# Microfluorometer assay to measure the expression of β-galactosidase and green fluorescent protein reporter genes in single Drosophila flies

A.C Jung, Marie-Claire Criqui, Sophie Rutschmann, Jules Hoffmann,
Dominique Ferrandon

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A.C Jung, Marie-Claire Criqui, Sophie Rutschmann, Jules Hoffmann, Dominique Ferrandon. Microfluorometer assay to measure the expression of β-galactosidase and green fluorescent protein reporter genes in single Drosophila flies. Biotechniques, 2001, 30 (3), pp.594-601. 10.2144/01303rr04. hal-05255362

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# Microfluorometer Assay to Measure the Expression of β-Galactosidase and Green Fluorescent Protein Reporter Genes in Single *Drosophila* Flies

*BioTechniques* 30:594-601 (March 2001)

A.C. Jung, M.-C. Criqui, S. Rutschmann, J.A. Hoffmann, and D. Ferrandon

Centre National de la Recherche Scientifique, Strasbourg, France

## ABSTRACT

β-galactosidase and green fluorescent protein (GFP) are among the most commonly used reporter genes to monitor gene expression in various organisms including Drosophila melanogaster. Their expression is usually detected in a qualitative way by direct microscopic observations of cells, tissues, or whole animals. To measure in vivo the inducibility of two antimicrobial peptide genes expressed during the Drosophila innate immune response, we have adapted two reporter gene systems based on the β-galactosidase enzymatic activity and GFP. We have designed a 96-well microplate fluorometric assay sensitive enough to quantify the expression of both reporter genes in single flies. The assay has enabled us to process efficiently and rapidly a large number of individual mutant flies generated during an ethylmethane sulfonate saturation mutagenesis of the Drosophila genome. This method may be used in any screen that requires the quantification of reporter gene activity in individual insects.

## INTRODUCTION

The Human Genome Sequencing Project is now well underway and is leading to the identification of scores of previously unknown genes (9,17). One of the major challenges will be to understand the function of these new genes. One approach to solve this problem is to investigate the function of homologous genes in genetically tractable model organisms such as *Caenorhabditis elegans* or *Drosophila melanogaster* (19). Indeed, the genome sequence of these two organisms is now available (1,27). The detailed analysis of several hundred genes in these organisms has already been performed. However, these genes are often analyzed more qualitatively than quantitatively (e.g., genes that control development) (14,20). For instance, novel developmental genes have been identified with an enhancer-trap strategy according to their histochemical expression patterns using the bacterial *LacZ* gene that encodes β-galactosidase as a reporter of gene expression (3,28). It is likely that some genes would be more adequately studied using quantitative methods, for instance, genes involved in regulating growth control (29). In this respect, reporter genes will be especially useful to study easily the expression levels of target genes in different conditions, whether genetic or environmental. Some quantitative techniques to monitor gene expression using β-galactosidase or chloramphenicol acetyltransferase reporter genes have been reported in *Drosophila* (2).

However, these techniques are relatively cumbersome to apply and require the preparation of bulk fly extracts. In this paper, we report a technique based on two reporter genes, β-galactosidase and green fluorescent protein (GFP), that allows the semi-quantitative monitoring of the expression levels of two genes induced during immune response in single *D. melanogaster* flies.

The *Drosophila* innate host defense relies on the synthesis of potent antibacterial or antifungal peptides in response to microbial infection (11,12). Interestingly, flies mutant for the *immune deficiency (imd)* gene synthesize only reduced amounts of Dipticicin and other antibacterial peptides and are sensitive to bacterial infections (15), whereas mutants of the Toll pathway, such as flies mutated in the *Dorsal-related immunity factor (Dif)* gene are unable to express the antifungal peptide Drosomycin and are susceptible to fungal infections (16,23). Our goal was to systematically identify all the genes required for the regulation of the *Drosophila* humoral immune response using ethylmethane sulfonate (EMS) saturation mutagenesis. To screen the mutated *Drosophila* lines, we used transgenic flies expressing the *LacZ* gene encoding β-galactosidase under the control of the *Diptericin* promoter and *GFP* under the control of the *Drosomycin* promoter (both transgenes are carried on the X chromosome). The expression of the reporter genes in these
strains has been shown to mimic that of
the endogenous peptides (10,21). Here,
we describe a powerful assay that
turned out to be invaluable in process-
ing a large number of mutated lines and
in isolating new mutants of the *Dro-
sophila* immune response.

