386
REPORTERGENES
[34]
[34] L u c i f e r a s e R e p o r t e r G e n e A s s a y in M a m m a l i a n Cells
By ALLAN R. BRASIER and DAVID RON The firefly luciferase reporter gene has been widely applied in both transient and stable transfection protocols in eukaryotic cells to measure transcriptional regulation of DNA elements. 1-6 Firefly luciferase (EC 1.13.12.7, luciferin 4-monooxygenase) produces light by the ATP-dependent oxidation of luciferin. In the presence of excess substrate, light output from samples containing firefly luciferase becomes proportional to enzyme concentration in the assay, thus allowing for its quantitation. Characteristics of the luciferase reporter system that make it especially suitable for gene regulation studies include the speed and sensitivity of the assay, compatibility of measurement with other transfected reporter genes such as alkaline phosphatase,7 and the use of nonradioactive reagents. Furthermore, the rapid turnover of the luciferase mRNA and protein allows for interrupted cycloheximide incubations to quantitate transcriptional induction mediated by preformed transcriptional activators. 8'9 Accumulation of transfected reporter activity can be measured subsequent to a stimulus delivered in the presence of protein synthesis inhibition. Luciferase enzymatic activity correlates with changes in reporter mRNA abundance, thus obviating tedious RNA analysis. 6'8'1° We describe in this chapter conventional harvest and assay of transfected luciferase reporter activity, the use of cotransfected reporter gene alkaline phosphatase to monitor changes in plate-to-plate transfection efficiency and, finally, a modified "minilysate" protocol that allows for simultaneous measurement of both luciferase reporter activity and assay of nuclear DNA-binding activity within the same plate of transfected cells. I S. J. Gould and S. Subramani, Anal. Biochem. 175, 5 (1988). 2 A. R. Brasier, J. E. Tate, and J. F. Habener, BioTechniques 7, 1116 (1989). 3 j. Alam and J. L. Cook, Anal. Biochem. 188, 245 (1990). 4 T. M. Williams, J. E. Buerlein, S. Ogden, L. J. Kricka, and J. A. Kant, Anal. Biochem. 176, 28 (1989). 5 S. K. Nordeen, BioTechniques 6, 454 (1988). 6 D. Ron, A. R. Brasier, K. A. Wright, J. E. Tate, and J. F. Habener, Mol. Cell. Biol. 10, 1023 (1990). 7 p. Henthorn, P. Zervos, M. Raducha, H. Harris, and T. Kadesch, Proc. Natl. Acad. Sci. U.S.A. 85, 6342 (1988). 8 A. R. Brasier and D. Ron, Methods Neurosci. 5, 181 (1991). 9 M. Subramanian, L. J. Schmidt, C. E. Crutchfield, and M. J. Getz, Nature (London) 340, 64 (1989). 10 D. Ron, A. R. Brasier, K. A. Wright, and J. F. Habener, Mol. Cell. Biol. 10, 4389 (1990).
METHODS IN ENZYMOLOGY, VOL. 216
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
[34]
LUCIFERASE REPORTER GENE ASSAY
387
Luciferase Reporter Vectors deWet and colleagues reported the sequence of the luciferase gene from the firefly P h o t i n u s pyralis and constructed pSV2-based reporter vectors that directed immunologic and enzymatic luciferase expression in transfected CV-1 cells. ~1These workers identified two potential translation initiation sites and observed that deletion of the 5'-most initiation codon increased luciferase reporter activity by twofold and reduced luciferase activity in CV-I cells transfected with promoterless plasmids. Nordeen introduced bidirectional multiple cloning sites into the pSV232AL-AA5' plasmid containing the simian virus 40 (SV40) polyadenylation signal upstream of the multiple cloning site to lower background from cryptic promoters within the pBR322 plasmid. 5 We inserted the LAY cDNA into a modified pGEM3 vector, which has the advantage of being a high-copy plasmid that yields greater amounts ofplasmid DNA. 2Although this vector lacks the polyadenylation signal sequences upstream of the multiple cloning site, primer extension analysis of poly(A) ÷-selected RNA from transfected hepatoma cells has demonstrated correct transcript initiation. 6 We have also introduced an SV40 polyadenylation cassette into the pGEM3based pOLUC plasmid to generate the plasmid designated pOALUC. This was done to minimize any spurious effect of cryptic promoter activity arising from within the plasmid using an identical strategy as described by Maxwell et al. ~2 The map of this vector is given in Fig. IA, and the sequence of the multiple cloning site in Fig. lB. Unique restriction sites are indicated by an asterisk for use in cloning promoters to test transcriptional regulatory elements. Cellular Transfections with Luciferase Reporters Luciferase reporter plasmids are prepared by cesium chloride density ultracentrifugation and are introduced into cultured cells using conventional techniques, with the optimal method of transfection being determined empirically for individual cell lines. We currently transfect human hepatoma HepG2 cell lines with calcium phosphate coprecipitation. Routinely, precipitates are prepared in triplicate (for statistical purposes) containing 20 /zg of supercoiled plasmid DNA (total) consisting of I0 /zg of luciferase reporter, 3.5/~g pSV2APAP (alkaline phosphatase internal It j. R. de Wet, K. V. Wood, M. DeLuca, D. R. Helinski, and S. Subramani, 114o1.Cell. Biol. 7, 725 (1987). 12 I. H. Maxwell, G. S. Harrison, W. M. Wood, and F. Maxwell, BioTechniques 7, 276 (1989).
388
REPORTER GENES
[34]
A.
B* ~* H*
LUC cDNA(LA5') Sac1'~
pOALUC 5.5 KB
B.
Bam H1
Sma ~, ~
POLY A
/
Hind III ~'
+31 I
!
~GGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTGCATT~ LUC c D N A
FIG. 1. (A) Restriction map of pOALUC, a promoterless luciferase reporter vector. The
vector contains a trimerized SV40 polyadenylation cassette 12 ligated as a BamHI/BglII fragment into the BarnHI site of pOLUC. 2 The SV40 cassette upstream of the multiple cloning site reduces background from plasmid-initiated transcripts. 12The vector is 5.5 kbp in size. (B) Sequence of the multiple cloning site 5' to the luciferase LA5' cDNA from Photinus pyralis. Restriction endonuclease sites are indicated as B, BamHI; S, SmaI; H, HindIII; Sc, SacI. Nucleotide positions over the luciferase cDNA (LA5') refer to coordinates as described by deWet. H
control reporter7), and 6.5 p.g of carrier p G E M plasmid D N A . Thus, each 60-mm plate is transfected with a total of 6.7/~g of total D N A , of which 3.3/~g is luciferase reporter, 1.25/~g is p S V 2 A P A P alkaline p h o s p h a t a s e r e p o r t e r plasmid, and 2.2 /~g is carrier p G E M 3 plasmid. The triplicate precipitates are p r e p a r e d in 5-ml snap-cap Falcon (Becton Dickinson, Oxnard, CA) tubes and the D N A mix is brought to a final volume of 225 /A with 0.45-/.Lm m e m b r a n e filtered distilled water. Twenty-five microliters of 2.5 M CaCI z is added, followed by 250/~1 of 2 × H B S [ H E P E S - b u f f e r e d saline; 275 m M NaCI, 10 m M KCI, 1.4 m M sodium p h o s p h a t e m o n o b a s i c , 11 m M dextrose, 42 m M N - 2 - h y d r o x y t e h y l p i p e r a z i n e - N ' - 2 - e t h a n e s u l f o n i c acid ( H E P E S ) , p H 7.5]. T h e precipitate is allowed to stand at r o o m temper-
[34]
LUCIFERASE REPORTER GENE ASSAY
389
ature for 20 min after mixing and 167/zl of this precipitate is added dropwise to a monolayer of 1 × HBS-washed cells. The precipitate is allowed to incubate on the cell monolayer for 15 min, followed by the addition of 3 ml of culture medium. The glycerol shock after 4 hr yields the same transfection efficiency as that obtained by allowing the precipitates to stand on the plate overnight. Cos-I cells (used in the subsequent section) are transfected by the DEAE-dextran technique, 13 shocked in phosphatebuffered saline (PBS)/10% (v/v) dimethyl sulfoxide (DMSO) and harvested 48 hr after transfection. Cell Lysis Reporter enzymatic activity accumulates within transfected cells in a time-dependent fashion for 48-72 hr. The optimal time from transfection until harvest for luciferase reporter activity may vary among different cell types. HepG2 cells are harvested 48-60 hr after transfection, a time at which the luciferase expression directed by the viral SV40 early region promoter or angiotensinogen promoter is maximal.2 For cellular extraction of luciferase reporter activity, cells are washed in PBS and then lysed in situ by the addition of Triton lysis buffer [1% (v/v) Triton X-100, 25 mM glycylglycine, pH 7.8, 15 mM MgSO4, 4 mM ethylene glycolbis(flaminoethyl ether) -N, N, N',N'-tetraacetic acid (EGTA), 1 mM dithiothreitol (DTT, added immediately before use)]. The presence of reducing agent is critical to maintain luciferase enzymatic activity. The cells are scraped, transferred to 1.5-ml microfuge tubes, and spun at room temperature in a microcentrifuge at maximum speed (10,000 rpm) for 5 min. One hundred microliters of each supernatant is immediately assayed for luciferase activity. Luciferase Reporter Assay The ATP concentration is an important variable in the luciferase assay, as ATP is both a substrate and a regulator of luciferase enzymatic activity. 14ATP (1-2 raM) is optimal for assay of transfected luciferase activity in HepG2 cells. 2 We assay 100 tzl of each lysate by adding 360 tzl of luciferase assay buffer (25 mM glycylglycine, pH 7.8, 15 mM MgSO4, 4 mM EGTA, 15 mM potassium phosphate, pH 7.8, 2 mM ATP, 1 mM DTT; concentrated phosphate buffer and DTT are added immediately before 13 M. A. Lopata, D. W. Cleveland, and B. Sollner-Webb, Nucleic Acids Res. 12, 5707 (1984). |4 M. DeLuca and W. D. McElroy, Biochem. Biophys. Res. Commun. 123, 764 (1984).
390
REPORTER GENES
[34]
use). The assay is initiated by injection of 200 /~1 of 0.2 mM luciferin solution (0.2 mM synthetic crystalline luciferin in 25 mM glycylglycine, pH 7.8, 15 mM M g S O 4 , 4 mM EGTA, 2 mM DTT) diluted from concentrated aqueous 1 mM luciferin stock solution in the same buffer frozen at - 70° in the dark. Unused diluted luciferin is discarded. We quantitate luciferase activity using an automated luminometer [we use LKB (Rockville, MD) 1251 luminometer driven by an IBM-compatible PC with term emulator program and a dot-matrix printer]. A program written in BASIC programming language that we have designed is reproduced in Fig. 2. The data output is continuously printed each second, as well as peak and integrated luminescence values between 1 and 16 sec after luciferin injection into the sample. Thus, this program allows for collection of data without the additional cost of purchasing a chart recorder. With the addition of luciferin in the presence of ATP, magnesium ions, and oxygen, luciferase produces a peak flash of light followed by a plateau that decays to 80% of the initial peak values within 16 sec. 2,~3Both peak values and integrated light output values between 1 and 16 sec are proportional to luciferase enzyme concentrations. ~'2 We have found integrated values to have a lower intraassay variation than peak values and therefore prefer to use the former measurement in the evaluation of our data. The luciferase assay, in our hands, is linear over a wide range of proteins (up to 200/zg total cellular protein from detergent lysates), thus avoiding the need for sample dilutions. 2 A more complete discussion of interfering substances in the luciferase assay is given by Thore. ~5
Measurement of Cotransfected Reporter Activity Provided that the transfected cells do not express endogenous alkaline phosphatase enzyme, aliquots of cellular lysate from the above-mentioned Triton lysis-prepared extracts can be assayed for phosphatase activity exactly as described by Henthorn and Kadesch. 7 Assay blanks are reconstituted by addition of the same amount of Triton lysis buffer to the alkaline phosphatase (PAP) assay buffer. Test luciferase reporter activity can then be normalized for plate-to-plate variations in transfection efficiency. Chloramphenicol acetyltransferase ( C A T 16) has also been used as a cotransfected reporter to monitor transfection efficiency2; however, the presence of Triton in the cytoplasmic extract is inhibitory in the CAT assay and thus lowers its sensitivity. 15 A. Thore, Sci. Tools 26, 30 (1979). 16 C. M. Gorman, L. F. Moffat, and B. H. Howard, Mol. Cell. Biol. 2, 1044 (1982).
[34]
LUCIFERASE REPORTER GENE ASSAY
391
LUCIFERASE PROGRAM FOR PC-DRIVEN LUMINOMETER 10
MIXER PULSE
20
I DELAY 1
30
I TIME 15
31
C FACTOR 0.1
32
P FACTOR 0.1
33
I FACTOR 0.1
40
FIRST IN
50
LOOP 25
60
PRINT POSITION
70
DISPENSER 3.1
80
START I
90
START P
100
LOOP 16
110
PRINT C;
120
WAIT 1
130
END LOOP
140
WRITE
150
WRITE ' NO. INTEGRAL PEAK'
155
PRINT POSITION;
160
PRINT I;
170
PRINT P
180
P RESET
190
I RESET
191
WAIT5
200
NEXT SAMPLE
210
END LOOP IF NO SAMPLE
FIG. 2. BASIC program for operation of the LKB term emulator program. As entered, the program will direct the LKB luminometer to inject luciferin, record continuous, peak, and integrated luminescence between 1 and 16 sec after injection of the luciferin, and advance to the next sample.
