[15] • -LACTAM AS E AS A REPORTER 2 2 1

sequences removed, the RNA-specific primer extension product will be shorter than any product that results from priming of the transfecting DNA. Knuchel et al. 44 have developed a CAT-specific RT-PCR assay that also allows for the quantitation of CAT mRNA but not CAT DNA. In this study, no intron was included in the CAT expression vector so a "tailed" RT-PCR approach was developed to distinguish between CAT mRNA and CAT DNA. This approach uses a tailed antisense primer for the RT step; 20 bp are complementary to the CAT mRNA and 27 bp are not complemen- tary to the CAT gene. Optimal conditions for this RT-PCR reaction were 2.0 Mg 2+ for the PCR buffer, an annealing temperature of 55 °, and 35 cycles.

CAT-TR tailed RT primer 5 ' C A T C G A T G A C A A G C T r A G G T A T C G A T A C C A T T C A T C C G C T r A T C Y

TAIL-R antisense primer specific for the tail sequence 5' C A T C G A T G A C A A A G C T F A G G T A T C G A T A Y

Underlined sequences are not complementary to CAT sequences. Based on this assay, CAT RNA produced a linear signal in between the range of 2 x 10 10 to 0.1 x 10 -16 pmol. The sensitivity of this assay was able to measure the equivalent of eight copies of CAT mRNA. The mRNA for CAT could be measured easily for 72 hr, and the stability of the CAT mRNA was determined to be at least 24 hr.

44 M. Knuchel, D. P. Bednarik, N. Chikkala, F. Villinger, T. M. Folks, and A. A. Anasri, J. Virol. Methods 48, 325 (1994).

[1 51 F u s i o n s t o / ~ - L a c t a m a s e a s a R e p o r t e r fo r G e n e

E x p r e s s i o n i n Live M a m m a l i a n Ce l l s

By G R E G O R Z L O K A R N I K

Introduction

/3-Lactamase has been developed as a reporter enzyme for monitoring gene expression in mammalian cells. 1,2 The enzyme is of bacterial origin without any mammalian homologues and permits quantitative analysis of

1 j. T. Moore, S. T. Davis, and I. K. Dev, Ana l Biochem. 247, 203 (1997). 2 G. Zlokarnik, P. A. Negulescu, T. E. Knapp, L. Mere, N. Burres, L. Feng, M. Whitney,

K. Roemer, and R. Y. Tsien, Science 279, 84 (1998).

Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

METHODS IN ENZYMOLOGY, VOL. 326 0076-6879/00 $30.00

222 GENE FUSIONS AS REPORTERS OF GENE EXPRESSION [15]

reporter expression in single mammalian cells without interference from an endogenous background. It can be fused to the N and C termini of proteins without loss of activity, which allows monitoring the expression levels of the tagged protein. 1'3 It is distinguished from popular luciferase and/3-galactosidase reporter enzymes 4'5 in that it can be detected sensitively in individual living cells under standard culture conditions. This is accom- plished with a fluorogenic membrane-permeable substrate ester that dif- fuses into cells and provides high intracellular concentrations of accumu- lated fluorescent substrate. The reporter is detected sensitively by its catalysis of substrate hydrolysis, which results in a highly amplified fluores- cent signal, allowing analysis of expression of proteins that are present in cells at low copy numbers. The fluorescent signal from live cells permits facile functional selection of reporter-expressing cells with flow cytometry or fluorescence microscopy. Selected cells usually remain viable and can be used for subsequent analyses that require intact cells.

The Reporter Enzyme

The fl-lactamase enzyme used as a reporter in mammalian cells is a member of a large and structurally diverse family of enzymes that cleave fl-lactam antibiotics such as penicillins and cephalosporins. The enzyme that was chosen as reporter in mammalian cells corresponds in part to the mature Escherichia coli enzyme encoded by the ampicillin resistance gene (Amp r) of the pUC18 and pBS plasmids. It differs from the published TEM-1/3-1actamase sequence 6 by conservative mutations V82I and A184V that are present in pUC18 and pBS plasmids and introduction of a new N terminus. The new N terminus was created by deletion of the first 23 amino acids, which comprise the signal sequence, and introduction of a methionine and mutation H24D, which provide an optimal Kozak sequence 7 for mam- malian expression. This enzyme is preferred as a reporter over its more distant cousins because it exhibits fast and simple kinetics with cephalospo- rin substrates, allowing its ready quantitation. The structural gene encoding the reporter/3-1actamase is referred to as bla.

Functional reporter fusion proteins may be constructed by replacing either the ATG initiator codon or the termination codon with the appro-

3 M. Whitney, E. Rockenstein, G. Cantin, T. Knapp, G. Zlokarnik, P. Sanders, K. Durick, F. F. Craig, and P. A. Negulescu, Nature Biotechnol. 16, 1329 (1998).

4 j. R. de Wet, K. V. Wood, M. DeLuca, D. R. Helinski, and S. Subramani, Mol. Cell. Biol. 7, 725 (1987).

5 p. A. Norton and J. M. Coffin, Mol. Cell Biol. 5, 281 (1985). 6 j. G. Sutcliffe, Proc. Natl. Acad. Sci. U.S.A. 75, 3737 (1978). 7 M. Kozak, Nucleic Acids Res. 12, 857 (1984).

[15] /3-LACTAMASE AS A REPORTER 223

priate sequence encoding the protein polypeptide chain.* The unmodified reporter enzyme has a half-life of approximately 3 hr in living mammalian cells, 2 permitting the monitoring of events that lead to a decrease in reporter gene expression and result in lower cellular reporter enzyme levels.