MATERIAL AND METHODS

Fly Strains

Fly cultures and crosses were grown
on standard fly medium at 25°C. y w
DD1 flies carry both P[ w*mC Dipt::
LacZ = pDipt-LacZ] and P[ w*mC
Drom::GFP = pDrom-GFP S65T] re-
porter transgenes (10,21). We used both
y w DD1;DTS91/CyO and y w DD1;cn
bw/cn bw flies as wild-type controls.
No significant difference in the induc-
tion of the transgenes could be ob-
served between these two strains. The
DTS91 chromosome is described in
Reference 20. Mutants in the imd gene
exhibit an impaired activation of the
*Diptericin* gene (15). Mutants in the Dif
gene are characterized by a low in-
ducibility of the *Drosomycin* (23). OrR
is a wild-type reference strain.

Immune Response Induction

Bacterial challenge was performed
by pricking flies with a tungsten needle
previously dipped into a concentrated
culture of *E. coli* 1106 and *Micrococ-
cus luteus* (CIP:A270).

Detection of Reporter Gene
Expression

After septic injury, flies were incu-
bated at 20°C in vials for 36 h before be-
ing frozen in microplates. Alternatively,
in a quicker procedure used during the
screens, flies were directly introduced
after immunization in single wells of a
black 96-well microplate kept on ice
(96-well black Cliniplates™; Labsys-
tems, Helsinki, Finland). (White plates
had also been tested: a stronger fluores-
cent signal was detected, but the back-
ground became unacceptably high). A
3-µL drop of a 1% sucrose solution was
then added in each well to allow the sur-
vival of immunized insects in the mi-
croplate. The plates were covered with

lids for thermowell plates (Costar, Bru-
math, France) and left at 20°C to allow
the flies to develop their immune re-
sponse. Thirty-six hours after induction,
the plates were frozen at -20°C. For
analysis, flies were crushed with a Mul-
tiple Homogenizer Type BA/MH96
(Burkard Scientific, Uxbridge, UK) in
50 µL 10 mM Tris (pH 8.4), 100 mM
NaCl, 1 mM MgCl₂, 10 mM dithiothre-
itol (homogenizing buffer described in
Reference 6). The final volume was ad-
justed to 100 µL with homogenizing
buffer. The plate was then analyzed with
a Fluoroskan Ascent® microfluorometer
type 374 (Labsystems). The plates were
first shaken (time: 30 s; diameter: 25
mm; speed: 120), and the fluorescence
of transgenic GFP-S65T was measured
[excitation filter: 485 nm; emission fil-
ter: 520 nm; moving type: 3; plate accel-
eration: 10; integration time: 20 ms
(varying the integration time between 1
ms and 1 s did not make a significant
difference)]. In initial experiments and
for the screens, a less sensitive emission
filter (510 nm) was used. In this case, the
GFP reporter was not accurately detect-
ed in males. We then obtained better re-
sults using males that carried two copies
of the GFP transgene (unpublished
data). The results were processed with
the Fluoroskan Ascent software (Lab-
systems) and given in arbitrary units for
each single fly. To measure the expres-
sion of the *pDipt-LacZ* transgene, 10 µL
of a 20 mM 4 methyl umbelliferyl-β D
galactopyranoside (MUG) (Sigma, St.
Quentin Fallaviers, France) in dimethyl
sulfoxide (DMSO) were then added in
each well by the machine (dispenser 1;
moving type: 3; plate acceleration: 10;
dispenser speed: 18). MUG is a nonflu-
orescent β-galactosidase substrate that
produces, after enzymatic cleavage,
methyl umbelliferone (MU), a fluores-
cent molecule. The plates were shaken
again after addition of the MUG solution
(time: 30 s; diameter: 25 mm; speed:
120), and the kinetics of appearance of
MU was monitored in each well every
25 s for a total of 15 measures (excita-
tion filter: 355 nm; emission filter: 460
nm; moving type: 3; plate acceleration:
10; integration time: 20 ms). The soft-
ware plotted the results as a graph and
was also able to determine the maxi-
mum slope of the curve, thus providing
a quantitative estimate of β-galactosi-
dase activity in the wells. It usually takes 10-15 min to process a plate. We routinely prepare the next microplate while the machine is reading another plate.

Northern blot analysis was carried out as previously described (23).

