Protocol

Spatiotemporal Gene Expression Targeting with the TARGET and Gene-Switch Systems in Drosophila

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Science's STKE  17 Feb 2004:
Vol. 2004, Issue 220, pp. pl6
DOI: 10.1126/stke.2202004pl6

Abstract

Targeted gene expression has become a standard technique for the study of biological questions in Drosophila. Until recently, transgene expression could be targeted in the dimension of either time or space, but not both. Several new systems have recently been developed to direct transgene expression simultaneously in both time and space. We describe here two such systems that we developed in our laboratory. The first system provides a general method for temporal and regional gene expression targeting (TARGET) with the conventional GAL4-upstream activator sequence (UAS) system and a temperature-sensitive GAL80 molecule, which represses GAL4 transcriptional activity at permissive temperatures. The second system, termed Gene-Switch, is based on a GAL4-progesterone receptor chimera that is hormone-inducible. We have used both systems for simultaneous spatial and temporal rescue of memory dysfunction in the rutabaga (rut) memory mutant of Drosophila. In this protocol, we provide guidelines for the use of these two novel systems, which should have general utility in studying Drosophila biology and in using the fly as a model for human disease.

Introduction

Targeted gene expression is a powerful approach to understanding and regulating the function of specific genes, cells, and tissues in the intact organism. The concept of targeted gene expression implies the ability to direct the expression of a transgene in a spatially or temporally restricted fashion in the living organism, and to analyze the consequences of this directed gene expression. A broad array of experimental questions can be addressed with this approach. These questions range from classic genetic rescue experiments, which provide the strongest proof of the relationship between a particular gene and a phenotype, to newer RNA interference experiments, which provide for loss of function of a particular gene product, to the expression of cellular toxins, which facilitates analysis of the role of particular cells or whole tissues in the normal function of the organism.

Until recently, it was necessary to choose between having temporal control or spatial control over the expression of a transgene in the fruit fly Drosophila melanogaster. Two methods have predominantly been used to achieve these ends (Fig. 1). One approach utilizes the hsp70 heat shock promoter which, when cloned upstream of a gene of interest, can be combined with heat shock to control the temporal pattern in which that gene is expressed (1) (Fig. 1A). The advantage of this method lies in the ability to control the timing of gene expression with a simple temperature shift. However, a limitation of this system is that expression of the transgene is induced ubiquitously, which may lead to pleiotropic effects that complicate the analysis.

Fig. 1.

The heat shock promoter, GAL4-UAS, Gene-Switch, and TARGET systems. (A) The heat shock promoter system. In this system, a copy of your favorite gene (YFG) is cloned downstream of the hsp70 heat shock promoter. The expression of YFG is induced ubiquitously by exposure of the fly to a heat shock regimen. (B) The GAL4-UAS System. In the conventional GAL4-UAS system, the yeast transcriptional activator GAL4 is driven in a specific spatial pattern by either a defined promoter or an endogenous enhancer. The GAL4 protein, in turn, binds to its cognate UAS binding site and constitutively activates the transcription of YFG cloned downstream of the UAS. (C) Ligand-inducible GAL4 chimeras. In these systems, the DNA-binding domain of the GAL4 protein is fused either to the p65 activation domain and a mutant progesterone receptor ligand binding domain (Gene-Switch, shown here) or to the ER to generate the ligand-inducible chimeric activators. In the absence of hormone, the Gene-Switch is in the "off" state. In the presence of hormone, the Gene-Switch molecule changes to an active conformation in which it can bind to a UAS sequence and activate transcription of YFG. (D) The TARGET System. In the TARGET system, the conventional GAL4-UAS system is conditionally regulated by a temperature-sensitive allele of GAL80. At 19°C, transcription of YFG is repressed, whereas this repression is relieved by a temperature shift to 30°C, leading to high levels of expression of YFG in a specific tissue. Both the Gene-Switch and TARGET systems provide spatial and temporal control over gene expression. This figure is modified from (12) and reproduced with permission.