392
REPORTERGENES
[34]
Assay for DNA-Binding Proteins and Luciferase Reporter Activity in Transfected Cells Changes in gene transcription can be correlated with alterations in the abundance or structure of nuclear DNA-binding proteins that interact with specific cis-regulatory sequences in the promoter region of the gene.17:8 These alterations can be detected by assaying in vitro for the sequencespecific DNA-binding activity of proteins isolated from nuclei of cells that have undergone a controlled physiological perturbation. It is often advantageous to be able to correlate, in the same cell, changes in reporter gene activity with the simultaneously occurring alterations in those DNAbinding proteins that are hypothesized to play a role in the regulatory event being studied. We have applied an existing protocol for the rapid preparation of nuclear extract from cultured cells 19to allow the simultaneous measurement of luciferase reporter gene activity in transfected cells. In the example presented in Fig. 3, this combined assay was used to evaluate DNA-binding activity and transcriptional activation potential of chimeric proteins generated by in-frame ligation of a truncated form of the activation domain of the cloned human transcription factor CREB and the DNA-binding domain of the yeast transcription factor Gal-4 [designated Gal(1-74); Ref. 20]. DNA (1-2/xg) prepared by alkaline lysis of Escherichia coli transformed with the ligation product CREB-GAL(1-74) in a replicating eukaryotic expression plasmid was used to transfect Cos-1 cells. The cells were cotransfected with 100 ng of reporter gene plasmid that contains the Gal-4 binding site (UAS) ligated upstream of a minimal promoter-luciferase reporter construct. Seventy-two hours following transfection, we simultaneously harvested cells for nuclear UAS-binding activity by electrophoretic gel mobility shift assay (EMSA), and cytosol for luciferase activity directed by the cotransfected reporter gene. The strategy is depicted in Fig. 3A. To harvest the transfected Cos-1 cells for simultaneous assay of DNAbinding activity and reporter activity, transfected 60-mm plates are washed twice with 3 ml of cold phosphate-buffered saline (PBS) and scraped in 0.5 ml of PBS/0.5 mM ethylenediaminetetraacetic acid (EDTA) on ice after a 5-min equilibration. The cells are scraped with a rubber policeman and then transferred to a microfuge tube and pelleted by spinning for 2 min at 4500 g at 4° in a tabletop microcentrifuge. The cell pellet is resuspended 17 A. R. Brasier, D. Ron, J. E, Tate, and J. F. Habener, EMBO J. 9, 3933 (1990). 18 C. Q. Lee, Y. Yun, J. P. Hoeffler, and J. F. Habener, EMBO J. 9, 4455 (1990). 19 E. Schreiber, P. Matthias, M. Muller, and W. Schaffner, Nucleic Acids Res. 17, 6419 (1989). 20 j. Ma and M. Ptashne, Cell (Cambridge, Mass.) 51, 113 (1987).
[34]
LUCIFERASE REPORTER GENE ASSAY
393
in 0.4 ml of buffer A [10 mM HEPES, pH 7.8, 10 mM KCI, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM DTT] and cells are allowed to swell on ice for 15 min. Lysis of the cells is effected by adding 20/zl of 10% (v/v) Nonidet P-40 (NP-40) in HzO, rapidly vortexing the cells for 10 sec, and immediately pelleting the nuclei by spinning for 30 sec at maximal speed (10,000 g, 4 °) in a tabletop microcentrifuge. The supernatant containing the cytosolic fraction with the luciferase enzymatic activity is transferred to a different microcentrifuge tube and stored on ice for further analysis. In a typical transfection experiment as little as 25-50 t~l of this lysate is sufficient to generate a reliable signal. To the nuclear pellet, 2 vol of buffer C [10 mM HEPES, pH 7.8,400 mM KC1, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM PMSF, 1 mM DTT, 0.1% (v/v) NP-40] is added, resuspended by vigorous vortexing for 15-30 sec, followed by 15 min of gentle agitation at 4 ° to allow for lysis of the nuclei and extraction of the DNA-binding proteins. The insoluble chromatin and membranes are removed by centrifugation for 5 rain in a microfuge at maximal speed and the supernatant is saved for analysis of DNA-binding activity. Typically for a confluent 60-mm plate of cells, we obtain 30-50 tzl of nuclear extract at a concentration of 3-10 tzg//zl that is suitable for EMSA, Western/Southern blot analysis, or immunoprecipitation. The autoradiogram of the EMSA and data from the luciferase assay are shown in Fig. 3B. Cells transfected with reporter gene only contain no nuclear proteins that bind the UAS (lane 1, Fig. 3B, consistent with the fact that mammalian cells do not have a Gal-4 homolog). Luciferase activity from the UAS-containing reporter gene is consistently low. Transfection of cells with a positive control Gal-4 chimera that contains the fulllength activating domain of CREB (lane 7, Fig. 3B) induces a novel UASbinding species and activates the reporter gene. The ligations used to transfect the Cos-1 cells represented in lanes 2 and 4 (Fig. 3B) were successful and gave rise to a chimeric UAS-binding protein that activates the reporter gene. Consistent with the smaller size of the encoded protein, the gel-shifted bands generated by the ligations being tested are of greater mobility than the positive control shown in lane 7 (Fig. 3B). The minipreparation DNA transfected and assayed in lanes 3, 5, and 6 (Fig. 3B) is from unsuccessful ligations and failed to encode a DNA-binding protein. Using this assay we have detected DNA-binding proteins from the b-ZIP class (CREB,C/EBP), zinc finger class (Gal-4), and Rel-like proteins (NFKB). Furthermore, comparison of luciferase reporter gene activity from transfected cells harvested with this combined technique with that from plates harvested by the plate lysis method demonstrates comparable levels of enzymatic activity. There are three major limitations of this technique. The first lies in the method of preparation of the nuclear extract,
394
REPORTER GENES
[34]
A
LIGATE CREB TRANSACTIVATION DOMAIN TO GAL 4 BINDING DOMAIN
TRANSFORM E. COLI AND GROW MINIPREP DNA OF CHIMERIC TRANSACTIVATOR
TRANSFECT COS-1 CELLS WITH: 1). INDIVIDUAL MINIPREP DNA 2). LUCIFERASE REPORTER DNA
HYPOTONIC/DETERGENT LYSE COS-1 CELLS
CYTOPLASMIC LYSATE
ASSAY FOR LUCIFERASE
NUCLEAR LYSATE
EXTRACT DNA BINDING PROTEINS
SPIN
SUPERNATANT
DISCARD CHROMATIN
ASSAY DNA BINDING