## RESULTS AND DISCUSSION

### Detection of the β-galactosidase Reporter Gene Activity

The kinetics of appearance of the

fluorescent cleavage product of the β-galactosidase substrate MUG was monitored to estimate the activity of the *pDipt-LacZ* transgene in single flies (see Materials and Methods) as shown in Figure 1 and Table 1. The fluorescent product accumulates in immune-challenged wild-type females (boxes A1-A6) and males (boxes C1-C6), but not in control noninjured flies (B1-B6 and D1-D6) or in wells with buffer only (E1-E6 and F1-F6). The fluorescence observed in induced insects is usually

stronger in females than in males; this discrepancy can be explained by the fact that females are bigger and have proportionally a more extended fat body than males. To increase the sensitivity of the assay in males, it may be necessary to follow the kinetics of MU accumulation over a longer time period (e.g., 15 min instead of 6 min) (data not shown).

In the course of screening, we could sometimes observe important individual variations between flies of the same genotype. For instance, almost no β-galactosidase activity can be detected in males put in wells C7 or C10 (Table 1). This lack of activity may be due to the premature death of the fly in the well or to a fly that was not perfectly crushed. Therefore, we found that it was usually better to test at least five to six flies per genotype or condition of induction of the immune response to obtain a good sensitivity.

In our assay, we could distinguish between wild-type flies and flies of the mutant *imd* strain that were unable to express the *Diptericin* gene in response to an immune challenge (15). The *imd*-induced females (squares E7-E12, Figure 1 and Table 1) and males (squares G7-G12) showed a dramatically reduced expression of the *pDipt-LacZ* reporter transgene, similar

<figure><img src="image_2.png" /><figcaption>**Figure 1. Detection of the pDipt-LacZ transgene expression in single adult flies.** Immunized and nonimmunized flies were put in single wells of the microplate before processing, as described in Materials and Methods. Each box in the figure corresponds to a well, its number indicated at the top right corner of the box. The curve in each box corresponds to the kinetics of appearance of a fluorescent product generated by β-galactosidase enzymatic activity. The machine gives measurements in arbitrary units in the 0-6000 range (Y-axis). To get a better resolution, especially with males, we usually limited the values of the Y-axis to 600-1200. Note that induced females easily attain this value and often saturate the detector by reaching values over 6000 (e.g., boxes A3 and A6). X-axis: time; the kinetics was stopped after 15 measurements (i.e., around 6 min). Boxes A1-A6; B1-B6: y w *DD1*;cn bw/cn bw females (used as wild-type controls). Boxes C1-C6; D1-D6: y w *DD1*;cn bw/cn bwmales. Boxes E1-E6; F1-F6: only homogenizing buffer was added to these wells. Boxes G1-G6; H1-H6: empty wells. Boxes A7-A12; B7-B12: *Dif* mutant females. Boxes C7-C12; D7-D12: *Dif* mutant males. Boxes E7-E12; F7-F12: *imd* mutant females. Boxes G7-G12; H7-H12: *imd* mutant males.</figcaption></figure>
<table><caption>Table 1. Detection of the *pDipt-LacZ* Transgene Expression in Single Adult Flies</caption><thead><tr><th></th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>Mean value</th></tr></thead><tbody><tr><td>A</td><td>Induced wild-type ♀</td><td>256.2</td><td>356.8</td><td>958.6</td><td>494.5</td><td>217.7</td><td>1055.2</td><td>556.5</td></tr><tr><td>B</td><td>Noninduced wild-type ♀</td><td>60.2</td><td>53.8</td><td>-47.5</td><td>28.1</td><td>-20.0</td><td>30.3</td><td>28.7</td></tr><tr><td>C</td><td>Induced wild-type ♂</td><td>115.6</td><td>197.1</td><td>300.9</td><td>129.6</td><td>111.8</td><td>262.8</td><td>186.2</td></tr><tr><td>D</td><td>Noninduced wild-type ♂</td><td>27.7</td><td>21.8</td><td>18.8</td><td>27.3</td><td>49.3</td><td>-18.2</td><td>24.1</td></tr><tr><td>E</td><td>Buffer only</td><td>-3.5</td><td>-47.9</td><td>-37.1</td><td>-45.6</td><td>9.0</td><td>-6.0</td><td>1.5</td></tr><tr><td>F</td><td>Buffer only</td><td>-4.5</td><td>-9.3</td><td>-9.1</td><td>-12.1</td><td>-14.4</td><td>-13.7</td><td>0</td></tr><tr><td>G</td><td>Empty well</td><td>-1.2</td><td>-0.6</td><td>-0.8</td><td>0.6</td><td>-1.2</td><td>0.5</td><td>0.2</td></tr><tr><td>H</td><td>Empty well</td><td>-9.7</td><td>-4.9</td><td>0.3</td><td>1.1</td><td>-0.5</td><td>0.9</td><td>0.2</td></tr></tbody></table>