The Substrate and Its Ester

/3-1actamase is detected sensitively within living cells with the fluorescent substrate coumarin cephalosporin fluorescein No. 2 (CCF2). 2 CCF2 is a cephalosporin derivative, which is labeled with a fluorescent donor 7-hy- droxycoumarin derivative and an acceptor fluorescein fluorophore. In the intact substrate, excitation of the donor leads to efficient resonance energy transfer to the acceptor fluorescein, which emits green fluorescence. /3- Lactamase catalyzes the hydrolysis of the cephalosporin, which results in the separation of the donor fluorophore from the acceptor. After hydro- lysis, excitation of the donor 7-hydroxycoumarin fluorophore results in emission of blue fluorescence. The substrate is delivered into live cells via its membrane-permeable substrate ester derivative, CCF2/AM (AM is the acronym for an acetoxymethylester). In this substrate derivative the moieties in CCF2 are modified as a butyrate ester (hydroxycoumarin), acetoxymethylester (cephalosporin), and diacetate ester (fluorescein). This substrate ester diffuses into cells and is converted to the free substrate, CCF2, by endogenous mammalian cytoplasmic esterases. The free substrate carries several negative charges at physiological pH, which help retain it in the cytoplasm of the cell.

Data Analysis

Cells loaded with the/3-1actamase substrate CCF2 fluoresce green in the absence of the reporter. In cells expressing the reporter,/3-1actamase converts the green fluorescent substrate to its blue fluorescent hydrolysis product. The substrate, which exhibits an easily visible color change on hydrolysis, has some distinct advantages over intensity-based substrates. The original fluorescence allows verification of whether the substrate has indeed been delivered into the cell. The change in the color of fluorescence provides a simple and sensitive way of visualizing whether a reporter con- struct is expressed and whether a cell population is homogeneous. The dual wavelength readout allows for ratiometric analysis, which is preferred, as it is less affected by experimental fluctuations than single wavelength mea- surements. Ratiometric analysis of CCF2 hydrolysis uses the fluorescence emission intensity value measured for the donor fluorophore and divides it by the fluorescence intensity value measured for the acceptor emission to give an "intensity ratio." This ratio is a measure of the progression of

224 GENE FUSIONS AS REPORTERS OF GENE EXPRESSION [ 15]

the substrate hydrolysis reaction, increasing in value as donor fluorescence increases with hydrolysis of the substrate. Ratiometric readouts (also re- ferred to as "ratioing") reduce the noise in assay measurements substan- tially, as factors that affect fluorescence intensities at both wavelengths cancel out in the ratio. Experimental variables whose impact on the assay results are reduced substantially by ratioing include variations in cell size, cell number, cellular probe concentration, and fluctuations in excitation in- tensity.

Typical Experimental Conditions for Live Cell Assays

In its simplest form, the ]3-1actamase reporter assay is performed as an end point assay. First, the bla reporter gene is introduced into cells either transiently or stably, followed by an experiment in which expression of the reporter is modulated over several hours or even days under physiological conditions. Then, at the end of the experiment, cellular/3-1actamase activity is determined by fluorescence measurement under separate optimized con- ditions outlined later.

Cells are typically loaded with a serum-free loading buffer containing 1 IzM CCF2/AM at ambient temperature (18-22 °) for 60-90 min. Intracellu- far fluorescence is usually first visible about 15 min after loading and in- creases steadily for about 60 minutes. Longer incubation times lead to little further increase in cellular fluorescence as equilibrium between dye uptake and leakage from cells is reached. This equilibrium may be reached at times and fluorescence intensities that differ from the ones depicted in Fig. i and will depend on the cell line, the specific experimental conditions, and the

.C

o

30 60 90 120 150 180

time in presence of CCF2/AM (rain)

FIG. 1. T ime course of cellular substrate loading at 22 ° measured by fluorescence intensity. Jurkat cells were kept in the presence of 1 /zM CCF2/AM, and fluorescence intensity was measured at 535 +-- 12.5 n m with excitation at 395 +-- 12.5 n m on a Cytofluor 4000 fluorescence microtiter plate reader at 10-min intervals. Each data point represents the average of four sample wells and each well contained 105 cells. Error bars represent s tandard error.

[151 /~-LACTAMASE AS A REPORTER 225

concentration of CCF2/AM in the loading medium. Results of loading Jurkat ceils with 1/zM CCF2/AM at ambient temperature (22 °) are shown in Fig. 1.

Commonly used assay cell lines were also loaded with 1 /zM CCF2/ AM at ambient temperature for 1 hr. The following increases in cellular fluorescence relative to endogenous background were measured on an epifluorescence microscope equipped for quantitative image analysis: Jurkat (85-fold), HeLa (120-fold), HEK 293 (75-fold), GH3 (56-fold), COS-7 (44-fold), CHO (38-fold), and CV-1 (28-fold). Suspension cells were loaded at cell densities up to 1-2 x 10 6 cells/ml. Adherent cells were loaded at 60-80% confluence, as loading was significantly less efficient when cells were completely confluent. Loading cells at higher temperatures, such as 37 °, led to more rapid loading of the substrate with the equilibrium being reached earlier, but for all cell lines tested, equilibrium concentrations of intracellular substrate were significantly lower at 37 ° than at ambient temperatures (data not shown). This is explained with a disproportionately larger effect of temperature on increase in dye export than on the processes involved in dye loading.

Substrate-Loading Protocols

The substrate ester CCF2/AM is dissolved to 1 mM in dry dimethyl sulfoxide (DMSO). This stock solution can be frozen and be kept protected from light for future use (for about 1 month). To prepare 1 ml of 1 tzM CCF2/AM loading buffer, 1/zl of the 1 mM CCF2/AM stock solution in DMSO is diluted with 9 ~1 of a DMSO solution containing 100 mg/ml of the dispersant Pluronic-F127 and 0.1% acetic acid. The resulting DMSO solution is added with vigorous agitation to 1 ml of serum-free medium containing HEPES (25 mM, pH 7.3) or a suitable buffer such as HBS (pH 7.3). The resulting medium is referred to as the loading buffer. Because CCF2/AM has limited stability in aqueous solutions, the loading buffer should be used within 1 hr of its preparation.

For loading adherent cells the medium is aspirated from cells and re- placed with loading buffer. After the desired loading period (typically 1 hr), the loading buffer is aspirated and replaced with fresh medium.

