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Adaptable doxycycline-regulated gene expression systems for Drosophila
Gene 270 (2001) 103±111
www.elsevier.com/locate/gene
Adaptable doxycycline-regulated gene expression systems for Drosophila Michael J. Stebbins a,b, Jerry C.P. Yin a,* a
Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA b Program in Genetics, SUNY at Stony Brook, Stony Brook, NY 11790, USA
Received 11 December 2000; received in revised form 6 March 2001; accepted 19 March 2001 Received by R. Di Lauro
Abstract We have engineered two new versions of the doxycycline (dox) inducible system for use in Drosophila. In the ®rst system, we have used the ubiquitously expressed Drosophila actin5C promoter to express the Tet-Off transactivator (tTA) in all tissue. Induction of a luciferase target transgene begins 6 h after placing the ¯ies on dox-free food. Feeding drug-free food to mothers results in universal target gene expression in their embryos. Larvae raised on regular food also show robust expression of a target reporter gene. In the second version, we have used the Gal4-UAS system to spatially limit expression of the transactivator. Dox withdrawal results in temporally- and spatiallyrestricted, inducible expression of luciferase in the adult head and embryo. Both the actin5C and Gal4-UAS versions produce more than 100fold induction of luciferase in the adult, with virtually no leaky expression in the presence of drug. Reporter gene expression is also undetectable in larvae or embryos from mothers fed dox-containing food. Such tight control may be due to the incorporation of Drosophila insulator elements (SCS and SCS 0 ) into the transgenic vectors. These systems offer a practical, effective alternative to currently available expression systems in the Drosophila research community. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Inducible; Transgenic; Spatial/temporal; Tetracycline; Tet-Off transactivator
1. Introduction With the completion of the genome sequencing projects, two new hurdles will be the assignment of gene function at the cellular and organismal levels. Inducible expression systems are essential tools for approaching these questions and any other ones where reverse genetics can be applied. Historically, induction of wild-type and mutant gene products has been invaluable in assigning gene function to complex phenotypes. In Drosophila, there have been two main systems used to induce genes. The ®rst approach uses the heat-shock 70 gene promoter to control expression of the cDNA of interest (Lindquist, 1986; Petersen and Lindquist, 1988, 1989). Induction is rapid and robust, but there is no spatial limitation on where induction occurs. Also, the stress conditions used to induce expression cause a range of pleiotropic effects that affect biological processes like aging, circadian rhythms, fertility, development, learning, and memory Abbreviations: tTA, Tet-Off transactivator; dox, doxycycline; Gal4UAS, Gal4 Upstream Activating Sequence; TetO, 7 £ , tetracycline operators; b-gal, beta-galactosidase * Corresponding author. Tel.: 11-516-367-8878; fax: 11-516-367-8880. E-mail address: [email protected] (J.C.P. Yin).
formation (Lindquist, 1986; Tully et al., 1994; Yin et al., 1994; Bello et al., 1998; Bieschke et al., 1998). In addition, sustained expression of the transgene can be problematic. Finally, the use of heat-shock precludes studies on heatshock proteins themselves. This last limitation has been partially circumvented by using a metallothionine promoter, but expression with this promoter is limited to a subset of cells in the gut (Otto et al., 1987). The second approach is the bipartite Gal4-UAS system (Brand and Perrimon, 1993), which allows spatial regulation of expression. The ®rst transgene contains the cDNA of interest placed under the control of the binding sites (UAS sequences) for the yeast Gal4 transcription activator. The second transgene contains the Gal4 coding region under the control of a spatially-delimited enhancer. When the two transgenes are brought together, Gal4 is made in a spatially-determined pattern, depending upon where and when the enhancer is active, and activates the UAScontrolled target gene. This system is very tightly regulated, easily adaptable, and a large collection of Gal4 driver lines with a variety of expression patterns is available in the Drosophila research community. However, temporal control of the system is determined by the regulatory sequences used to drive Gal4. Since most promoters and enhancers are expressed at multiple stages of development
0378-1119/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(01)00447-4
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as well as in the adult, interpretation of adult phenotypes can be particularly confounded by the earlier expression of the cDNA of interest. The next step is the development of an adaptable, spatially- and temporally-controlled induction system. One promising approach is to combine the tetracycline-dependent and Gal4-UAS systems (Gossen and Bujard, 1992; Bello et al., 1998). The `Tet-Off' version of the tetracycline system consists of one transgene that expresses a tetracycline-responsive transactivator (tTA) protein, and a second that contains the gene of interest under the control of the binding sites for tTA (Gossen and Bujard, 1992; Furth et al., 1994; Girard et al., 1998). tTA is a hybrid between the Escherichia coli tetracycline repressor protein and the activation domain from the Herpes Simplex Virus VP16 protein. In the absence of tetracycline, tTA binds to a series of tetracycline operator sequences (TetO) placed just upstream of a minimal promoter, allowing activation of transcription of the target gene of interest. When tetracycline or its analog, doxycycline (dox), is delivered into cells, it binds to the tTA protein, causing a conformational change that results in decreased DNA binding and gene inactivation. Therefore induction occurs when the antibiotic is withdrawn. The ®rst use of this system in ¯ies used the enhancer region of the eyeless gene to drive expression of the tTA protein in the developing eye disc (Bello et al., 1998). This report demonstrated that tetracycline could be fed to ¯ies by adding it to normal ¯y food, and that expression could be regulated. However, the approach is not generally useful to the Drosophila research community due to the limitations of the promotor being used to drive tTA expression. In other studies, female speci®c promoters have been used to drive expression of genes that are lethal when over-expressed in larvae (Heinrich and Scott, 2000; Thomas et al., 2000). These systems were designed speci®cally for use in insect population control, but are again not easily adaptable for most other purposes due to the limitations of the sex-speci®c promoters used to drive tTA expression. In this study, we describe two versions of a generally adaptable tetracycline expression system in Drosophila. One version uses the Drosophila actin5C promoter to ubiquitously express the tTA protein. The second version uses currently available Gal4 driver lines to spatially-restrict expression of the tTA protein. Our constructs incorporate the use of the Drosophila insulator (boundary) elements SCS and SCS 0 (Kellum and Schedl, 1991, 1992). We characterize the functional properties of both systems, discuss their uses, and provide a universal vector for target gene expression.