The second frequently used approach is based on the use of the GAL4-upstream activator sequence (UAS) system adapted from yeast (2) (Fig. 1B). [Note: Researchers who are not familiar with Drosophila nomenclature may obtain information at the Bloomington Stock Center website (http://flybase.bio.indiana.edu/docs/nomenclature/lk/nomenclature/html). Briefly, genes and transposon/transgene constructs are indicated in italics, proteins are indicated in roman letters, P indicates a P element transposon, and {} indicates a transposon construct. Yeast genes that have been introduced into Drosophila retain their names by yeast convention.] This system utilizes a cell-type specific cloned promoter or endogenous enhancer to direct the expression of the yeast transcriptional activator GAL4 in a spatially restricted fashion. The GAL4 transactivator then drives (thus is sometimes called the "GAL4 driver") the expression of any gene of interest that has been cloned downstream of a UAS binding site. The advantage of this system is that the transcriptional activator ("driver") and the UAS-based transgene ("target") are carried in different parental lines, thus ensuring their viability and enabling a combinatorial approach with different driver and target lines to the biological question of interest. Once generated, a line expressing GAL4 in a given spatial pattern can be crossed with any UAS target line, allowing the GAL4 line to be used as a general resource. Similarly, when a given UAS target line is generated, the target gene can be transcribed anywhere in the fly by crossing it to the appropriate GAL4 line.

One limitation of the GAL4-UAS system is the lack of experimenter-defined temporal control over the expression of the target transgene. Recently, several newer alternative gene expression systems were developed that address this issue, including the tetracycline (tet) system, the hormone-inducible chimeric GAL4 activators, and, more recently, the temporal and regional gene expression targeting (TARGET) system.

Our laboratory recently introduced into Drosophila a GAL4-progesterone receptor chimera, known as Gene-Switch (Fig. 1C). In this system, the DNA-binding domain of the GAL4 protein is fused to the p65 activation domain and a mutant progesterone receptor ligand binding domain to generate ligand-stimulated chimeric activators. In the absence of hormone, the Gene-Switch is in the "off" state. In the presence of hormone, the Gene-Switch molecule changes to an active conformation in which it can bind to a UAS sequence and activate transcription of a transgene. We demonstrated the ability of this chimeric molecule to regulate the expression of UAS-based transgenes (3). Osterwalder et al. independently developed this system for use in Drosophila (4). A similar system based on a GAL4-estrogen receptor fusion (GAL4-ER) has also been described (5). These systems provide tight temporal control over gene expression through exposure to the hormones mifepristone (RU486; Gene-Switch) or estrogen (GAL4-ER). In addition, they allow spatial restriction of gene expression using a cloned promoter or endogenous enhancer just like the conventional GAL4 system, with the benefit of being able to capitalize on the existing array of UAS lines. A large-scale effort to generate new driver lines expressing the Gene-Switch activator in unique patterns is underway in our laboratory.

A recent survey of FlyBase (http://flybase.bio.indiana.edu/) found 1095 characterized GAL4 lines and 238 stocks bearing UAS transgenes. An additional 2,300 UAS lines exist in the Rorth EP collection (6, 7), and 613 UAS lines exist in the Gene Search collection (8). A useful reagent would allow one both to capitalize on this existing universe of well-characterized GAL4 and UAS transgenic lines and to add temporal control of target gene expression. We have recently developed such a system based on a temperature-sensitive GAL80 protein.

In yeast, transcriptional activation by GAL4 in the absence of galactose is repressed by GAL80. In the presence of galactose, the GAL80-mediated repression of GAL4 activity is relieved, allowing GAL4 to activate genes required for galactose metabolism [reviewed in (9)]. Recently, wild-type GAL80 was shown to repress GAL4 activation of UAS-based transgenes in Drosophila (10). Matsumoto and colleagues previously reported a temperature-sensitive phenotype for yeast growth on galactose (11). At 25°C these yeast failed to grow on galactose as a sole carbon source, whereas at 35°C they demonstrated robust growth. To determine whether this phenotype depended on a temperature-sensitive mutation in GAL80, we cloned and characterized the GAL80 locus from one of these yeast strains. We demonstrated that this GAL80ts molecule conferred temperature-sensitive regulation of GAL4 in a heterologous yeast strain lacking GAL80. We subsequently introduced this GAL80ts molecule into Drosophila and showed that it can regulate GAL4 activity in a temperature-dependent manner in this context. This approach provides a general method for achieving combined temporal and regional gene expression targeting (that is, TARGET) with the conventional GAL4-UAS system in Drosophila (12) (Fig. 1D).

