[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.

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REPORTER GENES

[35]

response over eight decades of dilution. Although firefly luciferase is normally transported to and expressed in peroxisomes, 6 this localization is not an absolute requirement as the enzyme can be assayed in vivo in bacteria and in cell-free extracts. There are several advantages of luciferase over the commonly used CAT: detection is 10-1000 times more sensitive 4'7 ( a s little a s l 0 -2° mol can be detected), the assays are much cheaper and quicker, assays do not require hazardous radioactive chemicals, and there is no inherent background light production in cells. A disadvantage of using firefly luciferase is the cost o f a luminometer, although some assay methods permit the use of a standard scintillation counter to monitor activity. Firefly luciferase has been widely used in many organisms to monitor gene expression. The luciferase cDNA has been successfully expressed in bacteria, 8 yeast,9 Dictyostelium, lOmonocot and dicot plants, H and mammalian tissue culture c e l l s . 4 Most applications are based on the assay of luciferase enzyme in cell extracts. It is also possible to analyze luciferase expression in vivo because the substrate luciferin will diffuse across biological membranes. This approach has allowed identification of E. coli cultures expressing luciferase8 and direct video imaging of tobacco protoplasts. 12 Transgenic tobacco plants have been constructed in which luciferase expression has been localized and quantitated in individual organs and tissues. 11,13,14Thus, the firefly luciferase gene is a sensitive and versatile reporter gene. Plasmids Incorporating Firefly Luciferase Reporter Gene To investigate the effects of changing various components of a transcriptional unit on the level of transient gene expression in plant protoplasts, we designed and constructed modular expression vectors. These vectors have at least one unique restriction site between each functional component of the transcriptional unit so that individual segments can 6 S. J. Gould, G. A. Keller, M. Schneider, S. H. Howell, L. J. Garrard, J. M. Goodman, B. Distel, H. Tabak, and S. Subramani, EMBO J. 9, 85 (1990). 7 T. M. Williams, J. E. Burlein, S. Ogden, L. J. Kricka, and J. A. Kant, Anal. Biochem. 176, 28 (1989). 8 K. V. Wood and M. DeLuca, Anal. Biochem. 161, 501 (1987). 9 H. Tatsumi, T. Masuda, and E. Nakano, Agric. Biol. Chem. 52, 1123 (1988). to p. K. Howard, K. G. Ahern, and R. A. Firtel, Nucleic Acids Res. 16, 2613 (1988). 11 D. W. Ow, K. V. Wood, M. DeLuca, J. R. de Wet, D. R. Helinski, and S. H. Howell, Science 234, 856 (1986). iz D. R. Gallie, W. J. Lucas, and V. Walbot, Plant Cell 1, 301 (1989). t3 W. M. Barnes, Proc. Natl. Acad. Sci. U.S.A. 87, 9183 (1990). 14 M. Schneider, D. W. Ow, and S. H. Howell, Plant Mol. Biol. 14, 935 (1990).

[35]