For loading of suspension cells, cells are spun gently to a pellet by centrifugation, the supernatant removed, and the cells resuspended in load- ing buffer. After the desired loading period, cells are again spun gently into a pellet by centrifugation, the loading buffer aspirated, and the cells resuspended in fresh medium.

Adherent cells typically load best when 50-75% confluent. Most cell lines will accumulate more substrate when loaded at room temperature

226 GENE FUSIONS AS REPORTERS OF GENE EXPRESSION [15]

and kept at room temperature thereafter, as substrate loss in many cell fines increases significantly at 37 ° . Removing the solution containing sub- strate ester CCF2/AM after the desired loading period and replacing it with solution or media without dye ester reduces (removes) solution fluo- rescence./3-Lactamase nonexpressing cells will appear more greenish if left to convert the ester for another 15-30 min after loading and washout of the substrate ester.

Aqueous Substrate Ester Solutions

It is common for researchers to prepare concentrated aqueous reagent solutions for use in their experiments. This is to avoid adding too high a concentration of organic solvent to the preparation and to allow addition of accurate volumes of reagents, as the error in accuracy increases as addition volumes decrease. Unfortunately, this practice can cause problems when applied to CCF2/AM, which is a large hydrophobic molecule that has relatively low solubility in water. Making 100×, 10x, or even 5× aqueous loading solutions will usually lead to precipitation of CCF2/AM from solution (although this may not be visible to the unaided eye), resulting in inefficient loading.

To allow preparation of aqueous reagent stock solutions (CCF2/AM loading solutions), organic cosolvents were investigated for their ability to prevent precipitation of CCF2/AM from aqueous solution and their lack of interference with the cells' viability and/3-1actamase reporter activity. Polyethylene glycol with an average molecular weight of 400 g/mol (PEG- 400) is such a cosolvent. It will prevent CCF2/AM precipitation from aque- ous 6× or 11× loading solutions when present at a concentration of 24% (v/v). Many cell lines tolerate PEG-400 in the solution (at least for the purpose of determining intracellular/3-1actamase concentrations at the end of an experiment). For instance, Jurkat, HeLa, HEK 293, GH3, COS-7, CHO, and CV-1 will tolerate up to 5% PEG-400 in the final medium during substrate loading.

Table I gives the volumes of reagents needed to prepare 6x or l l x

TABLE I VOLUMES OF REAGENTS FOR 6 AND l l X CCF2/AM LOADING SOLUTIONS

Loading solution 6× 11 ×

1 mM CCF2/AM in DMSO 6/zl 12/zl DMSO, 100 mg/ml Pluronic F127, 0.1% acetic acid 54/zl 48/zl 24% (v/v) PEG-400 in serum-free medium or buffer 1 ml ] ml To 100 tzl cell medium, add: 20 tzl 10/zl

[151 fl-LACTAMASE AS A REPORTER 227

aqueous CCF2/AM loading solutions. (Note: Aqueous CCF2/AM loading solutions deteriorate quickly at ambient temperatures and should be used within 1 hr of preparation and unused portions discarded.) An appropriate volume of 1 mM CCF2/AM stock solution in DMSO is added to DMSO containing 100 mg/ml of the dispersant Pluronic-F127 and 0.1% acetic acid. The resulting DMSO solution is added with vigorous agitation to 1 ml of 24% (v/v) PEG-400 in serum-free medium containing HEPES (25 mM) or in a suitable buffer such as HBS (pH 7.3). The resulting solution is added to cells in their culture medium. After loading, the CCF2/AM containing medium is aspirated and replaced with fresh medium or, to avoid hypotonic shock, replaced with medium containing 4% PEG-400 (after loading with 6x solution) or medium containing 2% PEG-400 (after loading with 11x solution). (Note: Care should be taken with addition of the PEG-400 con- taining stock solutions and their removal. PEG-400 containing media and buffer solutions may be hypertonic and may cause cells to shrink initially. After a period of time in the presence of PEG-400, cells may regain original volume but at an increased intracellular tonicity. At this time, changing cells from PEG-400 containing solutions into nominally isotonic solutions may cause cells to swell. Cell lines not mentioned earlier, including primary cells, neurons, and so on, may not tolerate the change in tonicity or exposure to PEG-400.)

Blocking Active Substrate Export

If cells appear only dimly fluorescent by visual inspection on an epiflu- orescence microscope after following the substrate loading procedure just described, this may be because of active transport of the substrate CCF2 out of the cells. Active transport of CCF2 out of cells via anion transporters can be reduced by loading and keeping cells in the presence of a standard inhibitor of nonspecific anion transport, probenecid, 8 at a final concentra- tion of 2.5 mM. Probenecid (Sigma), an organic acid, is dissolved in aqueous solution with the addition of one equivalent of an aqueous base, such as aqueous sodium hydroxide. A 250 mM probenecid stock solution (100x) is prepared by the addition of 2.5 ml 1 N sodium hydroxide solution to a suspension of 2.5 mmol probenecid in 7.5 ml water and vigorous agitation to dissolve the organic acid. Adding this solution to the loading buffer and the medium used to wash the cells at a final concentration of 1% aids in retaining CCF2 and its enzymatic hydrolysis product.

s F. Di Virgilio, T. H. Steinberg, J. A. Swanson, and S. C. Silverstein, J. Immunol. 140, 915 (1988).

228 G E N E F U S I O N S A S R E P O R T E R S O F G E N E E X P R E S S I O N [15]

Discussion of Cell Loading

The fluorogenic/3-1actamase substrate ester, CCF2/AM, is designed to be hydrophobic to diffuse into mammalian cells where it is converted by mammalian cytoplasmic esterases to free the polar/3-1actamase substrate, CCF2. Ester protection groups used in CCF2/AM (butyrate, acetate, and acetoxymethyl ester) provide the necessary lability in the cytoplasmic envi- ronment for loading of the cells. These groups are well established for delivery of prodrugs as well as fluorescent probes into cells. Ester hydrolysis liberates CCF2, which is the substrate for/3-1actamase, and its hydrolysis gives rise to blue cytoplasmic fluorescence in cells (Fig. 2, path A).