2. Materials and methods 2.1. Transgenes and transgenic ¯ies A mini-white P-element transformation vector (Blocker5)
was used for all transgenic constructs (Kellum and Schedl, 1991, 1992). Four DNA constructs were assembled and subcloned into Blocker 5: (1) the actin5C promoter controlling expression of the tTA gene; (2) the UAS sequences controlling tTA expression; (3) the luciferase target gene under the control of TetO sequences; and (4) a universal vector for placing any cDNA under the control of TetO. The constructs were assembled so that the SCS and SCS 0 elements ¯anked the coding regions or the TetO cassette. Detailed information on plasmid construction is available upon request. 2.2. Doxycycline treatment Dox (Sigma) was added directly to our regular ¯y food as a 10 £ concentrate in phosphate-buffered saline (PBS) pH , 7.2. Solidi®ed ¯y food was heated in a microwave until it lique®ed and allowed to cool down to 508C. The dox concentrate was added, mixed thoroughly, and the food was allowed to re-solidify at room temperature. Since dox oxidizes rapidly in the light, the food is stored in the dark for a maximum of 24 h. To treat ¯ies with dox for longer than 24 h, ¯ies were transferred to fresh dox-containing food once a day. In the luminometer experiments, dox and luciferin concentrates were added to lique®ed agar. 2.3. Luciferase antibodies Western blot analysis was performed using a mouse monoclonal antibody raised against puri®ed Photinus pyralis luciferase protein (Sigma). Puri®ed luciferase protein was injected into mice and monoclonal antibodies were raised using standard techniques. Total antisera or hybridoma supernatant from one clone was used for Western blot analyses. 2.4. Luciferase measurements Total extracts were prepared from 25±50 ¯ies that were quickly frozen in liquid nitrogen and homogenized in RIPA buffer (Harlow and Lane, 1988). Total protein concentrations were determined using the BioRad Protein Assay (according to manufacturer's instructions). Twenty micrograms of total protein was loaded for each lane on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE), and Western blots probed with luciferase antibody (1:50) were performed as described previously (Belvin et al., 1999). As an additional control for Fig. 2C, we probed a second Western blot run in parallel with an antibody speci®c for the Drosophila ADF-1 protein (DeZazzo et al., 2000). Extracts and assays for in vitro luciferase assays were performed as recommended by the manufacturer (Promega, Luciferase Assay kit). Protein concentrations were determined using the BioRad Dc protein assay. In vivo luciferase assays were performed and analyzed as before with a few changes (Belvin et al., 1999). The top agar layer of each well contained 25 mM luciferin and 100 mg/ml dox. Flies
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were stored and tested at room temperature with no speci®c light±dark cycling. 2.5. Embryonic immunocytochemistry All in situ immunocytochemistry of embryos was performed as described (Patel et al., 1989). Mouse antisera (prior to hybridoma fusion) was diluted 1:50 and preadsorbed against 0±18 h-old 2202u embryos (Yin et al., 1994). Embryos were dechorinated, rinsed, ®xed in PEMFA (horse radish peroxidase), and incubated in diluted, preadsorbed antibody overnight at 48C. The secondary antibody was an HRP (100 mM PIPES 1 2 mM EGTA 1 1 mM MgSO4 1 4% formaldehyde)-conjugated goat-anti-mouse IgG and IgM (1:500) (Jackson ImmunoResearch). DABperoxidase (Sigma) or Ni-DAB peroxidase staining was used to visualize immune complexes as described previously (DeZazzo et al., 2000). 2.6. Larval histochemistry Wandering third instar larvae heterozygous for both an Actin5c-tTA and a TetO-lacZ transgene (Bieschke et al., 1998) were rinsed brie¯y in PBST to remove excess food. Larval brains and discs were dissected and ®xed in 4% paraformaldehyde (PBS). Dissected parts were then incubated in X-gal staining solution. After several brief rinses in PBST, discs and brains were dehydrated in an EtOH series, and cleared and mounted in Hemo-De. 2.7. RNA in situ hybridization RNA in situ hybridization of frozen head sections was performed using a digoxygenin-labeled antisense probe to luciferase (Roche, Dig RNA labeling Kit). The probes were diluted in hybridization buffer (Schaeren-Wiemers and Ger®n-Moser, 1993; Skoulakis and Davis, 1996) and stored at 2208C. Frozen tissue sections were prepared and ®xed in 4% paraformaldehyde-PBS for 10 min at room temperature (Skoulakis and Davis, 1996). The slides were washed three times for 5 min each in PBS. Sections were acetylated for 10 min at room temperature (295 ml H2O, 4 ml triethanolamine, 0.525 ml HCl, and.8 ml acetic anhydride) and washed for 5 min each in PBS. Slides were pre-hybridized for 6 h at room temperature in a humidi®ed chamber. Hybridization buffer with DIG-RNA probe at a concentration of 500 ng/ml was added to slides and incubated overnight at 728C. Slides were washed three times for 30 min each in 0.2 £ SSC at 748C. Anti-DIG complexes were detected according to manufacturer instructions. 3. Results 3.1. The actin system in adults Fig. 1 is a schematic picture of the actin system. The actin5C promoter is used to drive expression of the tTA
Fig. 1. (A) The ubiquitously-expressed Tet-Off system. Two separate Pelement constructs are made, one carrying the tTA gene under the control of the actin promotor, and the other carrying a gene of interest under the control of the tetracycline response elements (TetO). The two transgenes are crossed together into a single ¯y, and in the absence of doxycycline (2Dox), the transactivator protein binds to tetracycline operators (TetO), thereby controlling expression of a target gene. The mini-white gene is used to select transgenic lines. Boundary elements (labeled B) were used to control for position effects. (B) A schematic of our TetO-target P-element vector (pJY2000). cDNAs can be easily cloned downstream of the TetO sequences using XbaI or SpeI restriction sites.