We have used both the TARGET and Gene-Switch systems in Drosophila to achieve spatiotemporal rescue of rutabaga (rut) memory defect mutants, which lack expression of a Ca2+-calmodulin-dependent adenylyl cyclase (12, 13). These studies have allowed us to differentiate an acute role for adenylyl cyclase in learning and memory from a developmental role in the proper patterning of a specific region of the fly’s brain. In a more general sense, these two techniques will enable researchers to approach a wide range of questions that require the ability to regulate Drosophila transgene expression in both time and space. Our goal with this protocol is to provide general guidelines for the use of the TARGET and Gene-Switch systems at different developmental stages and in different tissues of the fly, so that both systems are used to their fullest capabilities to further our understanding of the biology and genetics of this organism, and as a model for human disease.

Materials

Kimwipes, 11.4 × 21.3 cm

Fly culture bottles, 6-ounce (Applied Scientific, AS-355), or vials (Applied Scientific, AS-516)

Fly Strains

P247 line (available from several laboratories, including the authors’)

c772 line (available from several laboratories, including the authors’)

P{MB-Switch}12-1 line, in which Gene-Switch may be selectively activated in the mushroom bodies (available from the authors)

7016 line, a GAL80ts transgenic line with the insertion on the X chromosome [Bloomington Stock Center at Indiana University, Bloomington, IN 47405, USA (http://fly.bio.indiana.edu)]

7017 and 7018 lines, GAL80ts transgenic lines with the insertions on chromosome 3 [Bloomington Stock Center at Indiana University, Bloomington, IN 47405, USA (http://fly.bio.indiana.edu)]

7019 and 7108 lines, GAL80ts transgenic lines with the insertions on chromosome 2 [Bloomington Stock Center at Indiana University, Bloomington, IN 47405, USA (http://fly.bio.indiana.edu)]

Vectors

Various vectors for cloning enhancers or promoters upstream of Gene-Switch (14). [Drosophila Genomics Resource Center]

Chemicals

Sucrose

80% Ethanol

Drosophila food

Food coloring

Mifepristone (RU486) (Sigma-Aldrich, M8046; store at 4°C)

Equipment

Incubator at 18°C

Incubator at 25°C and 60% humidity

Incubator or water bath at 30 to 32°C

Recipes

Recipe 1: RU486 Stock Solution
Dissolve 0.13 g of RU486 in 32 ml of 80% ethanol to make a 10 mM stock solution. This solution can be stored at 4°C for a few months.
Recipe 2: 2% (w/v) Sucrose
Dissolve 2 g of sucrose in 100 ml of ddH2O immediately before use.
Recipe 3: RU486-Sucrose
RU486 Stock Solution (Recipe 1)1 ml
2% (w/v) Sucrose (Recipe 2)19 ml
Food coloringAs desired
This ratio (1:19) makes a 500-μM RU486 solution. Place the 2% Sucrose (Recipe 2) into a small, clean beaker. Add the RU486 Stock Solution (Recipe 1) dropwise while stirring. Add a few drops of food coloring. Make just before use.
Note: Food coloring is added to monitor the uptake of the solution into the digestive system.
Note: Generally, 20 ml of solution is prepared, and 2 ml is distributed to each vial or bottle, with 10 flies fed per vial and 50 per bottle.
Recipe 4: RU486-Containing Food
RU486 Stock Solution (Recipe 1)20 ml
Molten Drosophila food980 ml
This ratio (1:49) makes a 200 μM RU486 solution. Add the RU486 Stock Solution (Recipe 1) and stir thoroughly. Pour immediately into food vials or bottles. Food containing RU486 can be stored at 4°C for several weeks.
Note: Researchers who are not familiar with fly culture may obtain information on food formulation at the Bloomington Stock Center site (View popup).