<table><thead><tr><th></th><th>7</th><th>8</th><th>9</th><th>10</th><th>11</th><th>12</th><th>Mean Value</th></tr></thead><tbody><tr><td>A</td><td>Induced Dif ♀</td><td>882.9</td><td>447.9</td><td>729.0</td><td>882.2</td><td>170.5</td><td>603.1</td><td>328.5</td></tr><tr><td>B</td><td>Noninduced Dif ♀</td><td>84.1</td><td>35.5</td><td>93.5</td><td>66.5</td><td>20.0</td><td>35.9</td><td>55.9</td></tr><tr><td>C</td><td>Induced Dif ♂</td><td>58.5</td><td>47.3</td><td>136.5</td><td>49.2</td><td>133.4</td><td>133.6</td><td>100.4</td></tr><tr><td>D</td><td>Noninduced Dif ♂</td><td>-8.8</td><td>19.6</td><td>6.2</td><td>14.5</td><td>12.7</td><td>23.6</td><td>12.8</td></tr><tr><td>E</td><td>Induced imd ♀</td><td>287.2</td><td>60.1</td><td>40.1</td><td>23.2</td><td>57.9</td><td>62.1</td><td>40.5</td></tr><tr><td>F</td><td>Noninduced imd ♀</td><td>-22.8</td><td>-34.0</td><td>-30.9</td><td>51.3</td><td>-88.6</td><td>68.3</td><td>19.9</td></tr><tr><td>G</td><td>Induced imd ♂</td><td>-23.1</td><td>60.1</td><td>36.3</td><td>14.7</td><td>30.9</td><td>6.44</td><td>24.7</td></tr><tr><td>H</td><td>Noninduced imd ♂</td><td>308.8</td><td>-84.7</td><td>-44.4</td><td>-11.8</td><td>35.5</td><td>-44.0</td><td>5.9</td></tr></tbody></table>

The level of induced expression of the *pDipt-LacZ* reporter transgene was measured in the same flies as those described in Figure 1. To this end, the program automatically determined the maximum slope of each curve using the three optimal consecutive points in the kinetics. A mean value of the six measures for each type of flies is given. However, these maximum slope values should be used with caution. Indeed, when the curve is irregular [e.g., A7 vs. A8 in Figure 1 (fly debris in the optical path?)] or when the slope is weak (D7–H12; note especially E7 and H7), the values can be misleading. The shaded values were not taken into account for mean calculations. Negative values were counted as 0. The differences observed between induced and noninduced wild-type males and females are statistically significant [Student's t test with *P*>0.01 (females) and *P*>0.001 (males)].

<table><caption>Table 2. Detection of the *pDrom-GFP* Transgene Expression in Single Adult Flies</caption><thead><tr><th></th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>Mean value</th></tr></thead><tbody><tr><td>A</td><td>Induced wild-type ♀</td><td>229.1</td><td>86.7</td><td>46.2</td><td>254.7</td><td>71.6</td><td>171.4</td><td>143.3</td></tr><tr><td>B</td><td>Noninduced wild-type ♀</td><td>28.9</td><td>23.6</td><td>67.0</td><td>4.2</td><td>13.6</td><td>9.1</td><td>24.4</td></tr><tr><td>C</td><td>Induced wild-type ♂</td><td>40.1</td><td>61.6</td><td>37.1</td><td>37.9</td><td>13.6</td><td>80.0</td><td>45.0</td></tr><tr><td>D</td><td>Noninduced wild-type ♂</td><td>9.9</td><td>12.5</td><td>5.5</td><td>1.5</td><td>9.8</td><td>3.7</td><td>7.1</td></tr><tr><td>E</td><td>Buffer only</td><td>-1.6</td><td>-1.1</td><td>-0.8</td><td>-1.2</td><td>-1.3</td><td>-0.9</td><td>-1.0</td></tr><tr><td>F</td><td>Buffer only</td><td>-2.7</td><td>-2.6</td><td>-2.8</td><td>-3.0</td><td>-2.3</td><td>-1.5</td><td>-2.5</td></tr><tr><td>G</td><td>Empty well</td><td>5.1</td><td>6.2</td><td>5.4</td><td>6.5</td><td>5.7</td><td>6.1</td><td>5.8</td></tr><tr><td>H</td><td>Empty well</td><td>5.3</td><td>5.6</td><td>3.4</td><td>4.9</td><td>5.7</td><td>5.3</td><td>5.0</td></tr></tbody></table>