gene, and this transgene is crossed into a ¯y containing the TetO-luciferase transgene, producing a double transgenic ¯y. Drug feeding (or withdrawal) represses (allows) induction of the target gene. 3.1.1. Dosage Dox feeding represses reporter expression in adult ¯ies. Transgenic ¯ies carrying two copies of both the actin-tTA and TetO-luciferase transgenes were fed varying concentrations of dox for 48 h. Equal amounts of total extract protein were loaded for Western analysis using a monoclonal antibody directed against luciferase. There is an inverse relationship between the amount of expression and the dosage of antibiotic in the food. Luciferase expression is very robust in the absence of antibiotic (Fig. 2A, lane 4), and undetectable if ¯ies are fed dox at 100 mg/ml (Fig. 2A, lane 1). Partial repression of expression occurs at the intermediate drug levels of 10 and 1 mg/ml, although the response does not appear to be linear. 3.1.2. Repression kinetics Target transgene expression is effectively shut down after 12 h of dox feeding, and chronic feeding can keep the transgene repressed for at least 3 days. Flies carrying two copies of both the actin-tTA and TetO-luciferase transgenes
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were fed dox at a concentration of 100 mg/ml. Western blots of whole ¯y extracts show that expression from the TetOluciferase transgene is repressed down to baseline levels after 12 h (Fig. 2B, lane 4). This baseline level is similar to the level of expression in ¯ies carrying two copies of the TetO-luciferase reporter in the absence of the transactivator (Fig. 2B, lane 2). Thus there is very little leakiness in this system, since expression in the presence of dox is similar to the expression in the absence of the transactivator protein. The data also shows that the half-life of the luciferase protein must be signi®cantly shorter than 12 h. 3.1.3. Induction kinetics The time course of luciferase expression upon dox withdrawal shows that detectable induction occurs within 6 h, but expression continues to increase out to 24 h. Flies were fed 100 mg/ml of dox for 48 h, to insure maximal target gene repression, as in Fig. 2B. The ¯ies were then transferred to food without antibiotic, and Western blot analysis shows a direct relationship between the amount of induction and the time on dox-free food. While induction increases for the ®rst 24 h after withdrawal, there is no further increase if ¯ies are fed drug-free food for 36 or 48 h (data not shown). This data shows that dox must clear from transactivator complexes and ¯y tissue in signi®cantly less than 24 h. The total
amount of luciferase protein in 20 mg of extract is estimated to be in the nanogram range. By extrapolation, we estimate that approximately 1 in 20,000 protein molecules is luciferase (not shown). We expect this number to range greatly depending upon the target protein being analyzed. A quantitative analysis of luciferase induction con®rms the Western blot analysis (Fig. 2D). From the same pool of ¯ies that were used for Western analysis, extracts were prepared in parallel and luciferase activity was measured in vitro. Induced activity increases directly as a function of withdrawal time, and there is greater than a 100-fold increase 24 h after removal from dox. Intermediate induction occurs when ¯ies are removed from dox for 6 or 12 h. Since the luciferase protein has a half-life that is shorter than 12 h, accumulation of protein cannot contribute signi®cantly to the increases that are detected. 3.1.4. Gene dosage Varying the gene dosage of the transactivator gene shows that the amount of transactivator protein is rate-limiting for the level of induced expression. Flies carrying either one or two copies of the transactivator transgene were compared directly for levels of induced expression 24 h after withdrawal of dox. Fig. 2E shows that there is roughly twice as much light production from ¯ies homozygous for the tTA Fig. 2. Analysis of the actin-tTA system in adults. (A) Dose curve of repression. Double transgenic ¯ies were maintained on regular ¯y food without dox (0), or food supplemented with 1, 10 or 100 mg/ml of drug for 48 h. Whole ¯y extracts were analyzed on Western blots using a luciferase monoclonal antibody. There is signi®cant repression when ¯ies are fed at 1 mg/ml, and nearly total repression at 100 mg/ml. (B) Kinetics of repression. Double transgenic ¯ies were transferred from regular food to vials containing food supplemented with 100 mg/ml of dox and collected immediately 0, 12, 24, 36 or 48 h later. Control ¯ies contain only the luciferase target transgene (2), or a transgene where luciferase is expressed under the control of a CREB-responsive promoter (1) (18). There is no detectable signal from the luciferase antibody on Western blots performed on wild type extracts (not shown). Extracts were prepared and analyzed on Western blots. Twelve hours of feeding at 100 mg/ml is suf®cient to repress transgene expression. (C) Induction kinetics upon dox withdrawal. Flies were maintained on regular ¯y food supplemented with 100 mg/ml of dox for 48 h, then transferred to food without drug. They were collected immediately 0, 3, 6, 12 or 24 h later. Whole ¯y extracts were made and analyzed on Western blots. Detectable induction occurs after 3±6 h of feeding, but continues to increase for 24 h. (D) Kinetics of in vitro luciferase activity upon dox withdrawal. In parallel with (C), ¯ies were fed dox for 48 h before being transferred to drug-free food. Extracts were made immediately 0, 3, 6, 12 or 24 h later and in vitro luciferase activity was measured. About ten ¯ies were used for each time point, and the values (with SEM) are shown. There is approximately a 100-fold induction when ¯ies are maintained on drugfree food for 24 h (relative to the t 0 time point). (E) Effect of transactivator gene dosage on luciferase activity. Flies containing one (tTA/CyO) or two (tTA/tTA) copies of the transactivator transgene and two copies of the luciferase transgene were used to measure in vivo luciferase activity using a luminometer. Approximately 20 ¯ies of each genotype were fed luciferin for 24 h before they were transferred to individual wells of a 96-well microtiter dish. Light output was measured directly from individual ¯ies and the SEM is shown. There is about twice as much light output when the transactivator gene dosage is doubled.
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Fig. 3. Analysis of the actin-tTA system in embryos. (A) Genotype of the embryos analyzed. (B) Embryo whose mother was fed dox. A typical embryo from mothers who were maintained on yeast paste supplemented with 1 mg/ml dox for 48 h. The embryo was ®xed and incubated with luciferase polyclonal sera. Detection and staining were completed with Ni-DAB in conjunction with an HRP-conjugated secondary antibody. There is no detectable luciferase expression when mothers are fed dox. (C) Embryo whose mother was not fed dox. A typical embryo from mothers treated in parallel to (B) except with no dox in the yeast paste. There is strong, ubiquitous expression of the transgene when mothers are not fed the drug.
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embryonic staining detected in older embryos is due to zygotic tTA-TetO gene expression. We were not able to detect any adverse effects on embryonic development when mothers were fed dox at 1 mg/ml. These embryos developed normally through larval and pupal stages, and enclosed to give normal sized adults. In contrast, larvae suffer clear effects on developmental morphology and behavior when fed concentrations of 100 mg/ml or greater (data not shown). The larvae generally appear smaller and spend less time burrowing in the food that contains the drug, consistent with previous ®ndings (Bello et al., 1998; Bieschke et al., 1998). However, we were able to fully repress expression of a TetO-LacZ transgene when larvae were raised on 10 mg/ml dox-containing food (Fig. 4), consistent with previous results using the TetOff system with a spatially-delimited promoter (Bello et al., 1998). There were no obvious effects on larvae when they were fed at concentrations of 10 mg/ml, or lower. At this concentration, repression of transgene expression is effective in a variety of tissues including larval brains (Fig. 4D), wing discs (Fig. 4C), leg discs, (Fig. 4B), gut, and salivary glands (data not shown). 3.3. Spatially-restricted induction in adults We combined the well-characterized Gal4-UAS (Brand and Perrimon, 1993) and Tet-Off expression systems (Gossen and Bujard, 1992) in a tripartite system (Fig. 5)
gene relative to ¯ies that carry only one copy of this gene. These measurements were made in living ¯ies using a luminometer, a technique with proven sensitivity and reliability (Belvin et al., 1999). This data indicates that in this range of transactivator expression, the amount of the transactivator protein is still rate-limiting for induction. 3.2. The actin system in embryos and larvae Embryonic reporter expression can be induced strongly if the mothers of the embryos are fed drug-free food. Embryos (6±15 h) were collected from mothers carrying two copies of the actin-tTA and TetO-luciferase transgenes. If the mothers were maintained on food supplemented with 1 mg/ml dox for 48 h prior to embryo collection, there is no detectable antibody staining (Fig. 3B). Apparently the antibiotic consumed by the mother is deposited into the embryo, repressing transgene expression. Very robust and ubiquitous staining occurs if the mothers were fed drug-free food (Fig. 3C). Early embryos (0±3 h) from the double transgenic mothers, embryos from wild-type mothers, or embryos lacking a tTA transgene did not produce any detectable staining (data not shown). These comparisons indicate that the doxcontaining embryos are totally repressed. This result is consistent with the expression data in adults (Fig. 2B, lane 2). The lack of early embryonic staining indicates that
Fig. 4. Analysis of the actin-tTA system in larvae. (A) Genotype of the larvae analyzed. (B±D) Leg discs, wing discs, and brains from wandering third instar larvae raised on dox free food (left) or food containing 10 mg/ml dox (right). Larvae were dissected, ®xed in 4% paraformaldehyde, and stained in X-gal solution. Staining is undetectable in larvae raised on dox food even when staining times are extended overnight. Robust staining was observed in all larval tissues tested when raised on dox-free food.
