA

76

450

NA

Bluescript SK(+)

125

285

NA

207

447

NA

Transfected DNA

N20.1 cells (6.57 × 105 per 60-mm dish) were cotransfected with a given luciferase construct and RSVlacZ or with Bluescript SK(+) DNA alone by the calcium phosphate procedure described in Materials and Methods. Lysates were prepared using 400 µL of reporter lysis buffer (Promega). Lysates were diluted 1/10 in the same lysis buffer, and luciferase or β-gal activities were determined in duplicate, from separate aliquots of lysate using the Luciferase Reporter System (Promega) or the GalactoLight Kit (Tropix), respectively. Determinations of both reporter enzyme activities from the same aliquot of lysate were made in duplicate for each transfection using the Dual-Light Assay System (Tropix). Luminescence was determined from 10 µL of diluted lysate and measured in a Monolight 2010 luminometer for 5 s following a 2-s delay, except for the Promega luciferase assay, where 20 µL of diluted lysate were used and luminescence was measured for 10 s following a 2-s delay. Results are presented as the average RLU between duplicate determinations of a given diluted lysate.The fold induction over poLUC represents the average luciferase RLU corrected for differences in transfection efficiency divided by the mean of the RLU obtained from poLUC-transfected cells. See Materials and Methods for detailed description of the calculations. NA indicates not applicable.

Okayama (8). The cells were incubated in an atmosphere of 3% CO2 for 24 h, followed by the removal of the DNA precipitate and incubation for an additional 40 h in 5% CO2. Lysates were made as per manufacturer’s specifications with 400 µL of reporter lysis buffer (Promega). Lysates were diluted 1/10 in the same lysis buffer and luciferase or β-gal activities were determined, in duplicate, from separate aliquots of lysate using the Luciferase Reporter System or the GalactoLight Kit, respectively. Determinations of both reporter enzyme activities from the same aliquot of lysate were made in duplicate using the Dual-Light Assay System. Luminescence

Figure 2. Detection of luciferase and β-gal with Dual-Light. Purified luciferase and β-gal were individually diluted in lysis solution and quantitated using the Dual-Light protocol as described in Materials and Methods. Ten microliters of each enzyme dilution were assayed. Measurements were performed using a Dynatech Laboratories ML2250 luminometer. 522 BioTechniques

was determined from 10 µL of diluted lysate and measured in a Monolight 2010 luminometer for 5 s following a 2-s delay. With the Promega luciferase assay, 20 µL of diluted lysate were used and luminescence was measured for 10 s following a 2-s delay. Results are shown as the average RLU between

Figure 3. Detection of luciferase and β-gal with Dual-Light, and levels of background signal. Purified luciferase and β-gal were diluted in cell lysis solution. A set of dilutions of each enzyme was assayed following the DualLight protocol as described in Materials and Methods. Ten microliters of each enzyme dilution were assayed. Measurements were performed in a Dynatech Laboratories ML2250 luminometer. Curves are described as follows: Luciferase—Signal intensity following injection of Buffer B into diluted luciferase. Luciferase residual signal—Signal intensity following injection of Accelerator-II into diluted luciferase. β-Galactosidase—Signal intensity following injection of Accelerator-II into diluted β-galactosidase. β-Galactosidase signal read-through—Signal intensity following injection of Buffer B into diluted β-galactosidase. Vol. 21, No. 3 (1996)