A

BtO 0 0 H AcO 0 OAc

/

ccF2/~ o ~ -c B

C

409 am 447 nm

\ /

5IO am 4~ . . FRET

TO H b 0 0

)9 nm 447 am

CYTOPLAS

CELLS ~ NSUFFICIENT

Estergse act iv i ty

4O9 . m 447 . m

\ / "o H o OAc

) ~ p - L a e t a m a s e

4O9 mm 447 nm ~o ~ ~ ~ OAc

\/ "0 0 H

e I ~ . + "~oLC°;

FIG. 2. Schematic of the various fates of CCF2/AM during cell loading. (A) Loading into healthy, esterase expressing cells. (B) Loading of compromised cells or cells lacking esterase. (C) Hydrolysis in medium.

[1 51 j~-LACTAMASE AS A REPORTER 229

The reason for the bluish appearance of compromised or overgrown cells is not entirely apparent. One can speculate that these cells may have decreased esterase activity, which results in inefficient removal of the ace- tate esters of fluorescein (Fig. 2, path B). Cells in which the donor is deprotected much more readily than the fluorescein acceptor will appear bluish. This is because, unlike the free fluorescein in CCF2, the esterified fluorescein acceptor in CCF2/AM is not a quencher of the free donor fluorophore. A suggested procedure is to load cells over extended periods of time (up to several hours), after which compromised cells often will have lost their dye and cells low in esterase activity will have had time to process the substrate ester. Cells that are not viable and do not exclude trypan blue may fluoresce blue during the loading procedure. Loading in the presence of trypan blue, with accumulation of the dye inside nonviable cells, efficiently reduces fluorescence from partially hydrolyzed CCF2/AM and stained cells appear nonfluorescent.

Although much more stable in aqueous medium than in the cytoplasm of cells, ester groups will hydrolyze to some degree in aqueous buffer at neutral pH. This occurs over the course of many hours for the butyrate ester or hours to days for fluorescein acetates and AM-ester. Buffer compo- nents such as albumin or serum speed this hydrolysis and are therefore not recommended during cell loading. The butyrate protection group of the hydroxycoumarin donor fluorophore is the most prone to hydrolysis in aqueous solution (Fig. 2, path C). A solution of CCF2/AM in which the butyrate protection group has been lost will have some blue fluorescence. In the cell, where fluorescein acetates are removed by cellular esterases, fluorescein quenches the donor efficiently. The partial hydrolysis of the butyrate ester is usually not a concern for loading of the substrate into cells, as under the recommended loading conditions (30-90 min, room temperature, absence of albumin or serum), the predominant amount of CCF2/AM remains esterified and available for loading.

Quantitative Analyses

Typically, reporter gene expression is reported relative to a set level, such as the fold induction or repression of reporter activity compared to background activity. Rarely, the number of reporter enzymes present per cell is determined quantitatively. 9 This is somewhat unfortunate because it does not allow quantitative comparisons of reporter expression levels between unrelated experiments or between different biological back-

9 G. P. Nolan, S. Fiering, J. F. Nicolas, and L. A. Herzenberg, Proc. Natl. Acad. ScL U.S.A. 85, 2603 (1988).

230 GENE FUSIONS AS REPORTERS OF GENE EXPRESSION [1 51

grounds. The lack of quantitative information is mainly due to the fact that for most reporter enzyme-substrate combinations the kinetics have not been determined to allow quantitative determinations in the sample, the enzyme kinetics are complicated, or the signal is difficult to calibrate. The alternative, which is to prepare a standard curve with purified enzyme and assess the amounts present in the sample by comparison, requires commercial availability of the reporter enzyme with known purity and activity. 1° This is not the case in most instances.

The kinetics of the fl-lactamase reporter enzyme with its fluorescent substrate CCF2 have been determined. 2 Kinetic data allow quantitative determination of the reporter actlvity in a sample and the experimental procedures are outlined.

l

~-Lactamase Activity in Live Cells

As the concentration of substrate in individual cells is typically unknown, the progress of the fl-lactamase-catalyzed substrate hydrolysis reaction is determined from the intensity ratio of donor-to-acceptor fluorescence emis- sions. This intensity ratio is less affected by variations in intracellular sub- strate concentrations than measurements at the individual donor and ac- ceptor wavelengths and is therefore better suited to assess the progression of the enzyme reaction in cells. Because the increase in blue fluorescence and the drop in green fluorescence with substrate hydrolysis are mostly linear with the amount of substrate hydrolyzed, a plot of the intensity ratio against substrate conversion has a hyperbolic shape. This is due to the nonlinear contribution of the numerator and denominator to the ratio. The two procedures that follow allow the progression of the substrate hydrolysis reaction to be related to the observed intensity ratios.

A linear relationship between the amount of substrate hydrolyzed and the ratio can be achieved experimentally by measuring the fluorescence intensity at the crossover point of substrate and product emission spectra (around 500 nm), which is independent of substrate conversion. When this value is used as the denominator, instead of acceptor fluorescence intensity at its 520-nm peak, a linear relationship of the progression of substrate hydrolysis and ratio value is obtained. This experimental modification pro- vides the benefit of reducing noise in the assay, as perturbations that affect emission intensities at both wavelengths to similar degrees cancel out in the ratio, although it does result in a smaller numerical range of the ratio values.

Alternatively, one can calculate the fraction (f) at time (t) of hydrolyzed substrate from experimentally determined ratio values using the following

10 V. K. Jain and I. T. Magrath, Anal. Biochem. 199, 119 (1991).

[1 51 fl-LACTAMASE AS A REPORTER 231

equation. Additional calibration values needed are obtained readily by measuring the fluorescence intensities with excitation of the hydroxycou- marin donor of unhydrolyzed and fully hydrolyzed substrate at the blue and green emission wavelengths.