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Fig. 5. The spatially-restricted tTA system. This tripartite system relies upon three separate transgenes that are crossed into a single ¯y. The ®rst transgene (A) expresses the Gal4 protein under the spatially restricted control of any genomic enhancer element. The second transgene (B) expresses the transactivator (tTA) under the control of Gal4 binding sites (UAS). The ®nal transgene expresses a target gene under the control of tetracycline operators (TetO). Withdrawal of dox from the food results in temporal induction of the target gene. The availability of Gal4 drivers with the desired expression pattern is the only limitation on the adaptability of this system.
to achieve tissue-speci®c control of tTA expression. Gal4UAS transgenes are used to control tTA expression in a spatially-restricted manner. Any of the widely available Gal4 driver lines (transgene A in Fig. 5) is combined with an UAS-tTA transgenic line (B in Fig. 5) to limit tTA to a particular expression pattern. Both of these transgenes are combined with a third transgene (C in Fig. 5) which contains the tetO-target cassette. Again, target gene expression is achieved upon withdrawal of dox from the food. The boundary elements SCS and SCS 0 were used to ¯ank the UAS-tTA and TetO-luciferase transgenes. Using an in vivo assay for luciferase activity, we detect greater than a 100-fold induction that is spatially and temporally regulated (Fig. 6B). Flies homozygous for the UAStTA and TetO-luciferase transgenes were crossed to ¯ies carrying the Gal4 gene under the control of the sevenless gene promoter (sev-Gal4). Flies were fed luciferin for 12 h then loaded into a 96-well microtiter dish containing agar. The drug-fed ¯ies were maintained on agar with 100 mg/ml dox and luciferin for 24 h, while the drug-free ¯ies were maintained on agar and luciferin. Activity measurements were made in the live animal using a luminometer. There is about a 115-fold (219/1.9) difference in activity between drug-free and drug-fed ¯ies that contain all three transgenes. There is no induction if ¯ies are missing the sev-Gal4 driver transgene, and a 1.9-fold increase when the Gal4 driver is present and the ¯ies are maintained on dox-containing food. Similar results were obtained with three other combinations of UAS-tTA and TetO-luciferase lines when they were combined with the sev-Gal4 driver. Therefore, the induction that is seen is not a function of speci®c lines that might
express at particularly high levels, or are particularly easy to induce due to their surrounding chromatin structure. The use of insulators may have contributed to the equivalent induction that we see with different combinations of the UAS-tTA and TetO-luciferase transgenes. The adaptability of this system was tested using other Gal4 drivers. The glass enhancer is known to express exclusively in the adult eye, while the elav enhancer is ubiquitously expressed in the nervous system (Kidd et al., 1998). These drivers produced a range of activation from approximately 30-fold (glassGal4) to 250-fold (elav-Gal4)). Therefore, the induction is also independent of the particular driver that is used and the particular combinations of driver, UAS-tTA, or TetO-luciferase lines. In situ hybridization of adult head sections shows that luciferase expression is spatially-restricted to the correct pattern for the sevenless enhancer. Luciferase mRNA is detected in the adult eye in a driver-dependent and doxdependent manner (Fig. 6C, middle panel). This pattern of in situ expression matches the known pattern of expression of the sev-Gal4 driver. There is undetectable background staining when triple transgenic ¯ies are fed dox for 24 h (Fig. 6C, left panel), or when ¯ies missing the Gal4 driver (-Gal4 driver) are not fed the drug (Fig. 6C, right panel). Since the induction occurs in the adult eye, is driver-dependent, and is dependent upon drug-free food, it is spatially and temporally regulated. 3.4. Spatially-restricted induction in embryos We used an elav-Gal4 driver to spatially limit tTA expression in embryos. The elav regulatory sequence drives expression in the central nervous system of embryos beginning at approximately stage13 eggs. Flies homozygous for UAS-tTA and TetO-luciferase were mated to homozygous elav-Gal4 ¯ies (Fig. 7A), and embryos were stained with an anti-luciferase antibody. We detect strong Gal4 driver-dependent and dox-responsive luciferase staining in embryos. In the presence of the elav-Gal4 driver, there is strong luciferase staining in the central nervous system of embryos from approximately stage 13 on (Fig. 7C) if their mothers were fed regular food. Embryos that do not have the elav-Gal4 driver, but contain the other two transgenes, do not show any staining (Fig. 7D). If the mothers were fed dox at 1 mg/ml for 48 h, there is no detectable staining in their triple transgenic embryos (Fig. 7B). Similar results were obtained when two other combinations of UAS-tTA and TetO-luciferase reporter transgenes were crossed to the elav-Gal4 driver. A second driver that uses the regulatory regions from the scabrous gene was used to con®rm the adaptability of the system (Kidd et al., 1998). The sca-Gal4 driver, in the presence of UAS-tTA and TetO-luciferase transgenes, produces luciferase expression in the early central nervous system, consistent with the known expression pattern of this driver. Dox feeding of the mothers abolishes this staining
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(data not shown). Therefore, in the embryo, as in the adult, induction occurs in the appropriate spatial pattern for the drivers that are used, is driver-dependent and requires a drug-free mother. 4. Discussion We describe two improved inducible systems for Drosophila, one resulting in ubiquitous expression, the other allowing spatially- and temporally-regulated expression. The second version is adaptable to currently available Gal4 driver lines. Combined with the induction properties of our systems, these changes represent major improvements to inducible technology in Drosophila. Four parameters characterize an inducible system: leakiness, the induction ratio, the kinetics of induction, and the pleiotropic effects of the inducer. Our data is consistent with the general consensus from tissue culture that Tet-Off
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systems are less leaky than Tet-On systems. In adults, total repression, or minimal leakiness, can be achieved with 12 h of feeding on drug-free food. If mothers feed for 48 h on drug-free food, their embryos are totally repressed. In both cases, the level of repressed expression is similar to the level of expression seen in the absence of the transactivator gene. Similar results are seen with the spatially-restricted system. In adults there is only a 1.9fold difference between ¯ies with- and without a Gal4 driver. These results suggest that almost all of the transactivator protein is inactive in the presence of drug. In spite of the strong repression, both systems exhibit high induction ratios, exceeding 100-fold in adults. Because of the instability of a luciferin-luciferase complex, luciferase accumulation is unlikely to contribute to an overestimation of the amount of induction. The actin system can be induced with 6±24 h of drug withdrawal. It is likely that the spatially-restricted system will have similar kinetics, since the fold-induction at 24 h is similar in both systems. However, the detailed pharmacokinetics of drug withdrawal may vary in different tissues, and needs to be empirically determined. We would like to stress that the magnitude and kinetics of induced expression for both the Actin5c and Gal4 based systems must be determined empirically for each target transgene. We fully expect that there will be differences based upon transgene insertion site, feeding protocol, the pharmacokinetics of drug delivery to the tissue of interest, and the mRNA and protein stability of the target gene. There are no obvious harmful effects of feeding dox up to 1±2 mg/ml in adult ¯ies, nor does this empirically appear to