FIREFLY LUCIFERASE REPORTER GENE

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be easily exchanged. To facilitate the exchange of reporter genes, we introduced an NcoI restriction site at the initiation codon of the firefly luciferase gene using oligonucleotide directed mutagenesis. The sequence at the initiation codon was changed from 5'-GGTAAAATGG-3' to 5'GGTACCATGG-3'. This maintained the consensus sequence as defined by Kozak for a eukaryotic translational start site 15'16 and introduced an NcoI site (5'-CCATGG-3') and an overlapping KpnI site (5'-GGTACC3'). The altered sequence is present in the luciferase cassettes carried in pJD250 and pJD251 (Fig. 1) and does not affect expression levels in maize protoplasts (J. R. de Wet, unpublished observations, 1990). The luciferase cassettes in pJD261, pJD293, and pJD294 (Fig. 1) carry an alternative sequence upstream of the luciferase initiation codon. This alternative sequence (5'-GTCGACCATGG-3') was obtained from the /3-glucuronidase vector pRAJ2751 and consists of a SalI site overlapping the NcoI site; this sequence maintains a consensus translational start site. The firefly luciferase cassette in pJD293 also features a different arrangement of restriction sites at its 3' end. In constructing our plant expression vectors we chose to use the cauliflower mosaic virus (CaMV) 35S promoter; this is a strong promoter in both dicots and monocots. 17-19The CaMV 35S promoter fragment we used extends from - 363 to + 1 relative to the transcriptional start site2°; this is a region responsible for the activity of the promoter. 21A restriction fragment carrying the polyadenylation signal of the nopaline synthase (nos) gene from the Ti plasmid ofAgrobacterium tumefaciens 18,22forms the 3' end of the transcriptional unit. Restriction maps of the basic CaMV 35S promoter-nos 3' vectors, pJD288 and pJD290, are shown in Fig. 2. The CaMV 35S promoter vector pJD290 was constructed as follows: the restriction fragment from the DdeI site at base 7069 to the HphI site at base 7444 from CaMV strain 184123 was isolated and treated with T4 DNA polymerase in the presence of all four deoxyribonucleoside triphosphates (dNTPs). The resultant blunt-ended fragment was 426 bases in 15 M. Kozak, Cell (Cambridge, Mass.) 44, 283 (1986). 16 M. Kozak, J. Cell Biol. 108, 229 (1989). 17j. Callis, M. Fromm, and V. Walbot, Genes Dev. 1, 1183 (1987). 18 M. Fromm, L. P. Taylor, and V. Walbot, Nature (London) 319, 791 (1986). 19 j. T. Odell, S. Knowlton, W. Lin, and C. J. Mauvais, Plant Mol. Biol. 10, 263 (1988). ~0 H. Guilley, R. Dudley, K. G. Jonard, E. Balazs, and K. E. Richards, Cell (Cambridge, Mass.) 30, 763 (1982). 2I R.-X. Fang, F. Nagy, S. Sivasubramanian, and N.-H. Chua, Plant Cell 1, 141 (1989). 22 H.-J. Fritz, in " D N A Cloning: A Practical Approach" (D. M. Glover, ed.), p. 151. IRL Press, Oxford, 2985. 23 R. C. Gardner, A. J. Howarth, P. Hahn, M. Brown-Luedi, R. J. Shepherd, and J. Messing, Nucleic Acids Res. 9, 2871 (1982).

400

REPORTER GENES

Kp

pJD250 (pUC118)

H3~I

II [] Sp

Kp I-I3[]Bg~

pJD251 (pUC118)

I

II RI Sp

Sa

pJD261 (pSP64) luciferase

II

~ RISsKpSmBaBgSsRI

II

NcXb

[] Sp

Ps.~l Sa

pJD293 (pUC18) luciferase

XbSm a

II

Nc Xb Sa

RI Kp Ba Kp RI Ss Sm Sm Ss

luciferase

Nc Xb

H3

~.RI SsKpSmBaSmKpSs[]

lueifemse

Nc Xb

H3

[35]

Ss

Kp[]

RI Sp pJD294 (pUC13)

H3 KP.~l

luciferase

~RIss[]

II Ne Xb

RI Sp

I

I I I base pairs

I

I I 500

I

I

I

I 1000

FIG. 1. Restriction maps of the firefly luciferase cassettes. Only the firefly luciferase gene and flanking restriction sites are shown. The cloning vector carrying the diagrammed fragments is in parentheses following the name of each of the luciferase plasmids. The NcoI restriction site that was introduced into the luciferase gene by mutagenesis occurs at - 2 relative to the translational start site. The shaded box in pJD261 represents the tobacco mosaic virus (TMV) f~ sequence, pSP64 is a product of Promega Corporation. The restriction sites are abbreviated as follows: BamHI, Ba; Bglll, Bg; EcoRI, RI; HindlII, H3; KpnI, Kp; NcoI, Nc; PstI, Ps; SalI, Sa; SstI, Ss; Srnal, Sm; Xbal, Xb.