R(/)FsG - FsB f"} = (FpB - FsB) + R,}(Fso - FpG) (1)

Progression of CCF2 hydrolysis reaction f(t) derived from ratio values R(t ) is shown in Eq. (1) in which f(t) is fraction of substrate converted to product at time (t), R(/) is ratio value of blue/green fluorescence intensities at time (t), Fsc is fluorescence emission intensity of substrate in the green channel, FsB is fluorescence emission intensity of substrate in the blue channel, Fp~ is fluorescence emission intensity of product in the blue chan- nel, and Fpc is fluorescence emission intensity of product in the green channel.

In vitro, the product is obtained by the incubation of substrate with a suitable concentration of the/3-1actamase enzyme to achieve full substrate hydrolysis. In experiments with cells, fluorescence intensity values for CCF2-1oaded cells with no/3-1actamase activity (e.g., wild-type cells) and cells with fully hydrolyzed CCF2 [e.g., clonal cells expressing bla under viral (e.g., CMV, SV40) promoter control] need to be determined.

~-Lactamase Expressing Cells in Populations

Epifluorescence microscopes and flow cytometers display single-cell resolution and permit direct quantitative determination of the fraction of reporter enzyme or reporter-fusion expressing cells in a population with high fidelity. However, many other instruments for fluorescence analysis do not have single-cell resolution capabilities, such as plate readers and fluorescence spectrophotometers. To determine the fraction of reporter enzyme-expressing cells in a population with an instrument lacking single- cell resolution, intensity ratios for donor and acceptor emission intensities can be determined for substrate-loaded cells in a cell population. Typically, donor and acceptor fluorescence intensity values determined in the mea- surement show a linear dependence with the fraction of blue cells in the population. The plot of a simple blue/green ratio has a hyperbolic shape due to the nonlinear contribution of the numerator and denominator. In general, one can accurately discriminate 5-10% changes in the percentages of blue cells in a population-based assay without spatial resolution (such as in a fluorescence microtiter plate reader) (Fig. 3).

To determine the fraction of/3-1actamase expressing blue cells [f{B~] in the population accurately, this value can be computed from the intensity

232 GENE FUSIONS AS REPORTERS OF GENE EXPRESSION [ 15]

A

c :

09

0)

o f - • blue s o~ 0 green signal

20 40 60 80 100

percentage blue cells in population

B

-- 3

E 82 CO ,q.

o3

~ 04,oO°°. ° 0 20 40 60 80 100

percentage blue cells in population

FIG. 3. (A) Change in blue and green emission intensities as a function of percentage blue cells in a 96-well plate. Wild-type and CMV-bla Jurkat cells (clonal Jurkat cell line with bla under CMV promoter control, expressing approximately 1.5 × 104 enzymes/cell) were mixed and the percentage of blue cells present was verified by flow cytometry. Each data point represents the average of six sample wells. Each well contained 105 cells. Data were obtained on a Cytofluor 4000 plate reader with 395 -+ 12.5 nm excitation and 460 _+ 20-nm (blue) and 535 __ 12.5-nm (green) emission filters. (B) Numerical ratio of blue and green intensity values graphed against the percentage of blue ceils in the population.

ratio using Eq. (2). I t permits determinat ion of the fraction of blue cells in a populat ion f rom experimental ly determined ratio values. For calibration purposes, this computat ion also requires fluorescence intensity measure- ments of a repor ter negative cell populat ion (e.g., wild-type cells) and a cell populat ion in which all cells express the enzyme (e.g., positive control such as clonal cells with CMV-bla or SV40-bla). Also, for accuracy, cells should be kept with substrate for sufficient t ime before fluorescence mea- surement to allow for complete conversion of intracellular substrate to product in cells expressing the reporter.

R(p)FNG -- FNB f(B) = (FpB -- FNB) -t- R(p)(FNG - FpG)

(2)

Fraction of blue cells f(B) in a populat ion derived for ratio values R(p) is shown in Eq. (2) in which fS is fraction of blue cells in the population, R(p) is ratio value of blue/green fluorescence intensities of cell population, FN6 is green signal f rom a repor ter negative cell populat ion (e.g., wild type), FNB is blue signal f rom a repor ter negative cell populat ion (e.g., wild type), FpB is blue signal f rom a repor ter positive control (e.g., clonal CMV- bla population), and Fp6 is green signal f rom a repor ter positive control (e.g., clonal CMV-bla population).

[15] ~3-LACTAMASE AS A RZPORTZR 233

"~ 100

8o

6o

40

g 20

E o 0 20 40 60 80 1 O0

percentage blue cells (actual)

FIG. 4. Percentage of blue cells derived from ratio values plotted against the percentage of blue cells determined by flow cytometry for the same samples.

Figure 4 demonstrates the advantages of dual wavelength analysis in reducing noise stemming from variations in cell number and cellular sub- strate loading. The percentages of blue cells determined mathematically from the ratio values plotted in Fig. 1B are graphed against the percentages of blue cells confirmed for the same cell populations using flow cytometry. As can be seen, the scatter around the regression line found for individual wavelength readings in Fig. 3A is substantially smaller after ratioing and mathematical conversion of ratios back to apparent percentages of blue cells present. Equation (2) computes the fraction of blue cells, a variable whose value is not readily obtained from individual emission intensity readings of cell populations alone.

Quantitation in L ysates

Concentrations of active/3-1actamase can be determined accurately in buffer solutions. This permits the average intracellular/3-1actamase concen- tration in reporter positive cells to be determined from the/3-1actamase activity in lysates from a known numbers of cells.