Instructions

Regulating Transgene Expression with the TARGET System

In the TARGET system, the conventional GAL4-UAS system is combined with GAL80ts (12) to provide temporal control over the transcriptional activity of GAL4. The system can be induced at any time during development or adulthood. One to several copies of the P{tubP-GAL80ts} construct can be introduced into a fly carrying a GAL4 driver and a UAS transgene.

This can be accomplished by moving the {tubP-GAL80ts} construct into the same genetic background as the GAL4 driver (or a given UAS transgene) by mating, in which case the two elements will be located on different chromosomes. Alternatively, the elements can be recombined onto the same chromosome with the GAL4 driver (or UAS-based transgene), and carried as a homozygous stock ready to be crossed with any homozygous UAS (or GAL4) line. We have found it efficient to recombine a GAL80ts transposon with those GAL4 elements that are used frequently in the laboratory, selecting the recombinants by single-fly polymerase chain reaction (PCR) for the two transposable elements.

1. Construct flies, by standard genetic crosses, that carry at least a single copy of the {tubP-GAL80ts} construct, a GAL4 driver, and a UAS-based transgene.

2. Rear progeny that contain the GAL80ts, GAL4 driver, and UAS-based transgenes in vials or bottles kept in an incubator at 18 to 19°C to repress GAL4-mediated transcriptional activation of the UAS transgene.

3. Induce the system by exposing the animals to a 30 to 32°C heat pulse, either by placing the vials or bottles containing the flies in an air incubator at elevated temperatures, or by partly submerging vessels containing flies in a water bath held at the appropriate temperature.

Note: The length of the heat pulse depends on the amount of expression required and the length of the time window in which expression is required in order to observe an effect. In most cases, this heat pulse will vary between 3 and 48 hours.

Regulating Transgene Expression with the Gene-Switch System

Fly strains bearing Gene-Switch elements are becoming increasingly available. Many different lines characterized for expression patterns in the adult head, with Gene-Switch regulated by endogenous enhancers, have been isolated from genetic screens (3), and such screens are continuing. In addition, fly strains bearing the Gene-Switch element driven by promoters or enhancers for the elav gene (panneural expression), the myosin heavy chain gene (panmuscular expression), glass (photoreceptor), and D-mef2 (mushroom body neurons), have been constructed and used successfully (4, 13, 14). These strains are available from the researchers. In addition, an efficient set of vectors that employ cre-mediated in vitro recombination have been fashioned for cloning entire promoters or defined enhancers upstream from the Gene-Switch coding cassette (14). These vectors are available from the Drosophila Genomics Resource Center (http://dgrc.cgb.indiana.edu).

Feeding adult flies RU486-sucrose

Although the ingestion of pharmacologic reagents by Drosophila adults can be increased by brief starvation before feeding, starvation is unnecessary for adequate induction of Gene-Switch.

1. Place a single Kimwipe tissue in an empty fly culture bottle or vial and wet it with 2 ml of either RU486-Sucrose (Recipe 3) or 2% Sucrose (Recipe 2) as a control.

2. Add 10 healthy adult flies to each vial or 50 to each bottle.

Note: For olfactory conditioning, use flies that are 1-5 days of age.

3. Place flies in an incubator at 25°C and 60% humidity for an appropriate amount of time.

4. 2 hours before olfactory classical conditioning, transfer flies to regular food vials to allow them to clean themselves.

Note: Two days of incubation was used to rescue the rut mutant behaviorally (13), although shorter periods of incubation are probably adequate for many studies. If a feeding duration of more than 2 days is required, the flies should be transferred to freshly prepared vials or bottles every 2 days.

Feeding adult flies or larvae on RU486-food

Both adult flies and larvae can be fed with food that contains RU486. Moreover, in contrast to flies fed on sucrose, flies can be fed RU486 in standard fly food for periods longer than 7 days, during which Gene-Switch will be induced.