FIG. 3. (A) Cos-I cells were transfected by the DEAE-dextran technique with 1-2/~g of plasmid DNA encoding chimeric DNA-binding proteins that contain the Gal-4-binding domain and the CREB activation domain, along with 100 ng of a reporter plasmid that contains the Gal-4-binding site (UAS) upstream of an angiotensinogen minimal promoter-luciferase reporter plasmid (UASp59RLG). Seventy-two hours later, plates were processed for nuclear
[34]
,,
395
LUCIFERASE REPORTER GENE ASSAY
'N °atiwL ati°r' 'ein Tes~//~/ Bound complex ~ ---)
~Positive]
,~:,~;ii~ii%i!i!i4iliL~ii;iii!ili!il;i:ii:~:~;ii:~:
Free probe Lane : Luciferase activity :
2:1 1 140
49
9~
protein and luciferase enzymatic activity. (B) Autoradiogram after EMSA using 5-10 ~g of nuclear protein (contained in 2 t~l of nuclear extract) using as a probe the radioactively labeled oligonucleotide containing the UAS sequence. Complexes were resolved by 4.5% PAGE as described. 18 Luciferase activity was measured in 50 pJ of cytosolic extract and results are expressed in integrated light units after subtracting machine background. Lane 1, cells transfected with the reporter plasmid only; lanes 2-6, transfected with plasmids from the ligation product; lane 7, transfected with a positive control Gal-4-CREB chimera) 9
i.e., reliance on detergent lysis to disrupt the cellular membrane and leave the nuclei intact. The concentration of detergent used during the lysis step needs to be calibrated for the specific cell type and growth conditions. Second, the presence of detergent and the high salt concentration in the nuclear extract limits the amount of extract that can be used in a solutionphase binding assay to 10% of the reaction volume. Otherwise the final
396
REPORTERGENES
[34]
salt and detergent concentrations become inhibitory to DNA binding. Finally, a critical assumption in this combined luciferase reporter/DNAbinding assay is that one can measure the changes in DNA-binding proteins that occur in the transfected cell population. Transiently transfected cells, on which the analysis of reporter gene activity is being carried out, represent only a small minority of the total cell population of the plate (0.001-1% of cells contain transfected DNA3). Although reporter enzymes are not expressed in nontransfected cells, binding proteins that interact with DNA regulatory elements may be present in both transfected and nontransfected cells. Thus, the DNA-binding activity derived from an expression plasmid may be too faint to be distinguished from the background produced by nontransfected cells. The advantage of a system that relies on the SV40 large T antigen-expressing Cos-I cells to replicate eukaryotic expression vectors containing the SV40 origin of replication to high levels is that plasmid-encoded proteins may be detected. Thus, not all transiently transfectable cell lines can be used for this combined reporter/DNA-binding assay strategy.
Conclusions The firefly luciferase reporter gene is a highly versatile tool for studies of transcriptional regulation. The reporter assay is extremely sensitive, reproducible, and compatible with internal control alkaline phosphatase reporters. These features allow for measurements of low levels of transcription and correction for variations in transfection efficiency. Further, the use of nonradioactive reagents for detection is a significant advantage in an era where radioactive waste disposal is an issue. The rapid mRNA and protein turnover of luciferase in mammalian cells are properties that can be utilized to measure inducible inhancer activity by preformed DNA-binding proteins. The use of the previously described interrupted cycloheximide pulse-chase analysis 6,8 allows the investigator to measure reporter gene induction by stimulation with hormones in the presence of protein synthesis inhibition. This is followed by removal of hormone and protein synthesis inhibitor and subsequent measurement of reporter activity. Under these conditions, transcripts synthesized by preformed proteins correlate with luciferase enzymatic activity. This simplified strategy avoids the necessity of time-consuming mRNA purification and transcript analysis. We show here that the Triton lysis strategy can be easily adapted to assay both overexpressed (transfected) DNA-binding proteins or endogenous inducible DNAbinding activity. Thus, the cells from the same plate can be used to
[35]
FIREFLY LUCIFERASE REPORTER GENE
397
monitor reporter gene activity and DNA-binding activity. This allows a more precise correlation of the two activities. Acknowledgment We thank Joel F. Habener, in whose laboratory these experiments were conducted, for support.
[35] T r a n s i e n t E x p r e s s i o n A n a l y s i s in P l a n t s U s i n g F i r e f l y Luciferase Reporter Gene
By KENNETH R. LUEHRSEN, JEFFREY R. DE WET, and VIRGINIA WALBOT Introduction Reporter genes have been extensively used to study gene expression. The hallmark of an excellent reporter gene is the ease with which its product can be assayed. Widely used reporter genes typically include those encoding chloramphenicol acetyltransferase (CAT),/3-galactosidase (lacZ), and neomycin phosphotransferase (neo); however, each of these suffers from one or more disadvantages including high backgrounds, costly and tedious assay procedures, and low signal-to-noise ratio. A new generation of reporter genes has been developed, including those encoding Escherichia coli/3-glucuronidase (uidA gene) GUS activity 1 and bacterial (EC 1.14.14.3, alkanal monooxygenase) 2 and firefly luciferases (EC 1.13.12.7, luciferin 4-monooxygenase). 3'4 Here we describe the use of the firefly luciferase gene as a reporter in plant transient expression assays. The enzymatic properties of firefly luciferase have been well studied; the first luciferase gene cloned was from the North American firefly, Photinus pyralis. 3"4 The enzyme has a molecular mass of 62 kDa and requires only luciferin, ATP, Mg 2+ , and molecular oxygen for the production of yellow-green (560 nm) light. 5 The enzyme kinetics show a linear t R. A. Jefferson, Plant Mol. Biol. Rep. 5, 387 (1987). 2 E. A. Meighen, Microbiol. Rev. 55, 123 (1991). 3 j. R. de Wet, K. V. Wood, D. R. Helinski, and M. DeLuca, Proc. Natl. Acad. Sci. U.S.A. 82, 787 (1985). 4 j. R. de Wet, K. V. Wood, M. DeLuca, D. R. Helinski, and S. Subramani, Mol. Cell. Biol. 7, 725 (1987). 5 S. J. Gould and S. Subramani, Anal. Biochern. 175, 5 (1988).
METHODS IN ENZYMOLOGY,VOL. 216
Copyright © 1992by Academic Press, Inc. All rights of reproductionin any form reserved.