<table><thead><tr><th></th><th>7</th><th>8</th><th>9</th><th>10</th><th>11</th><th>12</th><th>Mean Value</th></tr></thead><tbody><tr><td>A</td><td>Induced Dif ♀</td><td>54.4</td><td>27.7</td><td>125.6</td><td>45.6</td><td>58.7</td><td>48.2</td><td>60.0</td></tr><tr><td>B</td><td>Noninduced Dif ♀</td><td>69.6</td><td>62.9</td><td>28.7</td><td>48.0</td><td>17.8</td><td>29.4</td><td>40.4</td></tr><tr><td>C</td><td>Induced Dif ♂</td><td>16.3</td><td>32.7</td><td>13.5</td><td>18.0</td><td>16.5</td><td>16.5</td><td>19.9</td></tr><tr><td>D</td><td>Noninduced Dif ♂</td><td>-1.7</td><td>5.1</td><td>8.1</td><td>11.8</td><td>4.2</td><td>5.0</td><td>5.4</td></tr><tr><td>E</td><td>Induced imd ♀</td><td>337.1</td><td>199.1</td><td>148.6</td><td>340.0</td><td>312.7</td><td>231.7</td><td>263.5</td></tr><tr><td>F</td><td>Noninduced imd ♀</td><td>30.6</td><td>20.8</td><td>89.7</td><td>20.7</td><td>24.6</td><td>63.1</td><td>42.6</td></tr><tr><td>G</td><td>Induced imd ♂</td><td>103.9</td><td>75.4</td><td>41.3</td><td>66.9</td><td>101.0</td><td>82.1</td><td>78.4</td></tr><tr><td>H</td><td>Noninduced imd ♂</td><td>13.9</td><td>10.0</td><td>6.1</td><td>13.8</td><td>6.3</td><td>3.8</td><td>8.9</td></tr></tbody></table>

The level of induced expression of the *pDrom-GFP* reporter transgene was measured in the same flies as those described in Figure 1. The GFP fluorescence is expressed in arbitrary units. A mean value of the six measures for each type of flies is given.
to that observed in noninduced wild-
type (B1–B6; D1–D6) or noninduced
*imd* flies (F7–F12; H7–H12).

*Dif* mutant display a wild-type ex-
pression of the endogenous *Diptericin*
gene (23). Accordingly, the β-galac-
tosidase activity observed in *Dif*-in-
duced females and males (A7–A12 and
C7–C12) was comparable to that of the
wild-type controls.

Similar results were obtained by
monitoring the expression levels of en-
dogenous *Diptericin* RNA by Northern
blot analysis on total RNA isolated from
bulk extracts of 20 females (Figure 2).
We conclude that the data obtained with
the *pDipt-LacZ* transgene using the mi-
croplate assay correctly reflected the ex-
pression of the *Diptericin* gene.

Detection of GFP Reporter Gene Expression

The fluorescence of GFP was mea-
sured to estimate the activity of the
*pDrom-GFP* transgene in single flies
(see Materials and methods) as shown
in Table 2. Wild-type induced females
are characterized by a GFP fluorescence
value usually sixfold higher than that of
noninduced females. Induced males dis-
play weaker fluorescence values than
induced females, a sex difference in
keeping with the results obtained with
the *pDipt-LacZ* reporter gene (see
above). However, the difference be-
tween induced and noninduced males
was also in the sixfold range. The ob-
served differences were statistically sig-
nificant (Student's *t* test with *P*<0.01).

We were able with that approach to
identify strains mutant for the in-
ducibility of GFP. *Dif* mutants are char-
acterized by a low inducibility of the
expression of the *Drosomycin* gene
(23). The results illustrated in Table 2
show that the GFP values of induced
*Dif* males and females range roughly in
the same interval as noninduced wild-
type and *Dif* flies. On the contrary, *imd*
mutant flies, in which endogenous *Dro-
somycin* inducibility is not impaired
(16), exhibit GFP values comparable to
those obtained in wild-type controls.