length and extended from - 363 to + 3 relative to the transcriptional start site 2° of the 35S promoter. This fragment was inserted into the S m a I site of pUC18 z4 to produce pJD264. The plasmid pJD264 was digested with E c o R I and the 5' overhangs were filled in. A 260-bp P s t I to X b a I restriction fragment containing the poly(A) addition region of the nos gene 25 was isolated from the plasmid p C a M V n e o 18and made blunt ended by treatment 24 C. Yanisch-Perron, J. Vieira, and J. Messing, Gene 33, 103 (1985). 25 R. T. Fraley, S. G. Rogers, R. B. Horsch, P. R. Sanders, J. S. Flick, S. P. Adams, M. L. Bittner, L. A. Brand, C. L. Fink, J. S. Fry, G. R. Galluppi, S. B. Goldberg, N. J. Hoffman, and S. C. Woo, Proc. Natl. Acad. Sci. U.S.A. 80, 4803 (1983).

[35]

FIREFLY LUCIFERASE REPORTER GENE

pJD288 (pUC18) la RI Kp ~ B g Ss XbI Ps Ba Sa Sp

pJD290 (pUC18) Xb

H3 Ps Ba ~t,vt/'2~ .~'a, Bg RI Sp Xb " 1 ~ ' " " JJ~ [] " ~ J r- Xb

I

Kp Ss

Kp/ Sa

pJD300 (pUC18)

H3 Ps Ba.j CaMV 35S dl Sp Xb /

luciferase

'r I

II

Nc Xb

RI Sp

Sa

/

401

RI Ss

pJD301 (pUC18)

H3 Ps Ba--..,i 35S I ~ Sp Xb / CaMV

-I I

hciferase

~ nos3'

II

Nc Xb

!

RI Sp

Kp.Sa B~ si H3 Ps Ba_j Sp Xb "'1 CaMV 35S AdhlIntron I

RI Ss pJD312 (pUC18) luciferase

I

II

I I

H3

Nc Xb

RI Sp

LBg RI ~ n°s3' I--" Xb /

RI Ss

pJD353 (pUC18) H3 Ps Ba Sp Xb

~ ~I'B

luciferase ---11 Nc Xb

g xRbI

I I RI Sp

I i i ) i I ) i i) base pairs

500

RI Xb

RI Ss

I 1000

FIG. 2. Restriction maps of the CaMV 35S promoter luciferase expression vectors. The name of each expression vector is followed in parentheses by the name o f the plasmid vector carrying the diagrammed transcription units. The shaded box in pJD301 represents the TMV f~ sequence. The black box in pJD353 is the Adhl untranslated leader region. The restriction sites are abbreviated as follows: BamHI, Ba; BgllI, Bg; EcoRI, RI; HindlII, H3; KpnI, Kp;

NcoI, Nc; PstI, Ps; SalI, Sa; SspI, Sp; SstI, Ss; Sinai, Sm; XbaI, Xb.

with T4 D N A polymerase in the presence of all four dNTPs. This fragment was then inserted into the blunt-ended EcoRI site of pJD264 to produce pJD287. The SalI site in the pUC18 polylinker region of pJD287 was eliminated by digesting the plasmid with SalI, removing the overhangs by digestion with mung bean nuclease, and recircularizing the plasmid with T4 D N A ligase to produce pJD290. The plasmid pJD288 was constructed using the same CaMV 35S promoter and nos poly(A)-containing restriction fragments as were used in the construction of pJD290. The CaMV 35S promoter fragment was inserted into the HinclI site of pUC18 to produce pJD265. The nos 3'