Fig. 6. Analysis of the spatially-restricted tTA system in adults. (A) Schematic of the cross used to generate all of the experimental ¯ies. They contained one copy of the eye-restricted Gal4 driver (sev-Gal4), one copy of the transactivator transgene (UAS-tTA), and one copy of the target transgene (TetO-Luc). The control ¯ies, which lacked the driver transgene (-Gal4 driver), were the same genotype as the mothers. (B) In vivo luciferase activity. Flies containing one (UAS-tTA/CyO) or two transgenes (UAStTA/sev-Gal4) on the second chromosome, and one copy of the reporter transgene on the third chromosome were individually placed into the wells of a microtiter dish. They were kept on regular agar with 25 mM luciferin (2Dox) or on agar supplemented (1Dox) with 100 mg/ml of dox and 25 mM luciferin for 24 h. Light production was measured using a luminometer. N , 20 and the SEM is shown. There is signi®cant induction of light output when ¯ies containing all three transgenes are fed drug-free food. Flies without the Gal4 driver had identical activity in the absence or presence of antibiotic. If this value is set to one, there is a 1.9-fold increase when a Gal4 driver is added and the ¯ies are fed the drug (leakiness) and a 219-fold increase when the ¯ies are drug-free. This results in a 115-fold increase in the absence versus presence of dox. (C) In situ hybridization of luciferase expression. Flies containing either two (2Gal4 driver) or three transgenes were maintained on regular food (2Dox) or food supplemented with 100 mg/ml of dox for 48 h before adult head cryostat sections were cut and probed with digoxygenin-labeled RNA probes. All ¯ies contained one copy of the TetO-luciferase target, and the UAS-tTA transgene. The Gal4 driver was a sev-Gal4 line that expresses in the adult eye. Eye-speci®c expression of luciferase is detected in ¯ies that were either not fed dox or lacked a Gal4 driver.
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Fig. 7. Analysis of the spatially-restricted tTA system in embryos. (A) Genotype of the cross used to generate ¯ies with three transgenes. Progeny from this cross contain a single copy of the Gal4 driver (elav-Gal4), the transactivator (UAS-tTA), and the target transgene (TetO-Luc). Flies missing the Gal4 driver (2Gal4 driver) had the mother's genotype. (B) A typical embryo from mother's fed dox (1Dox) at 1 mg/ml in yeast paste for 48 h. (C) A typical embryo from mothers fed regular yeast paste (2Dox). (D) A typical embryo from uncrossed ¯ies (2Gal4 driver) with the mother's genotype. All embryos were ®xed, treated with the luciferase monoclonal antibody, and the immunohistochemical signal was detected using an HRPconjugated secondary antibody in conjunction with DAB. A strong signal is detected in the central nerve cord of stage 15 embryos, consistent with the elav-Gal4 expression pattern.
affect egg laying and embryonic development. However, there are noticeable effects of feeding antibiotic to larvae, even at concentrations as low as 100 mg/ml. However, we were able shut off expression of a TetO-lacZ transgene with 10 mg/ml dox and observed no developmental abnormalities when larvae were fed at this lower concentration. We have heard anecdotally of a number of failures in implementing the tetracycline system in ¯ies using similar component parts to those that we have used here. Even in the reports where the Tet-Off system has been successfully used, its utility and induction capabilities are limited due to the spatial and temporal limitations of the promoters being used to drive tTA expression. We suspect that our use of the SCS and SCS 0 boundary elements has made a signi®cant contribution to the effectiveness of our systems. When
multiple transgenes are involved, the cumulative effects of random insertion sites can be quite signi®cant. By insulating our transgenes from position effect, we may have put tighter control on expression levels and patterns. Thus the use of boundary elements may have increased expression levels from both the tTA and TetO transgenes. In addition, the use of insulators has been shown to cause independent insertions of each transgene to have nearly identical properties (Chung et al., 1993; Felsenfeld et al., 1996). This may decrease the number of combinations of transgenes that need to be screened, and simpli®es the genetics when combining three transgenes into a single ¯y. In spite of the use of insulators, it is still possible that random insertion sites may affect the leakiness and magnitude of induction. Similarly, it is possible, if enough independent lines are screened, that insertions can be found that have no leaky expression, whether the target transgene is insulated or not (Baron and Bujard, 2000). Although we used insulators, the Tet-Off systems does show some leakiness. For applications involving expression of potentially toxic proteins we recommend that users consider an uninsulated vector and screen many independent target lines. For certain biological questions, the Tet-On system may be more practical than Tet-Off. The Tet-On system utilizes a mutant transactivator that binds to TetO sequences only in the presence of dox (Gossen et al., 1995). The currently available Tet-On system in Drosophila uses the Actin5c promotor to achieve ubiquitous expression of the rtTA (Tet-On) transactivator (Bieschke et al., 1998). Overall induction in this system is slow, taking approximately 5 days to achieve 10-fold induction of bgal, rendering it of limited use for most applications. Since bgal activity is very stable, accumulation of lacZ protein probably contributes to an overestimation of the actual induction. Recently, second generation Tet-On transactivators have been published (Urlinger et al., 2000). Experiments to evaluate whether these transactivators signi®cantly improve the currently available system are underway, and the preliminary results are promising (M. Stebbins et al., manuscript in preparation). Our improvements to the tTA expression system should open the door for a wide array of applications in Drosophila. The actin-based tTA system is now a viable alternative to the heat-shock induction system, thereby by-passing possible pleiotropic effects of heat-shock itself. The availability of tTA and heat-shock also allows the combinatorial manipulation of multiple transgenes. The tTA systems may be particularly useful for rescue of developmentally lethal genes, where the rescuing genes are expressed throughout development, or for the study of heat-shock proteins. The spatially-restricted system will facilitate detailed investigations of tissue and temporal requirements for gene function. Until now, this type of experiment has been technically dif®cult to perform due to the ubiquitous nature of the heat-shock inducible system and the temporal restrictions of the Gal4-UAS system. This will be particularly useful for