duplicate determinations for each transfection. The fold induction over poLUC was calculated by first subtracting the background mean RLU of mock-transfected cells (Bluescript SK+) from the average RLU obtained with the luciferase constructs for both luciferase and β-gal. These values were then corrected for differences in transfection efficiency by normalizing luciferase values to a constant value for β-gal. Finally, the corrected luciferase RLU was divided by the mean of the RLU obtained from poLUC-transfected cells to determine the relative fold over poLUC. RESULTS With the Dual-Light assay, detection of luciferase and βgal in a cell extract sample is performed sequentially in a single reaction vessel (Figure 1). An aliquot of cell extract is added to the tube or microplate well, followed by the addition of Buffer A containing buffer salts and components necessary for the enhanced luciferase reaction. Injection of Buffer B containing luciferin and the β-gal substrate Galacton-Plus initiates an immediate luminescence signal from the luciferin/ luciferase reaction, which decays with a half-life of approximately one minute. Production of light signal from GalactonPlus present in the sample during luciferase signal measurement is negligible due to the low pH and absence of Sapphire-II polymeric enhancer (Tropix). Following measurement of the luciferase light signal, the reaction is further incubated for 30–60 min. A light-emission accelerator containing Sapphire-II at alkaline pH is then injected, and the light signal from the Galacton-Plus/β-gal reaction is measured. The light signal from the decomposition of GalactonPlus persists as a steady glow that decays with a half-life of approximately 180 min. At this point, the residual luciferase light signal is extremely low due to the fast decay of the luciferase signal and quenching by the accelerator formulation. The remaining signal from the luciferase reaction is generally nonexistent, except in samples containing in excess of 1 ng of luciferase (Figure 3). The Dual-Light assay was utilized for the quantitation of purified luciferase and β-gal. Detection of 10-15 to 10-8 g of luciferase and β-gal was achieved (Figure 2). Detection of 2 fg (0.032 amol) of luciferase and 8 fg (0.059 amol) of β-gal was achieved with a signal-to-noise ratio of 2. The level of interference produced by one enzyme at the low-level detection of the other enzyme was measured by assaying individual enzyme dilution series for β-gal and luciferase and plotting the results (Figure 3). The data indicate that, in the absence of luciferase, presence of β-gal in amounts up to 1 ng has no impact on the signal from the luciferase portion of the assay over background. When β-gal is not present, quantities of luciferase below 1 ng do not increase the background signal in the β-gal assay. At very high luciferase levels, 1 ng or above, residual luciferase signal is measurable; however, this level of signal interferes only in an extremely low-level detection of β-gal. Dynamic range over 5 orders of magnitude of enzyme concentration is maintained. The Dual-Light assay was used to quantitate enzyme activity in cell lysates, and the data were compared to the results obtained from assaying the same extracts individually for luciferase and β-gal (Table 1). When lysates from transiently transfected N20.1 cells were assayed for luciferase and β-gal using either the individual assays or the Dual-Light system, Vol. 21, No. 3 (1996)

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similar fold inductions of PLP-2400LUC over poLUC were obtained. As shown in Table 1, when reporter enzyme activities were assayed either independently or together, the fold induction of PLP-2400LUC over poLUC was 3.02 compared with 3.05, or 3.35 compared with 3.48, respectively. The RLU obtained with the two luciferase assays could not be compared directly, because different amounts of lysate and different measurement times were used in these assays. Likewise, RLUs obtained for the β-gal assays could not be compared directly because the lysates used for individual assays with the Galacto-Light Kit had been heated to 48°C for 45 min to reduce endogenous enzyme activity. DISCUSSION Detection of femtogram quantities of both β-gal and luciferase is achieved with the Dual-Light Reporter Gene Assay System. Both enzymes are quantitated in a single sample of cell extract using an enhanced bioluminescent luciferase reaction followed by chemiluminescence detection of β-gal with Galacton-Plus substrate and Sapphire-II enhancer. A dynamic range for the assay of over seven orders of magnitude of both enzyme concentrations is achieved. By combining the detection of both enzymes into one assay system, measurement of reporter gene expression from cotransfected cells is greatly simplified, and normalization of transfection efficiency is more precise since a single extract sample is used. As shown in Table 1, cell lysates assayed for luciferase and β-gal activi-