Suspension cells are harvested by centrifugation, and adherent cells are collected by dissociation with a EGTA containing buffer. If proteases need to be used to dissociate cells, great care should be taken to ensure complete removal of protease activity with multiple cell washes with protease-free buffer (or addition of a protease inhibitor). Cells are resuspended in isotonic phosphate buffer, pH 7.3, to a cell density of 10 7 cells/ml in a small tube with cap. A 10-/zl sample of cell suspension is removed and diluted into

234 GENE FUSIONS AS REPORTERS OF GENE EXPRESSION [ 15]

10 ml of buffer suitable for cell counting on a Coulter counter. Alternatively, the cell count can be determined using a hemocytometer. The remainder of cells are lysed by shock freezing. The tube is exposed to liquid nitrogen until the content freezes and is warmed to 30 ° in a water bath. After three freeze/thaw cycles, the cell debris is spun into a pellet by centrifugation in a cooled (4 °) tabletop microcentrifuge at maximum permissible speed. The lysate solution (50/A) is placed into a well of a black clear-bottom micro- titer plate. The average activity in wells from two twofold serial dilutions of 20 nM purified /3-1actamase derived from pUC1811 (a kind gift of S. Mobashery, Wayne State University, Detroit, MI) in phosphate buffer (50 /zl) serves as a standard in the calculation of fl-lactamase activity for cell lysis samples. Wells with 50/xl buffer alone and wells with lysate of un- transfected cells serve as baseline controls.

As an alternative to the freeze/thaw method, cells may be lysed using a final concentration of 0.5% CHAPS, which apparently has little effect on /3-1actamase activity at this concentration.

Quantitation with Standard Curve. To the samples (50/.d volume), one adds 50/zl of 20/zM CCF2 in phosphate buffer, pH 7.3, for a final concentra- tion of 10/~M CCF2. The microtiter plate is transferred to a fluorescence plate reader, and fluorescence intensity measurement (447 nm, product channel, e.g., 460 _+ 20 nm) readings are taken, initially at 3- to 5-min intervals for the first hour and then hourly for another 4 hr to allow assess- ment of very low enzyme concentrations. Hydrolysis of CCF2 is complete within 1 hr at the highest/3-1actamase concentrations in the standard (10 nM enzyme), which gives the signal for 100% conversion. This value, sub- tracted by the value for blue fluorescence reading from a well containing CCF2 in buffer alone, gives the total fluorescence change associated with 1 nmol CCF2 (100/zl of 10 /xM) converted. It is used to calculate the amount of CCF2 hydrolyzed as the magnitude of the blue fluorescence signal is proportional to blue fluorescence from the sample wells. The rate of increase in blue well fluorescence, for less than 5% conversion of CCF2 in each well, is a measure of/3-1actamase activity in that well. The amount of/3-1actamase present in wells containing cell lysate can be determined from the standard curve calculated for the purified enzyme. This activity is then normalized to the number of cells in the lysate, which was determined by cell count. In addition, the percentage of/3-1actamase expressing cells in the population can be determined in cells before lysis by loading with CCF2/AM and analysis by visual inspection on a fluorescence microscope

11 G. Zafaralla, E. K. Manavathu , S. A. Lerner, and S. Mobashery, Biochemistry 31, 3847

(1992).

[1 51 ]~-LACTAMASE AS A REPORTER 235

or by flow cytometry and the cell count modified to represent reporter- expressing cells only.

Note: Care should be taken to ensure that the detector has a linear response in the intensity range chosen for the experiment.

Quantitation without Standard Curve. A simple estimate of cellular/3- lactamase concentration relies on fluorescence intensity values for the sub- strate and the fully hydrolyzed CCF2 sample and the published kinetic values for CCF2 hydrolysis by TEM-1 /3-1actamase. From the published values (/(cat = 29 ± 1 sec -1 and Km = 23 ___ 1/xM), e the expected hydrolysis rate for 10/xM CCF2 is about 9 sec ~ (mol substrate/mol enzyme). Steady- state values for hydrolysis of 10 /zM CCF2 solutions differ only slightly from initial rates of hydrolysis (steady state rates of 7.3 and 6.7 sec i after 5 hr were determined in two independent experiments). Reliance on these data allows analysis without preparation of a standard curve with purified enzyme. Commercially available preparations of enzyme (i.e., Sigma, Peni- cillinase E. coli 205 RTEM) are suitable to catalyze hydrolysis of the sub- strate in order to obtain the fluorescence intensity values for fully hy- drolyzed CCF2.

Maximizing the Per fo rmance of the Repor ter Assay

In a cell-based assay it is desirable to be able to stop or at least slow the enzymatic reaction of the reporter enzyme to avoid exhaustion of substrate in cells that have high concentrations of reporter enzyme. Phenylethylthio-/3-galactopyranoside is a competitive inhibitor of the re- porter enzyme /3-galactosidase 12 and has been used successfully to stop fl-galactosidase activity in cells prior to flow cytometry. 13 It would be just as desirable to be able to inhibit the reporter enzyme with an irreversible or suicide inhibitor because, in addition to using it as a stopping agent, it would also allow inhibition of background reporter activity present prior to an assay. To abolish background activity only, cells would be treated with the inhibitor, followed by washout and quantitative analysis of reporter enzyme synthesized de novo, after the treatment with the inhibitor.

Irreversible Inhibition of Intracellular /3-Lactamase

The reporter enzyme /3-1actamase can be inactivated irreversibly in living cells with clavulanic acid. Although clavulanic acid is a substrate for /3-1actamase, it also acts as an inhibitor because 1 out of 115 turnovers of the substrate results in cross-linking of residues in the active site of the

12 C. K. De Bruyne and M. Yde, Carbohydr. Res. 56, 153 (1977). t3 S. N. Fiering, M. Roederer, G. P. Nolan, D. R. Micklem, D. R. Parks, and L. A. Herzen-

berg, Cytometry 12, 291 (1991).