402

REPORTER GENES

[35]

fragment was then inserted into the HindIII site of pJD265, after the HindIII staggered ends had been filled in to produce pJD288. An NcoI site was introduced into the luciferase gene at the transcriptional start site using the gapped duplex method of oligonucleotide mutagenesis. 22 Briefly, the HindIII to BarnHI restriction fragment containing the luciferase gene was isolated from pJD2044 and inserted into pUC11826 that had been digested with HindIII and BamHI to produce pJD243. The single-stranded DNA form of pJD243 was propagated in the E. coli strain BW313 (dut ung thi-1 relA spoT1 F' I s y A ) . 27 The uracil-substituted singlestranded template was hybridized to pJD243 that had been digested with HindIII and XbaI to form the gapped duplex and to the phosphorylated oligonucleotide 5'-pCCGGTACTGTTGGTACCATGGAAGACGCCA-3'. The two underlined C's were A's in the original luciferase sequence. The circles were repaired with T4 DNA polymerase and T4 DNA ligase and transformed into a dut + ung + strain of E. coli. The mutagenesis should introduce a KpnI site overlapping a following NcoI site. A successfully mutagenized plasmid clone was identified by digestion with KpnI and NcoI and named pJD250, pJD251 was constructed in the same manner as pJD250 except that the H i n d I I I - B a m H I luciferase restriction fragment was obtained from pJD205. 4 The tobacco mosaic virus (TMV) 1) leader, shown previously to enhance expression in a number of plant species,28 was added to the luciferase gene as follows. The EcoRI site at the 3' end of the fl-glucuronidase gene in pRAJ275 ~was cut and filled. The plasmid was then digested with HindIII and the fl-glucuronidase-containing restriction fragment was inserted into pUC18 that had been digested with HindIII and HincII. The resultant plasmid was digested with SalI and BamHI, and the fl-glucuronidase restriction fragment was inserted into the TMV O-containing plasmid pJII10129 that had been digested with SalI and BamHI. This resulted in the introduction of an NcoI site immediately downstream from the SalI site at the 3' end of the 1) sequence in pJII101. The/3-glucuronidase gene was then removed by digestion with NcoI and BamHI and replaced by an N c o I - B a m H I fragment obtained from pJD250 to produce the fl-luciferase plasmid pJD261. The S a l I - B a m H I luciferase gene fragment from pJD261 was inserted into pUC133° that had been digested with SalI and BamHI to 26 j. Vieira and J. Messing, this series, Vol. 153, p. 3. 27 T. A. Kunkel, Proc. Natl. Acad. Sci. U.S.A. 82, 488 (1985). 28 D. R. Gallie, D. E. Sleat, J. W. Watts, P. C. Turner, and T. M. A. Wilson, Nucleic Acids Res. 15, 3257 (1987). 29 D. R. Gallie, D. E. Sleat, J. W. Watts, P. C. Turner, and T. M. A. Wilson, Nucleic Acids Res. 15, 8693 (1987). 3o j. Messing, this series, Vol. 101, p. 20.

[35]