REPORTERGENES
[34]
[34] L u c i f e r a s e R e p o r t e r G e n e A s s a y in M a m m a l i a n Cells
By ALLAN R. BRASIER and DAVID RON The firefly luciferase reporter gene has been widely applied in both transient and stable transfection protocols in eukaryotic cells to measure transcriptional regulation of DNA elements. 1-6 Firefly luciferase (EC 1.13.12.7, luciferin 4-monooxygenase) produces light by the ATP-dependent oxidation of luciferin. In the presence of excess substrate, light output from samples containing firefly luciferase becomes proportional to enzyme concentration in the assay, thus allowing for its quantitation. Characteristics of the luciferase reporter system that make it especially suitable for gene regulation studies include the speed and sensitivity of the assay, compatibility of measurement with other transfected reporter genes such as alkaline phosphatase,7 and the use of nonradioactive reagents. Furthermore, the rapid turnover of the luciferase mRNA and protein allows for interrupted cycloheximide incubations to quantitate transcriptional induction mediated by preformed transcriptional activators. 8'9 Accumulation of transfected reporter activity can be measured subsequent to a stimulus delivered in the presence of protein synthesis inhibition. Luciferase enzymatic activity correlates with changes in reporter mRNA abundance, thus obviating tedious RNA analysis. 6'8'1° We describe in this chapter conventional harvest and assay of transfected luciferase reporter activity, the use of cotransfected reporter gene alkaline phosphatase to monitor changes in plate-to-plate transfection efficiency and, finally, a modified "minilysate" protocol that allows for simultaneous measurement of both luciferase reporter activity and assay of nuclear DNA-binding activity within the same plate of transfected cells. I S. J. Gould and S. Subramani, Anal. Biochem. 175, 5 (1988). 2 A. R. Brasier, J. E. Tate, and J. F. Habener, BioTechniques 7, 1116 (1989). 3 j. Alam and J. L. Cook, Anal. Biochem. 188, 245 (1990). 4 T. M. Williams, J. E. Buerlein, S. Ogden, L. J. Kricka, and J. A. Kant, Anal. Biochem. 176, 28 (1989). 5 S. K. Nordeen, BioTechniques 6, 454 (1988). 6 D. Ron, A. R. Brasier, K. A. Wright, J. E. Tate, and J. F. Habener, Mol. Cell. Biol. 10, 1023 (1990). 7 p. Henthorn, P. Zervos, M. Raducha, H. Harris, and T. Kadesch, Proc. Natl. Acad. Sci. U.S.A. 85, 6342 (1988). 8 A. R. Brasier and D. Ron, Methods Neurosci. 5, 181 (1991). 9 M. Subramanian, L. J. Schmidt, C. E. Crutchfield, and M. J. Getz, Nature (London) 340, 64 (1989). 10 D. Ron, A. R. Brasier, K. A. Wright, and J. F. Habener, Mol. Cell. Biol. 10, 4389 (1990).
METHODS IN ENZYMOLOGY, VOL. 216
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
[34]
LUCIFERASE REPORTER GENE ASSAY
387
Luciferase Reporter Vectors deWet and colleagues reported the sequence of the luciferase gene from the firefly P h o t i n u s pyralis and constructed pSV2-based reporter vectors that directed immunologic and enzymatic luciferase expression in transfected CV-1 cells. ~1These workers identified two potential translation initiation sites and observed that deletion of the 5'-most initiation codon increased luciferase reporter activity by twofold and reduced luciferase activity in CV-I cells transfected with promoterless plasmids. Nordeen introduced bidirectional multiple cloning sites into the pSV232AL-AA5' plasmid containing the simian virus 40 (SV40) polyadenylation signal upstream of the multiple cloning site to lower background from cryptic promoters within the pBR322 plasmid. 5 We inserted the LAY cDNA into a modified pGEM3 vector, which has the advantage of being a high-copy plasmid that yields greater amounts ofplasmid DNA. 2Although this vector lacks the polyadenylation signal sequences upstream of the multiple cloning site, primer extension analysis of poly(A) ÷-selected RNA from transfected hepatoma cells has demonstrated correct transcript initiation. 6 We have also introduced an SV40 polyadenylation cassette into the pGEM3based pOLUC plasmid to generate the plasmid designated pOALUC. This was done to minimize any spurious effect of cryptic promoter activity arising from within the plasmid using an identical strategy as described by Maxwell et al. ~2 The map of this vector is given in Fig. IA, and the sequence of the multiple cloning site in Fig. lB. Unique restriction sites are indicated by an asterisk for use in cloning promoters to test transcriptional regulatory elements. Cellular Transfections with Luciferase Reporters Luciferase reporter plasmids are prepared by cesium chloride density ultracentrifugation and are introduced into cultured cells using conventional techniques, with the optimal method of transfection being determined empirically for individual cell lines. We currently transfect human hepatoma HepG2 cell lines with calcium phosphate coprecipitation. Routinely, precipitates are prepared in triplicate (for statistical purposes) containing 20 /zg of supercoiled plasmid DNA (total) consisting of I0 /zg of luciferase reporter, 3.5/~g pSV2APAP (alkaline phosphatase internal It j. R. de Wet, K. V. Wood, M. DeLuca, D. R. Helinski, and S. Subramani, 114o1.Cell. Biol. 7, 725 (1987). 12 I. H. Maxwell, G. S. Harrison, W. M. Wood, and F. Maxwell, BioTechniques 7, 276 (1989).
388
REPORTER GENES
[34]
A.
B* ~* H*
LUC cDNA(LA5') Sac1'~
pOALUC 5.5 KB
B.
Bam H1
Sma ~, ~
POLY A
/
Hind III ~'
+31 I
!
~GGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTGCATT~ LUC c D N A
FIG. 1. (A) Restriction map of pOALUC, a promoterless luciferase reporter vector. The
vector contains a trimerized SV40 polyadenylation cassette 12 ligated as a BamHI/BglII fragment into the BarnHI site of pOLUC. 2 The SV40 cassette upstream of the multiple cloning site reduces background from plasmid-initiated transcripts. 12The vector is 5.5 kbp in size. (B) Sequence of the multiple cloning site 5' to the luciferase LA5' cDNA from Photinus pyralis. Restriction endonuclease sites are indicated as B, BamHI; S, SmaI; H, HindIII; Sc, SacI. Nucleotide positions over the luciferase cDNA (LA5') refer to coordinates as described by deWet. H
control reporter7), and 6.5 p.g of carrier p G E M plasmid D N A . Thus, each 60-mm plate is transfected with a total of 6.7/~g of total D N A , of which 3.3/~g is luciferase reporter, 1.25/~g is p S V 2 A P A P alkaline p h o s p h a t a s e r e p o r t e r plasmid, and 2.2 /~g is carrier p G E M 3 plasmid. The triplicate precipitates are p r e p a r e d in 5-ml snap-cap Falcon (Becton Dickinson, Oxnard, CA) tubes and the D N A mix is brought to a final volume of 225 /A with 0.45-/.Lm m e m b r a n e filtered distilled water. Twenty-five microliters of 2.5 M CaCI z is added, followed by 250/~1 of 2 × H B S [ H E P E S - b u f f e r e d saline; 275 m M NaCI, 10 m M KCI, 1.4 m M sodium p h o s p h a t e m o n o b a s i c , 11 m M dextrose, 42 m M N - 2 - h y d r o x y t e h y l p i p e r a z i n e - N ' - 2 - e t h a n e s u l f o n i c acid ( H E P E S ) , p H 7.5]. T h e precipitate is allowed to stand at r o o m temper-
[34]
LUCIFERASE REPORTER GENE ASSAY
389
ature for 20 min after mixing and 167/zl of this precipitate is added dropwise to a monolayer of 1 × HBS-washed cells. The precipitate is allowed to incubate on the cell monolayer for 15 min, followed by the addition of 3 ml of culture medium. The glycerol shock after 4 hr yields the same transfection efficiency as that obtained by allowing the precipitates to stand on the plate overnight. Cos-I cells (used in the subsequent section) are transfected by the DEAE-dextran technique, 13 shocked in phosphatebuffered saline (PBS)/10% (v/v) dimethyl sulfoxide (DMSO) and harvested 48 hr after transfection. Cell Lysis Reporter enzymatic activity accumulates within transfected cells in a time-dependent fashion for 48-72 hr. The optimal time from transfection until harvest for luciferase reporter activity may vary among different cell types. HepG2 cells are harvested 48-60 hr after transfection, a time at which the luciferase expression directed by the viral SV40 early region promoter or angiotensinogen promoter is maximal.2 For cellular extraction of luciferase reporter activity, cells are washed in PBS and then lysed in situ by the addition of Triton lysis buffer [1% (v/v) Triton X-100, 25 mM glycylglycine, pH 7.8, 15 mM MgSO4, 4 mM ethylene glycolbis(flaminoethyl ether) -N, N, N',N'-tetraacetic acid (EGTA), 1 mM dithiothreitol (DTT, added immediately before use)]. The presence of reducing agent is critical to maintain luciferase enzymatic activity. The cells are scraped, transferred to 1.5-ml microfuge tubes, and spun at room temperature in a microcentrifuge at maximum speed (10,000 rpm) for 5 min. One hundred microliters of each supernatant is immediately assayed for luciferase activity. Luciferase Reporter Assay The ATP concentration is an important variable in the luciferase assay, as ATP is both a substrate and a regulator of luciferase enzymatic activity. 14ATP (1-2 raM) is optimal for assay of transfected luciferase activity in HepG2 cells. 2 We assay 100 tzl of each lysate by adding 360 tzl of luciferase assay buffer (25 mM glycylglycine, pH 7.8, 15 mM MgSO4, 4 mM EGTA, 15 mM potassium phosphate, pH 7.8, 2 mM ATP, 1 mM DTT; concentrated phosphate buffer and DTT are added immediately before 13 M. A. Lopata, D. W. Cleveland, and B. Sollner-Webb, Nucleic Acids Res. 12, 5707 (1984). |4 M. DeLuca and W. D. McElroy, Biochem. Biophys. Res. Commun. 123, 764 (1984).
390
REPORTER GENES
[34]
use). The assay is initiated by injection of 200 /~1 of 0.2 mM luciferin solution (0.2 mM synthetic crystalline luciferin in 25 mM glycylglycine, pH 7.8, 15 mM M g S O 4 , 4 mM EGTA, 2 mM DTT) diluted from concentrated aqueous 1 mM luciferin stock solution in the same buffer frozen at - 70° in the dark. Unused diluted luciferin is discarded. We quantitate luciferase activity using an automated luminometer [we use LKB (Rockville, MD) 1251 luminometer driven by an IBM-compatible PC with term emulator program and a dot-matrix printer]. A program written in BASIC programming language that we have designed is reproduced in Fig. 2. The data output is continuously printed each second, as well as peak and integrated luminescence values between 1 and 16 sec after luciferin injection into the sample. Thus, this program allows for collection of data without the additional cost of purchasing a chart recorder. With the addition of luciferin in the presence of ATP, magnesium ions, and oxygen, luciferase produces a peak flash of light followed by a plateau that decays to 80% of the initial peak values within 16 sec. 2,~3Both peak values and integrated light output values between 1 and 16 sec are proportional to luciferase enzyme concentrations. ~'2 We have found integrated values to have a lower intraassay variation than peak values and therefore prefer to use the former measurement in the evaluation of our data. The luciferase assay, in our hands, is linear over a wide range of proteins (up to 200/zg total cellular protein from detergent lysates), thus avoiding the need for sample dilutions. 2 A more complete discussion of interfering substances in the luciferase assay is given by Thore. ~5
Measurement of Cotransfected Reporter Activity Provided that the transfected cells do not express endogenous alkaline phosphatase enzyme, aliquots of cellular lysate from the above-mentioned Triton lysis-prepared extracts can be assayed for phosphatase activity exactly as described by Henthorn and Kadesch. 7 Assay blanks are reconstituted by addition of the same amount of Triton lysis buffer to the alkaline phosphatase (PAP) assay buffer. Test luciferase reporter activity can then be normalized for plate-to-plate variations in transfection efficiency. Chloramphenicol acetyltransferase ( C A T 16) has also been used as a cotransfected reporter to monitor transfection efficiency2; however, the presence of Triton in the cytoplasmic extract is inhibitory in the CAT assay and thus lowers its sensitivity. 15 A. Thore, Sci. Tools 26, 30 (1979). 16 C. M. Gorman, L. F. Moffat, and B. H. Howard, Mol. Cell. Biol. 2, 1044 (1982).
[34]
LUCIFERASE REPORTER GENE ASSAY
391
LUCIFERASE PROGRAM FOR PC-DRIVEN LUMINOMETER 10
MIXER PULSE
20
I DELAY 1
30
I TIME 15
31
C FACTOR 0.1
32
P FACTOR 0.1
33
I FACTOR 0.1
40
FIRST IN
50
LOOP 25
60
PRINT POSITION
70
DISPENSER 3.1
80
START I
90
START P
100
LOOP 16
110
PRINT C;
120
WAIT 1
130
END LOOP
140
WRITE
150
WRITE ' NO. INTEGRAL PEAK'
155
PRINT POSITION;
160
PRINT I;
170
PRINT P
180
P RESET
190
I RESET
191
WAIT5
200
NEXT SAMPLE
210
END LOOP IF NO SAMPLE
FIG. 2. BASIC program for operation of the LKB term emulator program. As entered, the program will direct the LKB luminometer to inject luciferin, record continuous, peak, and integrated luminescence between 1 and 16 sec after injection of the luciferin, and advance to the next sample.