236 GENE FUSIONS AS REPORTERS OF GENE EXPRESSION [ 1 5]

~ '3 o

62 c.o

.o

none 1 10 100 Clavulanate [~M]

F~G. 5. Inhibition of intracellular/3-1actamase reporter activity as a function of clavulanic acid concentration. CMV-bla Jurkat ceils (expressing approximately 1.5 × 104 enzymes/cell) were treated for 16 hr with clavulanic acid at 37 °. Ceils were then loaded with 1/zM CCF2/ AM for 1 hr, and population fluorescence intensity ratios were analyzed in a clear-bottom, 96-well plate using a Cytofluor 4000 plate reader with excitation at 395 -z-_ 12.5 nm (bandpass filter) and 460 -4- 20-nm and 535 -4- 12.5-nm emission filters. Each data point represents the average of four sample wells and each well contained 105 cells. Error bars represent standard error. A population of cells lacking the reporter (wt Jurkat) gives a ratio of 0.15 under similar experimental conditions (not shown).

enzyme. 14 Cross-linking leads to irreversible inhibition of the enzyme, which allows the inhibitor to be removed from the medium without reappearance of activity. The apparent ICs0 for inhibition of intracellular/3-1actamase in the cytoplasm of cells for overnight exposures to inhibitor is about 10/xM (Fig. 5), although in vitro it has a Ki for the enzyme of 0.4/xM. 15 This apparent lower potency of clavulanic acid in the cell-based assay is likely due to the limited access of the inhibitor to the highly concentrated intracellular reporter enzyme.

The protein synthesis inhibitor cycloheximide was used to arrest de

novo reporter synthesis and to assess how long the reporter enzyme remains inhibited after inhibitor washout (not shown). The inhibition persisted throughout the entire length of the experiment (5 hr), which is a longer period than the half-life of the enzyme in cells. This period is sufficient to allow clavulanic acid to be used to remove/3-1actamase activity that may be present in cells prior to performing an assay.

The lithium salt of clavulanic acid is available from U.S. Pharmacopeia (USP, Maryland).

14 j. Fisher, R. L. Chamas, and J. R. Knowles, Biochemistry 17, 2180 (1978). ~5 U. Imtiaz, E. Billings, J. R. Knox, E. K. Manavathu, S. A. Lerner, and S. Mobashery, J. Am.

Chem. Soc. 115, 4435 (1993).

[15] fl-LACTAMASE AS A REPORTER 237

Experimental Procedure to Reduce Background

One day prior to the assay, CHO cells in which/3-1actamase reporter expression is dependent on the activation status of a G-protein-coupled receptor (GPCR) are plated in six clear-bottom 96-well microtiter plates at 75% confluency in the presence or absence of 300/~M clavulanic acid. After 16 hr, all cells are washed once with clavulanic acid-free medium containing 0.1% bovine serum albumin. Cells are then exposed to various concentrations of agonist for the GPCR. Cells are incubated for 3 hr at 37 ° for reporter gene expression to occur, after which/3-1actamase activity in the cells is determined using CCF2/AM. Figure 6 shows the increase of /3-1actamase reporter activity in the cells with agonist concentration for cells with and without clavulanic acid pretreatment. The curve shapes of the plotted ratio values are very similar, with similar ECs0 concentrations for the agonist. However, background/3-1actamase activity is much lower in the assay using cells that are pretreated with clavulanic acid. The agonist- induced ratio change is about 2.5-fold for untreated cells, whereas clavulanic acid pretreatment gives a ratio change of 7-fold in the assay.

In cases where stimulation of cells occurs over much longer periods (10 hr or longer), background activity may not only be from a preexisting reporter enzyme, but also from a reporter enzyme synthesized de novo in the absence of stimulus. Clavulanic acid pretreatment may only reduce the former and not the latter, and therefore may not be as effective as in

g o

~ 2 =E

(:3

o l

E ' " 0

• untreated

-5 -4 -3 -2 -1 0

[Agonist ] in log (pM)

FIG. 6. Effect of clavulanic acid pretreatment on the dynamic range (fold ratio change) in an assay with background reporter activity. CHO cells expressing the/3-1actamase reporter gene under control of a G-protein-coupled receptor were incubated with agonist for 3 hr with or without prior overnight exposure to 300/xM clavulanic acid. B-Lactamase reporter activity was determined by 1-hr loading with 1/zM CCT2/AM, and population fluorescence intensity ratios were analyzed in a clear-bottom, 96-well plate using a Cytofluor 4000 plate reader. Each data point represents the average of four sample wells and each well contained 105 cells. Error bars represent standard error.

2 3 8 GENE FUSIONS AS REPORTERS OF GENE EXPRESSION [15]

the depicted example (Fig. 6) in curbing total background signal under those conditions.

Modulating the Dynamic Range

Cells that express high concentrations of the/3-1actamase reporter turn blue fluorescent with the loading of substrate, as substrate loading becomes rate limiting. One can use the inhibitor to shift the dynamic range of the cell-based reporter assay to higher reporter concentrations by inhibiting a fraction of the expressed reporter enzyme. In this application, the inhibitor clavulanic acid is added to cells after the gene expression experiment but prior to or concomitant with the addition of CCF2/AM. The inhibitor concentration is adjusted for partial inhibition of the intracellular reporter activity. With this treatment, substrate loading is no longer rate limiting and the assay can be used to monitor changes in reporter expression at high expression levels.

Instrumentat ion

Fluorescence Microscopy

An inverted microscope equipped for epifluorescence using either a xenon or mercury excitation lamp is suitable for use in viewing fluorescence from CCF2 and its fl-lactamase-catalyzed hydrolysis product in cells. For visual inspection of cells, a long-pass filter passing blue and green fluores- cence fight is needed to allow visual inspection to determine whether the cells fluoresce green or blue. For epifluorescence microscopes, a long-pass dichroic mirror is needed to separate excitation and emission light and should be matched to the excitation filter (i.e., maximally reflect the excita- tion light around 405 nm, yet allow good transmission of the emitted light).

A suitable filter set for observing fl-lactamase activity is Excitation filter: 405 _ 10 nm Emission filter: 435 nm long pass Dichroic mirror: 425 nm

Filters for microscopes are available through Chroma Technology Corp. and Omega Optical, Inc., both in Vermont.