FIREFLY LUCIFERASE REPORTER GENE

403

produce pJD291. A KpnI site was inserted 5' of the luciferase gene by digesting pJD291 with PstI, using T4 DNA polymerase in the presence of all four dNTPs to remove the 3' overhangs, ligating KpnI linkers (pGGGTACCC) to the blunt ends of the plasmid, digesting the DNA with KpnI, and recircularizing the plasmid. The resultant plasmid was named pJD294. The CaMV 35S luciferase expression vector pJD300 was constructed by inserting the KpnI-SstI luciferase gene fragment from pJD294 into pJD290 that had been digested with KpnI and SstI. The CaMV35S-f~-luciferase expression vectors were constructed as follows: pJD290 was digested with KpnI, the 3' overhangs were removed, and the DNA was then digested with SstI. The plasmid pJD261 was digested with HindlII, the 5' overhangs were removed by digestion with mung bean nuclease, and the f~-luciferase fragment was released from the vector by digestion with SstI. This fragment was inserted into the prepared pJD290 DNA producing the vector pJD301. The plasmid pMANC1, 3~ which has an NcoI site introduced at the translational start site, was used as a source of the untranslated leader from the Zea mays alcohol dehydrogenase-1 (Adhl) gene. A KpnI linker (pGGGTACCC) was inserted into the BanlI site base 29 bp upstream of the Adhl transcriptional start site after the BanlI 3' overhangs had been removed. The Adhl untranslated leader was then inserted into pJD300 as a KpnI to NcoI restriction fragment to produce pJD353, pJD353 is expressed approximately fourfold higher in maize than pJD300. 32 The presence of Adhl intron 1 in a transcription unit has been shown to increase reporter gene expression in m a i z e 17'33 and other m o n o c o t s . 34'35 The luciferase expression vector containing the first intron of the maize Adhl gene, pJD312, was constructed as follows: the 557-bp BclI-BamHI Adhl intron 1-containing restriction fragment ~7 was inserted into the BamHI site of pUC9 such that the BclI site of the intron 1 fragment was proximal to the SalI site of pUC9. The resultant plasmid was subsequently digested with BamHI, the 5' overhangs were filled in, and phosphorylated SalI linkers were ligated to the blunt ends. The plasmid DNA/SalI linker ligation mix was then digested with SalI, and the Adhl intron 1 fragment was isolated and inserted into the SalI site of pJD300 to produce pJD312. Other luciferase cassette vectors useful for creating translational fusions have also been reported. 36 In addition to the DNA-based vectors 31 L. Lee, C. Fenoll, and J. L. Bennetzen, Plant Physiol. 85, 327 (1987). 32 j. R. deWet and V. Walbot, unpublished data (1990). 33 K. R. Luehrsen and V. Walbot, Mol. Gen. Genet. 225, 81 (1991). 34 j. Kyozuka, T. Izawa, M. Nakajima, and K. Shimamoto, Maydica 35, 353 (1990). 35 j. H. Oard, D. Paige, and J. Dvorak, Plant Cell Rep. 8, 156 (1989). 36 C. D. Riggs and M. J. Chrispeels, Nucleic Acids Res. 15, 8115 (1987).

404

REPORTER GENES

[35]

described above, this laboratory has previously described a series of plasmids from which luciferase mRNA can be transcribed in vitro. ~2 The mRNAs from these vectors can be directly introduced into plant protoplasts. These vectors have been useful in assessing posttranscriptional controls such as the TMV 12 translational enhancer element and the effect of capping and poly(A) tails on translation and mRNA stability. Transient Expression Assay The transient assay protocol described below utilizes electroporation 37 for gene transfer and was developed for maize suspension cells. With minor modifications we have adapted it to maize callus tissue as well as rice, carrot, tobacco, and bean suspension cultures~2'38'39; firefly luciferase has been expressed in all these cell types. Instrumentation Required

The protocols described below require specialized instrumentation. We list the devices we utilize and suggest equivalent instruments or alternative sources. We have extensively used the Promega Corp. (Madison, WI) X-Cell 450 electroporation apparatus; however, this is no longer being marketed; an apparatus with similar electrical parameters is available from Hoefer (San Francisco, CA) (model PG101). The most convenient method to detect luciferase expression is with a luminometer; we have used models 2001 and 2010 from Analytical Luminescence Laboratory (San Diego, CA) and model 3010 from Analytical Scientific Instruments (Alameda, CA) and find these machines to be adequate. However, the high price might preclude the purchase of a luminometer. A scintillation counter can substitute 4° if expression is high enough, fl-Glucuronidase enzyme assays can be done spectrophotometrically but the more sensitive fluorescence assay is preferred. A full-featured fluorometer works well but is costly; we have found the low cost model TKO-100 minifluorometer from Hoefer performs well for GUS assays and, in a separate procedure, can be used to quantitate DNA concentration. Reagents

Black Mexican sweet (BMS) medium: One package Murashige and Skoog salt (GIBCO, Grand Island, NY)/liter, 1 ml 1000 x vitamins 37 M. Fromm, J. Callis, L. P. Taylor, and V. Walbot, this series, Vol. 153, p. 351. 3s p. Leon, F. Planckaert, and V. Walbot, Plant Physiol. 95, 968 (1991). 39 F. Planckaert and V. Walbot, Plant Cell Rep. 8, 144 (1989). 4o V. T. Nguyen, M. Morange, and O. Bensaude, Anal. Biochem. 171, 404 (1988).