392
REPORTERGENES
[34]
Assay for DNA-Binding Proteins and Luciferase Reporter Activity in Transfected Cells Changes in gene transcription can be correlated with alterations in the abundance or structure of nuclear DNA-binding proteins that interact with specific cis-regulatory sequences in the promoter region of the gene.17:8 These alterations can be detected by assaying in vitro for the sequencespecific DNA-binding activity of proteins isolated from nuclei of cells that have undergone a controlled physiological perturbation. It is often advantageous to be able to correlate, in the same cell, changes in reporter gene activity with the simultaneously occurring alterations in those DNAbinding proteins that are hypothesized to play a role in the regulatory event being studied. We have applied an existing protocol for the rapid preparation of nuclear extract from cultured cells 19to allow the simultaneous measurement of luciferase reporter gene activity in transfected cells. In the example presented in Fig. 3, this combined assay was used to evaluate DNA-binding activity and transcriptional activation potential of chimeric proteins generated by in-frame ligation of a truncated form of the activation domain of the cloned human transcription factor CREB and the DNA-binding domain of the yeast transcription factor Gal-4 [designated Gal(1-74); Ref. 20]. DNA (1-2/xg) prepared by alkaline lysis of Escherichia coli transformed with the ligation product CREB-GAL(1-74) in a replicating eukaryotic expression plasmid was used to transfect Cos-1 cells. The cells were cotransfected with 100 ng of reporter gene plasmid that contains the Gal-4 binding site (UAS) ligated upstream of a minimal promoter-luciferase reporter construct. Seventy-two hours following transfection, we simultaneously harvested cells for nuclear UAS-binding activity by electrophoretic gel mobility shift assay (EMSA), and cytosol for luciferase activity directed by the cotransfected reporter gene. The strategy is depicted in Fig. 3A. To harvest the transfected Cos-1 cells for simultaneous assay of DNAbinding activity and reporter activity, transfected 60-mm plates are washed twice with 3 ml of cold phosphate-buffered saline (PBS) and scraped in 0.5 ml of PBS/0.5 mM ethylenediaminetetraacetic acid (EDTA) on ice after a 5-min equilibration. The cells are scraped with a rubber policeman and then transferred to a microfuge tube and pelleted by spinning for 2 min at 4500 g at 4° in a tabletop microcentrifuge. The cell pellet is resuspended 17 A. R. Brasier, D. Ron, J. E, Tate, and J. F. Habener, EMBO J. 9, 3933 (1990). 18 C. Q. Lee, Y. Yun, J. P. Hoeffler, and J. F. Habener, EMBO J. 9, 4455 (1990). 19 E. Schreiber, P. Matthias, M. Muller, and W. Schaffner, Nucleic Acids Res. 17, 6419 (1989). 20 j. Ma and M. Ptashne, Cell (Cambridge, Mass.) 51, 113 (1987).
[34]
LUCIFERASE REPORTER GENE ASSAY
393
in 0.4 ml of buffer A [10 mM HEPES, pH 7.8, 10 mM KCI, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM DTT] and cells are allowed to swell on ice for 15 min. Lysis of the cells is effected by adding 20/zl of 10% (v/v) Nonidet P-40 (NP-40) in HzO, rapidly vortexing the cells for 10 sec, and immediately pelleting the nuclei by spinning for 30 sec at maximal speed (10,000 g, 4 °) in a tabletop microcentrifuge. The supernatant containing the cytosolic fraction with the luciferase enzymatic activity is transferred to a different microcentrifuge tube and stored on ice for further analysis. In a typical transfection experiment as little as 25-50 t~l of this lysate is sufficient to generate a reliable signal. To the nuclear pellet, 2 vol of buffer C [10 mM HEPES, pH 7.8,400 mM KC1, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM PMSF, 1 mM DTT, 0.1% (v/v) NP-40] is added, resuspended by vigorous vortexing for 15-30 sec, followed by 15 min of gentle agitation at 4 ° to allow for lysis of the nuclei and extraction of the DNA-binding proteins. The insoluble chromatin and membranes are removed by centrifugation for 5 rain in a microfuge at maximal speed and the supernatant is saved for analysis of DNA-binding activity. Typically for a confluent 60-mm plate of cells, we obtain 30-50 tzl of nuclear extract at a concentration of 3-10 tzg//zl that is suitable for EMSA, Western/Southern blot analysis, or immunoprecipitation. The autoradiogram of the EMSA and data from the luciferase assay are shown in Fig. 3B. Cells transfected with reporter gene only contain no nuclear proteins that bind the UAS (lane 1, Fig. 3B, consistent with the fact that mammalian cells do not have a Gal-4 homolog). Luciferase activity from the UAS-containing reporter gene is consistently low. Transfection of cells with a positive control Gal-4 chimera that contains the fulllength activating domain of CREB (lane 7, Fig. 3B) induces a novel UASbinding species and activates the reporter gene. The ligations used to transfect the Cos-1 cells represented in lanes 2 and 4 (Fig. 3B) were successful and gave rise to a chimeric UAS-binding protein that activates the reporter gene. Consistent with the smaller size of the encoded protein, the gel-shifted bands generated by the ligations being tested are of greater mobility than the positive control shown in lane 7 (Fig. 3B). The minipreparation DNA transfected and assayed in lanes 3, 5, and 6 (Fig. 3B) is from unsuccessful ligations and failed to encode a DNA-binding protein. Using this assay we have detected DNA-binding proteins from the b-ZIP class (CREB,C/EBP), zinc finger class (Gal-4), and Rel-like proteins (NFKB). Furthermore, comparison of luciferase reporter gene activity from transfected cells harvested with this combined technique with that from plates harvested by the plate lysis method demonstrates comparable levels of enzymatic activity. There are three major limitations of this technique. The first lies in the method of preparation of the nuclear extract,
394
REPORTER GENES
[34]
A
LIGATE CREB TRANSACTIVATION DOMAIN TO GAL 4 BINDING DOMAIN
TRANSFORM E. COLI AND GROW MINIPREP DNA OF CHIMERIC TRANSACTIVATOR
TRANSFECT COS-1 CELLS WITH: 1). INDIVIDUAL MINIPREP DNA 2). LUCIFERASE REPORTER DNA
HYPOTONIC/DETERGENT LYSE COS-1 CELLS
CYTOPLASMIC LYSATE
ASSAY FOR LUCIFERASE
NUCLEAR LYSATE
EXTRACT DNA BINDING PROTEINS
SPIN
SUPERNATANT
DISCARD CHROMATIN
ASSAY DNA BINDING