ties, either independently or together, generate identical results. The Dual-Light assay system for cells cotransfected with both luciferase and β-gal reporter genes is anticipated to have wide-spread use with many benefits, providing highly sensitive detection of both enzymes, decreased manual labor and increased accuracy in determining activities of both reporter enzymes. Development of additional dual and triple reporter gene assay systems is ongoing. ACKNOWLEDGMENTS We thank John Fortin, Corinne Olesen and John Voyta from Tropix for their assistance, and Tony Campagnoni for use of the N20.1 cells. These studies were supported in part by the National Multiple Sclerosis Society Grant RG2705A1 to P.A.W. REFERENCES 1.Alam, J. and J.L. Cook. 1990. Reporter genes: application to the study of mammalian gene transcription. Anal. Biochem. 188:245-254. 2.Brasier, A.R., J.E. Tate and J.F. Habener. 1989. Optimized use of the firefly luciferase assay as a reporter gene in mammalian cell lines. BioTechniques 7:1116-1122. 3.Bronstein, I., J.J.Fortin, P.E. Stanley, G.S.A.B. Stewart and L.J. Kricka. 1994. Chemiluminescent and bioluminescent reporter gene assays. Anal. Biochem. 219:169-181. 4.Bronstein, I., J.J. Fortin, J.C. Voyta, R.-R. Juo, B. Edwards, C.E.M. Olesen, N. Lijam and L.J. Kricka. 1994. Chemiluminescent reporter gene assays: sensitive detection of the GUS and SEAP gene products. BioTechniques 17:172-178. 5.Bronstein, I., J.J. Fortin, J.C. Voyta, C.E.M. Olesen and L.J. Kricka. 1994. Chemiluminescent reporter gene assays for β-galactosidase, β-glucuronidase and secreted alkaline phosphatase, p. 20-23. In A.K. Campbell, L.J. Kricka and P.E. Stanely (Eds.), Bioluminescence and Chemiluminescence: Fundamentals and Applied Aspects. John Wiley, Chichester, England. 6.Chalfie, M., Y. Tu, G. Euskirchen, W.W. Ward and D.C. Prasher. 1994. Green fluorescent protein as a marker for gene expression. Science 263:802-805. 7.Chang, A.C.Y. and D.G. Brenner. 1988. Cationic liposome-mediated transfection: a new method for the introduction of DNA into mammalian cells. Focus 10:66-68. 8.Chen, C.A. and H. Okayama. 1988. Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA. BioTechniques 6:632-638. 9.Cullen, B. and M. Malim. 1992. Secreted placental alkaline phosphatase as a eukaryotic reporter gene. Methods Enzymol. 216:362-368. 10.Gallagher, S.R. (Ed.) 1992. GUS Protocols: Using the GUS Gene as a Reporter of Gene Expression. Academic Press, San Diego. 11.Gorman, C.M., L.F. Moffat and B.H. Howard. 1982. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2:1044-1051. 12.Jain, V.K. and I.T. Magrath. 1991. A chemiluminescent assay for quantitation of β-galactosidase in the femtogram range: application to quantitation of β-galactosidase in lacZ-transfected cells. Anal. Biochem. 199:119-124. 13.Tanahashi, H., T. Ito, S. Inouye, F.I. Tsuji and Y. Sakaki. 1990. Photoprotein aequorin: use as a reporter enzyme in studying gene expression in mammalian cells. Gene 96:249-255. 14.Verity, A.N., D. Bredesen, C. Vonderscher, V.W. Handley and A.T. Campagnoni. 1993. Expression of myelin protein genes and other myelin components in an oligodendrocytic cell line conditionally immortalized with a temperature-sensitive retrovirus. J. Neurochem. 60:577-587. 15.Wight, P.A., C.S. Duchala, C. Readhead and W.B. Macklin. 1993. A myelin proteolipid protein-lacZ fusion protein is developmentally regulated and targeted to the myelin membrane in transgenic mice. J. Cell Biol. 123:443-454.

Address correspondence to Irena Bronstein, Tropix, Inc., 47 Wiggins Ave., Bedford, MA 01730-2314, USA. Vol. 21, No. 3 (1996)