1. Place 10 or 50 adult flies on RU486-Containing Food (Recipe 4) in a vial or bottle, respectively.

2. Incubate bottles or vials at 25°C and 60% humidity.

3. For larvae, clear the parents from the cultures within a week of seeding the vials or bottles.

4. Select larvae, pupae, or adult flies from the cultures as needed.

Note: Whereas the above steps describe inducing the Gene-Switch system during larval development or adulthood by feeding larvae or adults on food containing RU486 ( 3 , 4 , 13 , 14 ), the same effect can also be achieved by immersing larvae or adults in RU486 solution ( 3 , 4 , 14 ). Induction during embryonic development may be achieved by feeding mothers with RU486 before egg laying ( 4 ), or by directly treating embryos with RU486 by injection or by immersion ( 3 ).

Troubleshooting

TARGET System

The respective levels of expression of GAL4 and GAL80ts in a particular cell determine the effectiveness of UAS repression. Lines in which GAL4 is expressed at very high levels may require two or more copies of the GAL80ts construct to achieve adequate repression of GAL4-mediated UAS transgene expression. The level of UAS-based transgene expression can often be monitored by assays for protein or RNA expression by such methods as Western blotting or quantitative reverse transcription (RT)-PCR. Ideally, the expression level should be minimal or undetectable after low-temperature incubation.

Gene-Switch System

Using the strong myosin heavy chain promoter to drive expression of Gene-Switch in muscle, Osterwalder and colleagues observed reporter gene expression in late third instar larval muscle in the absence of RU486 (4). This stage coincides with a large surge in the level of the flies’ major steroid hormone, 20-HE. The same phenomenon was not observed with other Gene-Switch lines. The most reasonable explanation for this observation is that a muscle metabolite of 20-HE may be involved in activating Gene-Switch. Nevertheless, this exception points out that there are instances in which Gene-Switch will be active at restricted times or places in the absence of exogenous inducer.

Related Techniques

A system similar to Gene-Switch has been developed using an GAL4-ER chimera (5). In principle, this system should function identically to the Gene-Switch system except that estrogen is used as the inducing agent instead of RU486. Another approach to achieving spatial and temporal control of transgene activation is based on the tetracycline repressor from bacteria, which can be regulated by the presence or absence of tetracycline derivatives (15). The tet repressor has been fused with the VP-16 activation domain to create a transcriptional activator that specifically activates gene expression from the tet operator (16). The original tetracycline transactivator is constitutively active and is turned off in the presence of tetracycline or doxycycline (tet-off), whereas a newer version, known as the reverse tetracycline transactivator, is activated in the presence of tetracycline (tet-on) (17).

Notes and Remarks

Although GAL80ts strongly represses endogenous GAL4 activity in yeast at 25°C, in Drosophila we have observed some leakiness of repression at 25°C when using transgenes that provide for a sensitive assay of leaky expression (toxin genes). Stronger repression is observed at 19°C.

Examples of Transgene Induction During Development and Adulthood

Transgene induction can be achieved with either the TARGET or Gene-Switch systems at any time during development or adulthood. Therefore, these techniques can be used to study many different biological processes. The following examples illustrate some applications of the TARGET system. Figure 2 shows induction of a UAS-green fluorescent protein (GFP) reporter in embryos exposed to a brief heat pulse of 30°C. Uninduced embryos exhibit no fluorescence (Fig. 2A and Fig. 2B), whereas heat-shocked embryos exhibit fluorescence that is detectable even through the eggshell (Fig. 2C and Fig. 2D). Figure 3 demonstrates the result of induction of the TARGET system driving UAS-hid expression in the eye at different stages of larval and pupal development. Increasing amounts of eye tissue are lost with induction of the cell death gene hid at earlier times of development. Figure 4 demonstrates the result on olfactory memory (12, 13) of inducing a constitutively active heterotrimeric GTP-binding protein α subunit that can stimulate adenylyl cyclases (Gαs) in the mushroom bodies of the adult fly. Induction of wild-type Gαs has no effect on learning, whereas induction of the constitutively active form reduces memory by about half.

Fig. 2.