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studying complex behavioral phenotypes such as learning, memory, courtship and circadian rhythms. Finally, the graded response to dox suggests that it should be possible to sustain intermediate levels of induction simply by controlling drug dosage in the food, providing a controllable ªrheostatº for inducible gene expression in Drosophila. Acknowledgements We thank Jim DeZazzo for ADF-1 antibody, reagents, advice and comments on early versions of the manuscript. We also thank Grisha Enikolopov, Maurice Kernan, Paul Schedl and Sid Strickland for valuable discussions and Hong Zhou for technical assistance. This work was supported by grants from NIH (R01 NS35575), the McKnight Foundation, and start-up funds from Cold Spring Harbor Laboratory (to J.C.P.Y.). References Baron, U., Bujard, H., 2000. Tet repressor-based system for regulated gene expression in eukaryotic cells: principles and advances [in process citation]. Methods Enzymol. 327, 401±421. Bello, B., Resendez-Perez, D., Gehring, W.J., 1998. Spatial and temporal targeting of gene expression in Drosophila by means of a tetracyclinedependent transactivator system. Development 125, 2193±2202. Belvin, M.P., Zhou, H., Yin, J.C., 1999. The Drosophila dCREB2 gene affects the circadian clock. Neuron 22, 777±787. Bieschke, E.T., Wheeler, J.C., Tower, J., 1998. Doxycycline-induced transgene expression during Drosophila development and aging. Mol. Gen. Genet. 258, 571±579. Brand, A.H., Perrimon, N., 1993. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401±415. Chung, J.H., Whiteley, M., Felsenfeld, G., 1993. A 5 0 element of the chicken beta-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila. Cell 74, 505± 514. DeZazzo, J., Sandstrom, D., de Belle, S., Velinzon, K., Smith, P., Grady, L., DelVecchio, M., Ramaswami, M., Tully, T., 2000. Nalyot, a mutation of the Drosophila myb-related Adf1 transcription factor, disrupts synapse formation and olfactory memory. Neuron 27, 145±158. Felsenfeld, G., Boyes, J., Chung, J., Clark, D., Studitsky, V., 1996. Chromatin structure and gene expression. Proc. Natl. Acad. Sci. USA. 93, 9384±9388. Furth, P.A., St Onge, L., Boger, H., Gruss, P., Gossen, M., Kistner, A., Bujard, H., Hennighausen, L., 1994. Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proc. Natl. Acad. Sci. USA 91, 9302±9306. Girard, F., Bello, B., Laemmli, U.K., Gehring, W.J., 1998. In vivo analysis
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of scaffold-associated regions in Drosophila: a synthetic high-af®nity SAR binding protein suppresses position effect variegation. EMBO J. 17, 2079±2085. Gossen, M., Bujard, H., 1992. Tight control of gene expression in mammalian cells by tetracycline- responsive promoters. Proc. Natl. Acad. Sci. USA 89, 5547±5551. Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W., Bujard, H., 1995. Transcriptional activation by tetracyclines in mammalian cells. Science 268, 1766±1769. Harlow, E., Lane, D. (Eds.), 1988. Antibodies, A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Heinrich, J.C., Scott, M.J., 2000. A repressible female-speci®c lethal genetic system for making transgenic insect strains suitable for a sterile-release program. Proc. Natl. Acad. Sci. USA 97, 8229±8232. Kellum, R., Schedl, P., 1991. A position-effect assay for boundaries of higher order chromosomal domains. Cell 64, 941±950. Kellum, R., Schedl, P., 1992. A group of scs elements function as domain boundaries in an enhancer- blocking assay. Mol. Cell Biol. 12, 2424± 2431. Kidd, T., Russell, C., Goodman, C.S., Tear, G., 1998. Dosage-sensitive and complementary functions of roundabout and commissureless control axon crossing of the CNS midline. Neuron 20, 25±33. Lindquist, S., 1986. The heat-shock response. Annu. Rev. Biochem. 55, 1151±1191. Otto, E., Allen, J.M., Young, J.E., Palmiter, R.D., Maroni, G., 1987. A DNA segment controlling metal-regulated expression of the Drosophila melanogaster metallothionein gene Mtn. Mol. Cell Biol. 7, 1710±1715. Patel, N.H., Martin-Blanco, E., Coleman, K.G., Poole, S.J., Ellis, M.C., Kornberg, T.B., Goodman, C.S., 1989. Expression of engrailed proteins in arthropods, annelids, and chordates. Cell 58, 955±968. Petersen, R., Lindquist, S., 1988. The Drosophila hsp70 message is rapidly degraded at normal temperatures and stabilized by heat shock. Gene 72, 161±168. Petersen, R.B., Lindquist, S., 1989. Regulation of HSP70 synthesis by messenger RNA degradation. Cell Regul. 1, 135±149. Schaeren-Wiemers, N., Ger®n-Moser, A., 1993. A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: in situ hybridization using digoxigenin-labelled cRNA probes. Histochemistry 100, 431±440. Skoulakis, E.M., Davis, R.L., 1996. Olfactory learning de®cits in mutants for leonardo, a Drosophila gene encoding a 14-3-3 protein. Neuron 17, 931±944. Thomas, D.D., Donnelly, C.A., Wood, R.J., Alphey, L.S., 2000. Insect population control using a dominant, repressible, lethal genetic system. Science 287, 2474±2476. Tully, T., Preat, T., Boynton, S.C., Del Vecchio, M., 1994. Genetic dissection of consolidated memory in Drosophila. Cell 79, 35±47. Urlinger, S., Baron, U., Thellmann, M., Hasan, M.T., Bujard, H., Hillen, W., 2000. Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc. Natl. Acad. Sci. USA. 97, 7963±7968. Yin, J.C., Wallach, J.S., Del Vecchio, M., Wilder, E.L., Zhou, H., Quinn, W.G., Tully, T., 1994. Induction of a dominant negative CREB transgene speci®cally blocks long-term memory in Drosophila. Cell 79, 49± 58.
www.elsevier.com/locate/gene
Adaptable doxycycline-regulated gene expression systems for Drosophila Michael J. Stebbins a,b, Jerry C.P. Yin a,* a
Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA b Program in Genetics, SUNY at Stony Brook, Stony Brook, NY 11790, USA
Received 11 December 2000; received in revised form 6 March 2001; accepted 19 March 2001 Received by R. Di Lauro
Abstract We have engineered two new versions of the doxycycline (dox) inducible system for use in Drosophila. In the ®rst system, we have used the ubiquitously expressed Drosophila actin5C promoter to express the Tet-Off transactivator (tTA) in all tissue. Induction of a luciferase target transgene begins 6 h after placing the ¯ies on dox-free food. Feeding drug-free food to mothers results in universal target gene expression in their embryos. Larvae raised on regular food also show robust expression of a target reporter gene. In the second version, we have used the Gal4-UAS system to spatially limit expression of the transactivator. Dox withdrawal results in temporally- and spatiallyrestricted, inducible expression of luciferase in the adult head and embryo. Both the actin5C and Gal4-UAS versions produce more than 100fold induction of luciferase in the adult, with virtually no leaky expression in the presence of drug. Reporter gene expression is also undetectable in larvae or embryos from mothers fed dox-containing food. Such tight control may be due to the incorporation of Drosophila insulator elements (SCS and SCS 0 ) into the transgenic vectors. These systems offer a practical, effective alternative to currently available expression systems in the Drosophila research community. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Inducible; Transgenic; Spatial/temporal; Tetracycline; Tet-Off transactivator
1. Introduction With the completion of the genome sequencing projects, two new hurdles will be the assignment of gene function at the cellular and organismal levels. Inducible expression systems are essential tools for approaching these questions and any other ones where reverse genetics can be applied. Historically, induction of wild-type and mutant gene products has been invaluable in assigning gene function to complex phenotypes. In Drosophila, there have been two main systems used to induce genes. The ®rst approach uses the heat-shock 70 gene promoter to control expression of the cDNA of interest (Lindquist, 1986; Petersen and Lindquist, 1988, 1989). Induction is rapid and robust, but there is no spatial limitation on where induction occurs. Also, the stress conditions used to induce expression cause a range of pleiotropic effects that affect biological processes like aging, circadian rhythms, fertility, development, learning, and memory Abbreviations: tTA, Tet-Off transactivator; dox, doxycycline; Gal4UAS, Gal4 Upstream Activating Sequence; TetO, 7 £ , tetracycline operators; b-gal, beta-galactosidase * Corresponding author. Tel.: 11-516-367-8878; fax: 11-516-367-8880. E-mail address: [email protected] (J.C.P. Yin).