[35]

FIREFLY LUCIFERASE REPORTER GENE

405

(1000 x vitamin stock contains 1.3 mg/ml niacin, 250/zg/ml thiamin, 250/zg/ml pyroxidine, 250/zg/ml pantothenate; store at -20°), 130 mg/liter asparagine, 20 g/liter sucrose, 200 mg/liter inositol, 2 mg/liter 2,4-D (2,4-dichlorophenoxyacetic acid). The final solution is adjusted to pH 5.8 with 0.1 N NaOH and autoclaved to sterilize. This is a modification of the MS medium previously described 41 Protopalst isolation medium (PIM): 90 g/liter mannitol, 0.73 g/liter CaCI2-2H20, 0.97 g/liter MES [2-(N-morpholino)ethanesulfonic acid], final solution adjusted to pH 5.8 with 0.1 N NaOH PIM + enzymes: To 100 ml PIM add 0.3 g cellulase (CELF/Worthington, Freehold, N J), 1 g cytolyase (Genencor, South San Francisco, CA), 20 mg pectolyase Y23 (Seishin Pharmaceuticals, Tokyo, Japan), 0.5 g bovine serum albumin (BSA) (Sigma, St. Louis, MO), 50/zl 2mercaptoethanol. Centrifuge the mixture for 5 min at 3000 g to pellet any insoluble material and sterilize by passage through a 0.45-~m Nalgene filter (Nadge, Rochester, NY) Electroporation solution (EPH): 36.4 g/liter mannitol, 8.95 g/liter KCI, 0.58 g/liter NaC1, 0.59 g/liter CaCI2 • 2H20, 2.38 g/liter N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), final solution adjusted to pH 7.2 with 0.1 N NaOH Plating out medium for transients (POMT): To 800 ml of BMS medium add 54.6 g mannitol and add 200 ml of filter-sterilized conditioned BMS medium (below) Conditioned BMS medium: Centrifuge and recover the supernatant from a 2- to 4-day-old BMS culture. The medium is filter sterilized through a 0.45-/zm Nalgene filter

Routine Maintenance of Suspension Cultures The BMS suspension line we use has been in culture for several years, and the cells are densely cytoplasmic and homogeneous in size. Cultures are split 1 : 1 into fresh medium every 3-4 days. We use 40 ml of culture medium in a 100-ml flask and shake at 110 rpm at 25°.

Protoplast Preparation and Electroporation The BMS suspension is routinely maintained as described above. For the highest levels of transient gene expression, we find it best if the cells are in a logarithmic growth phase. A 1 : 1 transfer on each of 2 days before electroporation satisfies that requirement and results in a sufficient density of cells for several (about eight) gene transfers per flask. Cultured cells ( - 100 ml) are transferred to sterile 50-ml screw-cap plastic tubes (Corning, 41 T. Murashige and F. Skoog, Physiol. Plant. 15, 473 (1962).

406

REPORTER GENES

[35]