As with many fluorescent dyes, care should be taken to avoid photo- bleaching of dye-loaded cells. The fluorescent substrate is particularly sensi- tive to continuous illumination with UV, or any other wavelength of fight suitable to excite the dye, through a high magnification, high numerical aperture objective. In the case of CCF2, intense illumination can cause the acceptor fluorophore to be bleached (destroyed) with loss of fluorescence resonance energy transfer (FRET) and appearance of donor fluorescence.

[151 /~-LACTAMASE AS A REPORTER 239

This effect is progressive and nonreversible with an increase in light expo- sure leading to more acceptor destruction.

Photobleaching is reduced by limiting the exposure of cells to excitation light for brief fluorescence analyses lasting a few seconds at a time. Alterna- tively, the light exposure of the substrate can be reduced by use of a lower magnification objective, which spreads the excitation beam over a larger area. For photography, use of high-sensitivity film, such as 400 ASA or greater, is recommended.

Ratio-Imaging Microscopy

For microscope-based ratiometric analysis, blue and green fluorescence emissions are analyzed separately by filtering emitted light separately through two emission filters, passing either blue or green fluorescence. The ratio of blue fluorescence and green fluorescence intensities allows for numerical analysis of data (color is expressed as a numeric ratio of the intensities at the two wavelengths after background subtraction). Imaging microscopy and analysis can be used to monitor gene expression in single cells in real time. 2

A suitable filter set for ratiometric analysis is Excitation filter: 405 _+ 10 nm Dichroic mirror: 425 nm Emission filter (blue): 460 ___ 25 nm Emission filter (green): 535 + 25 nm

Fluorescence Microtiter Plate Readers

Most fluorescence microtiter plate readers are suitable for measuring/3- lactamase reporter activity with the substrate CCF2. Although plate readers with top reading geometry permit analysis of/3-1actamase reporter expres- sion, live-cell experiments are best performed in black-walled, 96-well mi- crotiter clear-bottom plates with low fluorescence background and the sig- nal read on a bottom-count instrument, as cells usually settle to or grow on the well bottom.

A suitable filter set for plate readers is Excitation filter: 405 + 10 nm (superior to 395 _+ 12.5 nm) Emission filter (blue): 460 _+ 20 nm (or 470 _+ 30 nm) Emission filter (green): 535 + 12.5 nm (or 535 + 15 nm)

Filters for plate readers are available through Chroma Technology Corp., Vermont.

After background subtraction, ratios can be calculated by dividing the 460-nm emission (blue channel) reading by the 535-nm emission (green channel) reading. True background values should be determined for each

240 GENE FUSIONS AS REPORTERS OF GENE EXPRESSION [ 15]

read, as these are highly dependent on instrument-specific factors and on the length of time the lamp in the instrument has been lit.

Flow Cytometers

Flow cytometers permitting UV or violet excitation and collection of two emission wavelengths are suitable for analysis of/3-1actamase expression in cells. Suitable excitation sources include an argon laser delivering UV excitation (351-364 nm), a krypton laser with excitation lines at 407 or 413 nm or multiline violet at 407-415 nm, or a mercury arc lamp with excitation through a filter centered at 405 nm (e.g., 405 nm with a 10-nm bandwidth). UV excitation (351-364 nm) from an argon laser is more than adequate, although for maximum signal over noise, excitation in the violet with a krypton laser is better (we have not compared these results with excitation from a mercury arc lamp). Optical filters and a dichroic beam splitter suitable for use with CCF2 on a flow cytometer are:

Emission filter (blue): 460 +__ 25 nm Emission filter (green): 535 +_ 20 nm Dichroic mirror: 490 nm long pass

Filters are available through Chroma Technology Corp. and Omega Opti- cal, Inc., both in Vermont.

Discussion

Reporter genes are often used as markers of the expression of a particu- lar protein. It is important to realize that in the process of replacing or modifying the structural gene of the protein of study with the reporter gene, many processing events, including splicing of mRNA and secondary protein modifications, are likely to differ from those experienced by the native protein. Conversely, fusion of a protein to the reporter may affect its kinetics with substrates. Barring these complications, which affect all reporter proteins to varying degrees, the ~qactamase reporter permits tracking of gene expression events, including expression of low abundance proteins in individual living ceils. Together with the finding that the reporter can be fused to N and C termini of proteins without loss of function, these properties make it ideally suited for gene-tagging and gene-trapping appli- cations. 3

The current fluorogenic substrate, CCF2, is freely diffusable in the cytoplasm and does not permit spatial localization of the enzyme to specific cytoplasmic locations. However, the simple Michaelis-Menten type enzyme kinetics of this reporter-substrate pair allow quantitative determination of reporter activity and permit comparison of reporter levels across different

[1 51 /~-LACTAMASE AS A REPORTER 241

experiments and biological backgrounds. The dual wavelength readout reduces susceptibility of the reporter assay to variations in cell number, substrate concentration, light intensity, and sample volume.

The/3-1actamase reporter enzyme can be inhibited irreversibly inside living cells, allowing/3-1actamase background activity (i.e., from "leaky" promoters) to be removed before an experiment. Inhibitor treatment may also allow/3-1actamase to be used as a live cell reporter of high protein expression levels, a domain currently occupied by the GFP reporter. This is achieved by partial inhibition of intracellular/3-1actamase activity until it falls within the linear range of the assay.

The /3-1actamase reporter assay is easy to use, with simple reagent incubations and nondestructive optical detection in live cells. This permits facile preparation of reporter cell lines through functional selection of clones by their optical properties and subsequent propagation of selected cells. It facilitates the transition from molecular biology, with the tagging of genes and elucidation of their regulation, to cell biology with its study of intracellular signals, cell differentiation pathways, and proliferation events in living cells.

Acknowledgments

I thank Torn Knapp for performing experiments involving flow cytornetry and Susanne Bayat for performing/3-1aetamase inhibition experiments.