formation (Lindquist, 1986; Tully et al., 1994; Yin et al., 1994; Bello et al., 1998; Bieschke et al., 1998). In addition, sustained expression of the transgene can be problematic. Finally, the use of heat-shock precludes studies on heatshock proteins themselves. This last limitation has been partially circumvented by using a metallothionine promoter, but expression with this promoter is limited to a subset of cells in the gut (Otto et al., 1987). The second approach is the bipartite Gal4-UAS system (Brand and Perrimon, 1993), which allows spatial regulation of expression. The ®rst transgene contains the cDNA of interest placed under the control of the binding sites (UAS sequences) for the yeast Gal4 transcription activator. The second transgene contains the Gal4 coding region under the control of a spatially-delimited enhancer. When the two transgenes are brought together, Gal4 is made in a spatially-determined pattern, depending upon where and when the enhancer is active, and activates the UAScontrolled target gene. This system is very tightly regulated, easily adaptable, and a large collection of Gal4 driver lines with a variety of expression patterns is available in the Drosophila research community. However, temporal control of the system is determined by the regulatory sequences used to drive Gal4. Since most promoters and enhancers are expressed at multiple stages of development
0378-1119/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(01)00447-4
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as well as in the adult, interpretation of adult phenotypes can be particularly confounded by the earlier expression of the cDNA of interest. The next step is the development of an adaptable, spatially- and temporally-controlled induction system. One promising approach is to combine the tetracycline-dependent and Gal4-UAS systems (Gossen and Bujard, 1992; Bello et al., 1998). The `Tet-Off' version of the tetracycline system consists of one transgene that expresses a tetracycline-responsive transactivator (tTA) protein, and a second that contains the gene of interest under the control of the binding sites for tTA (Gossen and Bujard, 1992; Furth et al., 1994; Girard et al., 1998). tTA is a hybrid between the Escherichia coli tetracycline repressor protein and the activation domain from the Herpes Simplex Virus VP16 protein. In the absence of tetracycline, tTA binds to a series of tetracycline operator sequences (TetO) placed just upstream of a minimal promoter, allowing activation of transcription of the target gene of interest. When tetracycline or its analog, doxycycline (dox), is delivered into cells, it binds to the tTA protein, causing a conformational change that results in decreased DNA binding and gene inactivation. Therefore induction occurs when the antibiotic is withdrawn. The ®rst use of this system in ¯ies used the enhancer region of the eyeless gene to drive expression of the tTA protein in the developing eye disc (Bello et al., 1998). This report demonstrated that tetracycline could be fed to ¯ies by adding it to normal ¯y food, and that expression could be regulated. However, the approach is not generally useful to the Drosophila research community due to the limitations of the promotor being used to drive tTA expression. In other studies, female speci®c promoters have been used to drive expression of genes that are lethal when over-expressed in larvae (Heinrich and Scott, 2000; Thomas et al., 2000). These systems were designed speci®cally for use in insect population control, but are again not easily adaptable for most other purposes due to the limitations of the sex-speci®c promoters used to drive tTA expression. In this study, we describe two versions of a generally adaptable tetracycline expression system in Drosophila. One version uses the Drosophila actin5C promoter to ubiquitously express the tTA protein. The second version uses currently available Gal4 driver lines to spatially-restrict expression of the tTA protein. Our constructs incorporate the use of the Drosophila insulator (boundary) elements SCS and SCS 0 (Kellum and Schedl, 1991, 1992). We characterize the functional properties of both systems, discuss their uses, and provide a universal vector for target gene expression.
2. Materials and methods 2.1. Transgenes and transgenic ¯ies A mini-white P-element transformation vector (Blocker5)
was used for all transgenic constructs (Kellum and Schedl, 1991, 1992). Four DNA constructs were assembled and subcloned into Blocker 5: (1) the actin5C promoter controlling expression of the tTA gene; (2) the UAS sequences controlling tTA expression; (3) the luciferase target gene under the control of TetO sequences; and (4) a universal vector for placing any cDNA under the control of TetO. The constructs were assembled so that the SCS and SCS 0 elements ¯anked the coding regions or the TetO cassette. Detailed information on plasmid construction is available upon request. 2.2. Doxycycline treatment Dox (Sigma) was added directly to our regular ¯y food as a 10 £ concentrate in phosphate-buffered saline (PBS) pH , 7.2. Solidi®ed ¯y food was heated in a microwave until it lique®ed and allowed to cool down to 508C. The dox concentrate was added, mixed thoroughly, and the food was allowed to re-solidify at room temperature. Since dox oxidizes rapidly in the light, the food is stored in the dark for a maximum of 24 h. To treat ¯ies with dox for longer than 24 h, ¯ies were transferred to fresh dox-containing food once a day. In the luminometer experiments, dox and luciferin concentrates were added to lique®ed agar. 2.3. Luciferase antibodies Western blot analysis was performed using a mouse monoclonal antibody raised against puri®ed Photinus pyralis luciferase protein (Sigma). Puri®ed luciferase protein was injected into mice and monoclonal antibodies were raised using standard techniques. Total antisera or hybridoma supernatant from one clone was used for Western blot analyses. 2.4. Luciferase measurements Total extracts were prepared from 25±50 ¯ies that were quickly frozen in liquid nitrogen and homogenized in RIPA buffer (Harlow and Lane, 1988). Total protein concentrations were determined using the BioRad Protein Assay (according to manufacturer's instructions). Twenty micrograms of total protein was loaded for each lane on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE), and Western blots probed with luciferase antibody (1:50) were performed as described previously (Belvin et al., 1999). As an additional control for Fig. 2C, we probed a second Western blot run in parallel with an antibody speci®c for the Drosophila ADF-1 protein (DeZazzo et al., 2000). Extracts and assays for in vitro luciferase assays were performed as recommended by the manufacturer (Promega, Luciferase Assay kit). Protein concentrations were determined using the BioRad Dc protein assay. In vivo luciferase assays were performed and analyzed as before with a few changes (Belvin et al., 1999). The top agar layer of each well contained 25 mM luciferin and 100 mg/ml dox. Flies