GAL80ts regulates GAL4 in a temperature-dependent fashion during embryonic development. (A) Bright field and (B) fluorescent images of embryos of the genotype elav-GAL4, UAS-GFP/+; tubP-GAL80ts/+ cultured at 19°C. (C) Bright field and (D) and fluorescent images of embryos of the same genotype cultured at 30°C.

Fig. 3.

Range of phenotypes produced as a function of timing of UAS-hid expression with the TARGET system. Flies were raised at 19°C and treated with different regimens of heat shock to stimulate expression of the proapoptotic gene head involution defect (hid) under control of the glass multiple repeat (gmr)-GAL4 driver. (A) Complete ablation of the eye in flies of the genotype GMR-GAL4/UAS-hid; GAL80ts/+ when the flies are shifted to 25°C at the second larval instar stage and maintained until adulthood. (B) Partial ablation of the eye when flies of the genotype GMR-GAL4/UAS-hid; GAL80ts/+ are exposed to 12 hours of heat shock at 30°C during late third larval instar stage through early puparium formation. (C) Normal size and shape of eye, with loss of pigment cells, when flies are heat shocked for 1 hour at 37°C during mid-pupal stage. (D) Normal appearance of eye in the absence of heat shock.

Fig. 4.

Spatiotemporal disruption of short-term olfactory memory. The two genotypes tested are GAL4c772;GAL80ts/UAS-Gαs, carrying a wild-type Gαs transgene, and GAL4c772;GAL80ts/UAS-Gαs*, carrying a constitutively active Gαs transgene. (A) In the absence of heat shock, flies of both genotypes show normal and equivalent levels of performance in an immediate memory assay. (B) Flies given a 12 hour heat shock at 32°C and tested 24 hour later. Flies of the GAL4c772;GAL80ts/UAS-Gαs genotype show normal levels of memory performance, whereas the performance of the GAL4c772;GAL80ts/UAS-Gαs* flies is significantly impaired.

Kinetics of Induction and Repression

We have determined the on-rate kinetics of the TARGET system by measuring the accumulation of messenger ribonucleic acid (mRNA) message for an UAS-GFP reporter driven by the panneuronal GAL4 driver c155 (12). We observed mRNA by RT-PCR as early as 30 min after initiation of a heat shock regimen of 32°C, with half-maximal levels achieved at 3 hours and steady state levels at 6 hours. We have also examined the off-rate kinetics by measuring mRNA levels of GFP by RT-PCR in flies maintained at 19°C for different lengths of time after a 6-hour heat shock at 32°C. Under these conditions, mRNA levels fell to half-maximal by about 15 hours, and by 36 hours mRNA levels were at baseline.

We have also utilized β-galactosidase (β-gal, using UAS-lacZ) as a reporter for determining the on- and off-kinetics at the protein level. β-gal is a large and relatively stable protein, and therefore provides a good reporter for the slow side of two important time windows: (i) the kinetics of transcriptional induction and protein synthesis required to detect a functional protein, and (ii) the kinetics for transcriptional repression and clearance of the protein through turnover. Figure 5A illustrates the expression of β-gal in the peduncle of adult mushroom bodies driven by the very strong GAL4 line c739 (12) and controlled temporally by a TARGET element GAL80ts. Flies exposed to elevated temperature (32°C) for 6 hours or longer produced detectable β-gal activity in their peduncles when assayed immediately after the heat pulse, whereas exposure for 3 hours did not result in detectable β-gal activity. Increasing β-gal activity is observed if the flies are kept at 32°C for periods longer than 6 hours. Figure 5B shows the β-gal activity in the mushroom bodies of TARGET flies that were returned to 18°C and assayed 24 hours after the beginning of the 3- or 6-hour incubation at 32°C. The activity of β-gal in this case is detectable even after the 3-hour induction. Thus, a 3-hour heat pulse is sufficient to derepress this very strong GAL4 driver. One GAL80ts element is sufficient to repress the GAL4 activity in the brain when flies are held at 18°C, although there is weak expression of the reporter in some loosely organized non-neuronal tissue (Fig. 5C).

Fig. 5.