As for the *pDipt-LacZ* reporter gene,
an important individual variability was
observed for the expression of the GFP
reporter gene. For example, the fluores-
cence intensity is between 46 and 254

in wild-type induced females (Table 2,
boxes A1–A7). It is therefore advisable
to test a high enough number of flies to
get clear results; we routinely tried to
test five to six females.

After immune challenge, endoge-
nous *Drosomycin* RNA accumulates to
high levels for at least 72 h. The fluores-
cence of the *pDrom-GFP* reporter gene
becomes detectable in our system 12–15
h after induction, but the induced/nonin-
duced signal ratio is low. Better repro-
ducibility of induced/noninduced ratio
was obtained after at least 36 h after in-
duction (best: 72 h). This time point is
still compatible with the detection of the
*pDipt-LacZ* reporter gene activation in
adult flies. The transcriptional level of
*Diptericin* gene peaks 12–24 h after sep-
tic injury, and transcription is still ob-
served after 48 h (23). The activity of β-
galactosidase is still easily detected 36 h
after induction since this enzyme is
rather stable in fly cells. For those rea-
sons, we attained a reasonable compro-
mise between the sensitivity of the two
reporters by testing the flies 36 h after
immune challenge.

As in the case of the *pDipt-LacZ* re-
porter gene, Northern blot analysis con-
firmed that the measurement of the
*pDrom-GFP* transgene expression in
the microplate assay accurately reports
the endogenous *Drosomycin* RNA ex-
pression levels (Figure 2).

CONCLUSIONS

Many large-scale assays have been
described to detect the expression of re-
porter genes in various organisms. The
sequencing of the yeast *Saccharomyces
cerevisiae* genome has spawned many
such assays: for example, the identifi-
cation of markers by random polypep-
tides fusions with GFP (25), the study
of the regulation of gene expression on
a genomic scale by DNA microarrays
(8,26), and large-scale genome analysis
by transposon tagging and gene disrup-
tion (22).

The realization that many of the
components of biological processes,
and the way they interact with each oth-
er, will be conserved between flies and
humans (19) will certainly lead to in-
creased requirements for high-through-
put assays to allow the functional analy-
sis of the *Drosophila* genome that has
become recently available. Here, we
have described a quick microfluorome-
ter assay to detect the activity of two re-
porter transgenes. We have used this as-
say in a large-scale mutagenesis screen
to isolate mutations affecting the in-
ducibility of two antimicrobial genes in
adult flies after immune challenge: *Diptericin* and *Drosomycin*. In this
process, about 8000 homozygous viable
EMS mutant lines have been generated
and screened, allowing the identifica-

<figure><img src="image_3.png" /><figcaption>**Figure 2.** Expression of endogenous *Diptericin* and *Drosomycin* in wild-type and mutant backgrounds. A Northern blot was probed successively with *Diptericin*, *Drosomycin*, and *Ribosomal Protein 49 (RP49)* probes. *RP49* RNA levels serve as loading controls. The time of incubation after infection is shown on top, and the phenotypes of the immunized flies are indicated at the bottom. *Or*<sup>R</sup> was used as a wild-type control strain in this experiment.</figcaption></figure>
tion of about 20 new mutations that represent several novel genes that control the humoral immune response (unpublished data) (23,24). We expect that this microplate assay can be adapted to detect a variety of mutant phenotypes or to screen different chemicals on insects. Indeed, transgenesis has been achieved in several insect orders (4,5,7,13,18), and it is expected that insects of economical or biomedical interest such as agricultural pests or disease vectors will soon be amenable to genetic transformation, thus opening new applications for this microplate assay.

## ACKNOWLEDGMENTS

The expert technical help of Martine Schneider and Raymonde Syllas is gratefully acknowledged. We are indebted to Jean-Luc Imler for critical reading of the manuscript. We thank our colleagues for stimulating discussions. This work has been supported by institutional funds from the CNRS, grants from the French Ministère de l'Éducation Nationale, de la Recherche et de la Technologie (Programme de Recherche Fondamentale en Microbiologie, Maladies Infectieuses et Parasitaires). D.F. wishes to acknowledge the support of the Fondation pour la Recherche Médicale (aide à l'implantation de nouvelles équipes).

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Received 29 March 2000; accepted 22 September 2000.

Address correspondence to:

Dr. Dominique Ferrandon
Institut de Biologie Moléculaire et Cellulaire
UPR 9022 du Centre
National de la Recherche Scientifique
15 Rue René Descartes
F. 67084 Strasbourg Cedex, France
e-mail: d.ferrandon@ibmc.u-strasbg.fr