Corning, NY) and centrifuged at 150 g for 2 min at room temperature, and the supernatant is discarded or used for POMT (above). The cells are washed once with 50 ml PIM and centrifuged as before. The cell pellet is dispersed in 50 ml of PIM + enzymes and 10 ml is aliquoted to each of 5 petri plates (100 × 15 mm); these are incubated at 25° on a rotary shaker at 50 rpm for 3-5 hr. The optimal time of digestion is variable and is terminated when the protoplasts are close to spherical yet still retain some cell shape (e.g., some cell wall) as monitored by light microscopy. The digested protoplasts are transferred to a 50-ml screw-cap tube and centrifuged as before. The pellet is washed twice with 50 ml PIM to remove the protoplasting enzymes and once with 50 ml EPH. Finally, the protoplasts are suspended in a total volume of 8-12 ml EPH ( - 2 - 4 x 106 protoplasts/ ml). For the highest levels of expression, an optional 10-min heat shock at 45 ° increases transient gene expression of luciferase or GUS approximately two- to fourfold. With or without the optional heat shock, the protoplasts are chilled on ice for >30 min before electroporation. Each transfection is set up by dispensing 0.5 ml of EPH containing 5-50 /xg of luciferase expression plasmid, 10-20 ~g of a GUS plasmid (optional; see below), and 75/zg of sheared, nondenatured salmon sperm DNA (as a carrier) into a 1-ml plastic semimicrocuvette (Cat. No. 58017847; VWR Scientific, Philadelphia, PA). BMS protoplasts (0.5 ml) are added to the cuvette and gently mixed. Within 2 min of protoplast addition, a flame-sterilized stainless steel electrode (0.4-cm plate separation; model E4 obtained from Prototype Design Services, Madison, WI) is inserted into the cuvette and a 12-msec pulse of 450 V/cm (capacitors charged to 1550/~F) is applied with an electroporation apparatus. For our laboratory cultures, the optimal conditions for tobacco protoplasts are 450 V/cm (1550 ~F) with a 5-msec pulse; carrot protoplasts are electroporated at 700 V/cm (1550 ~F) with a 6-msec pulse. A prior report describes the optimization procedure for a new cell source. 37 Immediately after discharge, the protoplasts are removed with a Pasteur pipette and gently dispensed into 7.5 ml POMT in a 100 x 15 mm petri plate. The transfected protoplasts are incubated statically at 25° for 20-40 hr. Generally, expression can be detected as early as 2 hr and is linear for the first 30-40 hr; expression decreases thereafter as a result of degradation of the input plasmid DNA and turnover of mRNA and luciferase protein. Luciferase A s s a y s

For assessing luciferase expression in plant cells, we have traditionally used an adaptation of the luciferase assay reported in de Wet et al. 4

[35]

FIREFLY LUCIFERASE REPORTER GENE

407

Promega Corporation has described an improved assay system, 42 composed of the cell culture lysis reagent (CCLR) and luciferase assay reagent (LAR) buffers described below; this is available in kit form (Cat. No. El500). We will describe each assay system and compare the efficacy of each.

Reagents Luciferase extraction buffers: Standard buffer is 100 mM potassium phosphate, pH 7.8, 1 mM ethylenediaminetetraacetic acid (EDTA), 7 mM 2-mercaptoethanol, 10% (v/v) glycerol; autoclave to sterilize. The alternative CCLR buffer is 25 mM Tris-phosphate, pH 7.8, 2 mM dithiothreitol (DTT), 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% (v/v) glycerol, 1% (v/v) Triton X-100 Luciferase assay buffers: The standard buffer is 25 mM Tricine, pH 7.8, 15 mM MgC12, 5 mM ATP, 7 mM 2-mercaptoethanol, 0.5 mg/ml BSA. We make this fresh from stock solutions of 0.1 M Tricine, pH 7.8, 1 M MgC12, 0.1 M ATP, pH 7.5, and BSA at 10 mg/ml. The alternative buffer LAR is 20 mM Tricine, pH 7.8, 1.07 mM (MgCO3)4Mg(OH) 2. 5H20, 2.67 mM MgSO 4, 0.1 mM EDTA, 33.3 mM DTT, 270 /zM coenzyme A, 470/zM luciferin, 530 tzM ATP. Store the LAR buffer in small aliquots at - 8 0 ° Potassium D-luciferin (10 mM): Potassium luciferin (Cat. No. 1600) from Analytical Luminescence Laboratory has a molecular weight of 318.41. To prepare, dissolve 32 mg of potassium luciferin in I0 ml water and store at 4 ° in the dark. The working solution is 250/zM and is made by dilution with distilled water Cell extracts are prepared by transferring the transfected protoplasts to a 15-ml conical screw-cap tube; a rubber policeman can be used to dislodge any protoplasts adhering to the petri dish. The protoplasts are spun at 150 g for 2 min at 4 ° and the supernatant is removed by aspiration. The protoplasts are resuspended in 0.4 ml extraction buffer and then transferred to a 1.5-ml microfuge tube. To disrupt the cells, we sonicate on ice for 10 sec at 75 W with an Ultrasonics (Long Island, NY) sonifier (model W185D) using the microtip; alternatively, using the CCLR buffer [containing 1% (v/v) Triton X-100], sonication can be omitted and a 10-sec vortexing at high speed will solubilize the enzyme. The disrupted protoplasts are centrifuged at 12,000 g for 5 min at 4° to pellet the cell debris. The luciferase activity remains stable for at least I month in extracts stored at - 80°. 4z K. V. Wood, in "Bioluminescence & Chemiluminescence: Current Status" (P. E. Stanley and J. Kricka, eds.), Wiley, Chichester, 1991.