FIG. 3. (A) Cos-I cells were transfected by the DEAE-dextran technique with 1-2/~g of plasmid DNA encoding chimeric DNA-binding proteins that contain the Gal-4-binding domain and the CREB activation domain, along with 100 ng of a reporter plasmid that contains the Gal-4-binding site (UAS) upstream of an angiotensinogen minimal promoter-luciferase reporter plasmid (UASp59RLG). Seventy-two hours later, plates were processed for nuclear
[34]
,,
395
LUCIFERASE REPORTER GENE ASSAY
'N °atiwL ati°r' 'ein Tes~//~/ Bound complex ~ ---)
~Positive]
,~:,~;ii~ii%i!i!i4iliL~ii;iii!ili!il;i:ii:~:~;ii:~:
Free probe Lane : Luciferase activity :
2:1 1 140
49
9~
protein and luciferase enzymatic activity. (B) Autoradiogram after EMSA using 5-10 ~g of nuclear protein (contained in 2 t~l of nuclear extract) using as a probe the radioactively labeled oligonucleotide containing the UAS sequence. Complexes were resolved by 4.5% PAGE as described. 18 Luciferase activity was measured in 50 pJ of cytosolic extract and results are expressed in integrated light units after subtracting machine background. Lane 1, cells transfected with the reporter plasmid only; lanes 2-6, transfected with plasmids from the ligation product; lane 7, transfected with a positive control Gal-4-CREB chimera) 9
i.e., reliance on detergent lysis to disrupt the cellular membrane and leave the nuclei intact. The concentration of detergent used during the lysis step needs to be calibrated for the specific cell type and growth conditions. Second, the presence of detergent and the high salt concentration in the nuclear extract limits the amount of extract that can be used in a solutionphase binding assay to 10% of the reaction volume. Otherwise the final
396
REPORTERGENES
[34]
salt and detergent concentrations become inhibitory to DNA binding. Finally, a critical assumption in this combined luciferase reporter/DNAbinding assay is that one can measure the changes in DNA-binding proteins that occur in the transfected cell population. Transiently transfected cells, on which the analysis of reporter gene activity is being carried out, represent only a small minority of the total cell population of the plate (0.001-1% of cells contain transfected DNA3). Although reporter enzymes are not expressed in nontransfected cells, binding proteins that interact with DNA regulatory elements may be present in both transfected and nontransfected cells. Thus, the DNA-binding activity derived from an expression plasmid may be too faint to be distinguished from the background produced by nontransfected cells. The advantage of a system that relies on the SV40 large T antigen-expressing Cos-I cells to replicate eukaryotic expression vectors containing the SV40 origin of replication to high levels is that plasmid-encoded proteins may be detected. Thus, not all transiently transfectable cell lines can be used for this combined reporter/DNA-binding assay strategy.
Conclusions The firefly luciferase reporter gene is a highly versatile tool for studies of transcriptional regulation. The reporter assay is extremely sensitive, reproducible, and compatible with internal control alkaline phosphatase reporters. These features allow for measurements of low levels of transcription and correction for variations in transfection efficiency. Further, the use of nonradioactive reagents for detection is a significant advantage in an era where radioactive waste disposal is an issue. The rapid mRNA and protein turnover of luciferase in mammalian cells are properties that can be utilized to measure inducible inhancer activity by preformed DNA-binding proteins. The use of the previously described interrupted cycloheximide pulse-chase analysis 6,8 allows the investigator to measure reporter gene induction by stimulation with hormones in the presence of protein synthesis inhibition. This is followed by removal of hormone and protein synthesis inhibitor and subsequent measurement of reporter activity. Under these conditions, transcripts synthesized by preformed proteins correlate with luciferase enzymatic activity. This simplified strategy avoids the necessity of time-consuming mRNA purification and transcript analysis. We show here that the Triton lysis strategy can be easily adapted to assay both overexpressed (transfected) DNA-binding proteins or endogenous inducible DNAbinding activity. Thus, the cells from the same plate can be used to
[35]
FIREFLY LUCIFERASE REPORTER GENE
397
monitor reporter gene activity and DNA-binding activity. This allows a more precise correlation of the two activities. Acknowledgment We thank Joel F. Habener, in whose laboratory these experiments were conducted, for support.
[35] T r a n s i e n t E x p r e s s i o n A n a l y s i s in P l a n t s U s i n g F i r e f l y Luciferase Reporter Gene
By KENNETH R. LUEHRSEN, JEFFREY R. DE WET, and VIRGINIA WALBOT Introduction Reporter genes have been extensively used to study gene expression. The hallmark of an excellent reporter gene is the ease with which its product can be assayed. Widely used reporter genes typically include those encoding chloramphenicol acetyltransferase (CAT),/3-galactosidase (lacZ), and neomycin phosphotransferase (neo); however, each of these suffers from one or more disadvantages including high backgrounds, costly and tedious assay procedures, and low signal-to-noise ratio. A new generation of reporter genes has been developed, including those encoding Escherichia coli/3-glucuronidase (uidA gene) GUS activity 1 and bacterial (EC 1.14.14.3, alkanal monooxygenase) 2 and firefly luciferases (EC 1.13.12.7, luciferin 4-monooxygenase). 3'4 Here we describe the use of the firefly luciferase gene as a reporter in plant transient expression assays. The enzymatic properties of firefly luciferase have been well studied; the first luciferase gene cloned was from the North American firefly, Photinus pyralis. 3"4 The enzyme has a molecular mass of 62 kDa and requires only luciferin, ATP, Mg 2+ , and molecular oxygen for the production of yellow-green (560 nm) light. 5 The enzyme kinetics show a linear t R. A. Jefferson, Plant Mol. Biol. Rep. 5, 387 (1987). 2 E. A. Meighen, Microbiol. Rev. 55, 123 (1991). 3 j. R. de Wet, K. V. Wood, D. R. Helinski, and M. DeLuca, Proc. Natl. Acad. Sci. U.S.A. 82, 787 (1985). 4 j. R. de Wet, K. V. Wood, M. DeLuca, D. R. Helinski, and S. Subramani, Mol. Cell. Biol. 7, 725 (1987). 5 S. J. Gould and S. Subramani, Anal. Biochern. 175, 5 (1988).
METHODS IN ENZYMOLOGY,VOL. 216
Copyright © 1992by Academic Press, Inc. All rights of reproductionin any form reserved.