TARGET kinetics of reporter expression. Flies carrying the mushroom body GAL4 driver c739, tubP-GAL80ts, and UAS-lacZ were cultured at 18°C and subjected to temperature shifts to 32°C for various periods of time as indicated. Frontal frozen sections were stained for β-gal activity. Photographs of frontal sections of two different flies for each condition taken at the level of the paired ocelli are shown. Arrows indicate the position of one peduncle of the mushroom bodies. (A) Increasing levels of β-gal activity in the peduncles occur with increasing periods of incubation at 32°C; activity is detectable at the 6, 12, and 24 hour time points. (B) A 3-hour incubation at 32°C is sufficient to derepress the c739 GAL4 activity if flies are incubated for additional time after heat shock at 18°C. (C) No β-gal activity is detectable in the brain with incubation only at 18°C.

It should be cautioned, however, that although these test experiments may provide a general guide to the on- and off-kinetics of the TARGET system, the specific kinetics in any experiment will vary depending on the strength of the particular GAL4 driver used, the particular UAS-transgene used, the stability of the expressed mRNA, the specific kinetics of protein translation and posttranslational modifications, and protein stability. In addition, the various GAL80ts transgenes may vary in their ability to repress GAL4 because of differing levels of expression secondary to position effects. Thus, several different GAL80ts transgenes should be employed with any particular GAL4 to find the optimum repression of GAL4 and the maximum amount of induction.

The β-gal expression controlled by Gene-Switch is rapid. β-gal expression in adult mushroom bodies is visible as early as 1.5 hours after feeding on RU486-containing sucrose solution in UAS-lacZ-P{MB-Switch}12-1 flies (13). However, the β-gal activity is more robust with longer feeding periods, and 24 to 48 hours are required for maximal levels as judged by histochemistry. In parallel, memory performance of flies with P{MB-Switch}12-1 and UAS-rut in rut mutant background showed that memory performance improved with longer durations of feeding, reaching wild-type levels only with a 24 to 48 hour feeding. Flies that are fed RU486 for 1 hour and then kept on regular food for 23 hour before histochemical assay displayed β-gal activity equivalent to that of those fed RU486 for 24 hours, suggesting that the uptake of RU486 reached saturating levels within the first hour of feeding (3). However, prolonged feeding for 1-2 days does not produce any detectable negative effects on viability and several different behaviors, including phototaxis, geotaxis, locomotion, escape responses, and memory performance after olfactory conditioning (3, 13). The decay of reporter activity appears to be slow: β-gal activity was detectable 6 days after withdrawal of RU486, although it remains unclear whether this is due to the stability of β-gal, slow metabolism of RU486, or other reasons (3).

Rescue and Misexpression Experiments

Both the TARGET and Gene-Switch systems have been used to rescue a genetic defect in time and space (12, 13). For the TARGET system, a 12-hour incubation of rut mutant flies carrying a mushroom body GAL4 driver, GAL80ts, and UAS-rut is sufficient to significantly rescue the memory deficit (12). Shorter times of incubation at elevated temperatures were not systematically studied. For Gene-Switch, feeding RU486 for 24 hours to rut mutant flies carrying the mushroom body-specific Gene-Switch element 12-1 and UAS-rut is sufficient to rescue the memory deficit (13). These experiments demonstrate that the specific expression of the rut-encoded type I adenylyl cyclase in the mushroom bodies of adult flies is both necessary and sufficient to rescue the rut memory defect.

The two systems have also been used to misexpress transgenes. The TARGET system has been used to drive the expression of the UAS-hid cell death gene at different times in the developing eye (Fig. 3). Similarly, the Gene-Switch system has been used to drive the expression of diphtheria toxin A chain in the fat bodies to achieve ablation of this tissue (3), and both UAS-hid and UAS-ricin genes have been used with Gene-Switch to disrupt eye development (14).

In summary, the data described here and elsewhere illustrate that the TARGET and Gene-Switch systems provide versatile tools for gene rescue and misexpression experiments in both time and space at any stage during development. These tools should prove useful to the general Drosophila community for tackling more complex biological questions that require both spatial and temporal resolution.

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