408

REPORTER GENES

[35]

Our standard assay is as follows: add 5-50/xl of extract to 200/zl assay buffer in a luminometer cuvette and allow the mixture to equilibrate to room temperature (about 15 min). The cuvette is placed in the counting chamber of a luminometer and 100/xl of 250/zM luciferin is injected into the cuvette to start the reaction. The photons emitted are integrated over a 10-sec period and are expressed as light units (lu)/10 sec. Few cell extracts contain a source of chemiluminescence, hence background can be ascribed to the luminometer. The machines we have tested have a background of 50-200 events/10-sec counting period. This is a very low background considering that 20/xl of extract from cells electroporated with 10/xg plasmid pJD300 and incubated for 20-40 hr routinely yields >20,000 lu/10 sec (see below and Table I). The quantity of light emitted per unit time falls off rapidly after an initial burst about 0.3 sec 5 after luciferin is added. Consequently, longer assay times do not accurately measure luciferase activity. Mechanistically, luciferase activity is inhibited by oxyluciferin, the reaction product, because this molecule remains in the active site. An alternative procedure extending the sensitivity of the assay takes advantage of a buffer system promoting a more rapid turnover of the luciferase enzyme. Using the LAR buffer described above, the rate of light emission is essentially constant for several minutes (tl/2 5 min), allowing longer assay times and hence increased sensitivity. The improved assay is as follows: add 20/xl of cell extract to 100/~1 of the LAR buffer (equilibrated to 22°), place in the luminometer, and measure the light produced for the desired time. Alternatively, using the luminometers described above, 100/zl of LAR buffer can be injected directly into the cell-free extract to initiate light production. The LAR assay is also well suited for use with a scintillation counter. Using the standard assay, the time required to initiate the reaction and to insert the vial in the counting chamber will miss the initial burst of light (t = 0.3 sec). The extended emission of light using the LAR buffer allows sufficient time for sample manipulation and detection using a scintillation counter. Maximal sensitivity requires turning off the coincidence counter to ensure that all photons are counted as events.

fl-Glucuronidase Important in comparing the expression of several luciferase constructs is the significant variation caused by differences in protoplast viability, protoplast recovery, and purity among luciferase plasmid preparations. A simple correction for protoplast recovery can be made by standardizing the luciferase activity with the total protein in each extract and representing expression as a specific activity (lu/lO sec//zg protein). To correct each

[35]

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12 h r . 43 These observations suggest that luciferase protein is not long lived in plant cells. Protein engineering could increase the enzyme half-life, but an alternative approach is now available. Inclusion of luciferin analogs in in vitro assay buffers or in cell culture media serves to increase the luciferase half-life severalfold. 48One advantage of a short enzymatic half-life that should not be forgotten is that it allows repetitive induction of luciferase activity from a regulatable promoter, without building up a high background of reporter gene expression. Consequently, experimental design may favor a luciferase reporter activity with a short or long half-life. Similarly, the luciferase mRNA can be engineered to give a short or long half-life by manipulating the composition of the 5' and 3' untranslated regions as required for particular experiments. Acknowledgments We thank Keith Wood and Peter Christie for helpful comments. Development ofluciferase reporter genes for maize cells was aided in part by support of K.R.L. by an American Cancer Society postdoctoral fellowship (PF2943), of J.R.D. by a National Institutes of Health (NIH) Postdoctoral Fellowship (GM11767-02), and by a grant from the NIH (GM 32422).

48 j. F. Thompson, L. S. Hayes, and D. B. Lloyd, Gene 103, 171 (1991).