M.J. Stebbins, J.C.P. Yin / Gene 270 (2001) 103±111
105
were stored and tested at room temperature with no speci®c light±dark cycling. 2.5. Embryonic immunocytochemistry All in situ immunocytochemistry of embryos was performed as described (Patel et al., 1989). Mouse antisera (prior to hybridoma fusion) was diluted 1:50 and preadsorbed against 0±18 h-old 2202u embryos (Yin et al., 1994). Embryos were dechorinated, rinsed, ®xed in PEMFA (horse radish peroxidase), and incubated in diluted, preadsorbed antibody overnight at 48C. The secondary antibody was an HRP (100 mM PIPES 1 2 mM EGTA 1 1 mM MgSO4 1 4% formaldehyde)-conjugated goat-anti-mouse IgG and IgM (1:500) (Jackson ImmunoResearch). DABperoxidase (Sigma) or Ni-DAB peroxidase staining was used to visualize immune complexes as described previously (DeZazzo et al., 2000). 2.6. Larval histochemistry Wandering third instar larvae heterozygous for both an Actin5c-tTA and a TetO-lacZ transgene (Bieschke et al., 1998) were rinsed brie¯y in PBST to remove excess food. Larval brains and discs were dissected and ®xed in 4% paraformaldehyde (PBS). Dissected parts were then incubated in X-gal staining solution. After several brief rinses in PBST, discs and brains were dehydrated in an EtOH series, and cleared and mounted in Hemo-De. 2.7. RNA in situ hybridization RNA in situ hybridization of frozen head sections was performed using a digoxygenin-labeled antisense probe to luciferase (Roche, Dig RNA labeling Kit). The probes were diluted in hybridization buffer (Schaeren-Wiemers and Ger®n-Moser, 1993; Skoulakis and Davis, 1996) and stored at 2208C. Frozen tissue sections were prepared and ®xed in 4% paraformaldehyde-PBS for 10 min at room temperature (Skoulakis and Davis, 1996). The slides were washed three times for 5 min each in PBS. Sections were acetylated for 10 min at room temperature (295 ml H2O, 4 ml triethanolamine, 0.525 ml HCl, and.8 ml acetic anhydride) and washed for 5 min each in PBS. Slides were pre-hybridized for 6 h at room temperature in a humidi®ed chamber. Hybridization buffer with DIG-RNA probe at a concentration of 500 ng/ml was added to slides and incubated overnight at 728C. Slides were washed three times for 30 min each in 0.2 £ SSC at 748C. Anti-DIG complexes were detected according to manufacturer instructions. 3. Results 3.1. The actin system in adults Fig. 1 is a schematic picture of the actin system. The actin5C promoter is used to drive expression of the tTA
Fig. 1. (A) The ubiquitously-expressed Tet-Off system. Two separate Pelement constructs are made, one carrying the tTA gene under the control of the actin promotor, and the other carrying a gene of interest under the control of the tetracycline response elements (TetO). The two transgenes are crossed together into a single ¯y, and in the absence of doxycycline (2Dox), the transactivator protein binds to tetracycline operators (TetO), thereby controlling expression of a target gene. The mini-white gene is used to select transgenic lines. Boundary elements (labeled B) were used to control for position effects. (B) A schematic of our TetO-target P-element vector (pJY2000). cDNAs can be easily cloned downstream of the TetO sequences using XbaI or SpeI restriction sites.
gene, and this transgene is crossed into a ¯y containing the TetO-luciferase transgene, producing a double transgenic ¯y. Drug feeding (or withdrawal) represses (allows) induction of the target gene. 3.1.1. Dosage Dox feeding represses reporter expression in adult ¯ies. Transgenic ¯ies carrying two copies of both the actin-tTA and TetO-luciferase transgenes were fed varying concentrations of dox for 48 h. Equal amounts of total extract protein were loaded for Western analysis using a monoclonal antibody directed against luciferase. There is an inverse relationship between the amount of expression and the dosage of antibiotic in the food. Luciferase expression is very robust in the absence of antibiotic (Fig. 2A, lane 4), and undetectable if ¯ies are fed dox at 100 mg/ml (Fig. 2A, lane 1). Partial repression of expression occurs at the intermediate drug levels of 10 and 1 mg/ml, although the response does not appear to be linear. 3.1.2. Repression kinetics Target transgene expression is effectively shut down after 12 h of dox feeding, and chronic feeding can keep the transgene repressed for at least 3 days. Flies carrying two copies of both the actin-tTA and TetO-luciferase transgenes
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were fed dox at a concentration of 100 mg/ml. Western blots of whole ¯y extracts show that expression from the TetOluciferase transgene is repressed down to baseline levels after 12 h (Fig. 2B, lane 4). This baseline level is similar to the level of expression in ¯ies carrying two copies of the TetO-luciferase reporter in the absence of the transactivator (Fig. 2B, lane 2). Thus there is very little leakiness in this system, since expression in the presence of dox is similar to the expression in the absence of the transactivator protein. The data also shows that the half-life of the luciferase protein must be signi®cantly shorter than 12 h. 3.1.3. Induction kinetics The time course of luciferase expression upon dox withdrawal shows that detectable induction occurs within 6 h, but expression continues to increase out to 24 h. Flies were fed 100 mg/ml of dox for 48 h, to insure maximal target gene repression, as in Fig. 2B. The ¯ies were then transferred to food without antibiotic, and Western blot analysis shows a direct relationship between the amount of induction and the time on dox-free food. While induction increases for the ®rst 24 h after withdrawal, there is no further increase if ¯ies are fed drug-free food for 36 or 48 h (data not shown). This data shows that dox must clear from transactivator complexes and ¯y tissue in signi®cantly less than 24 h. The total
amount of luciferase protein in 20 mg of extract is estimated to be in the nanogram range. By extrapolation, we estimate that approximately 1 in 20,000 protein molecules is luciferase (not shown). We expect this number to range greatly depending upon the target protein being analyzed. A quantitative analysis of luciferase induction con®rms the Western blot analysis (Fig. 2D). From the same pool of ¯ies that were used for Western analysis, extracts were prepared in parallel and luciferase activity was measured in vitro. Induced activity increases directly as a function of withdrawal time, and there is greater than a 100-fold increase 24 h after removal from dox. Intermediate induction occurs when ¯ies are removed from dox for 6 or 12 h. Since the luciferase protein has a half-life that is shorter than 12 h, accumulation of protein cannot contribute signi®cantly to the increases that are detected. 3.1.4. Gene dosage Varying the gene dosage of the transactivator gene shows that the amount of transactivator protein is rate-limiting for the level of induced expression. Flies carrying either one or two copies of the transactivator transgene were compared directly for levels of induced expression 24 h after withdrawal of dox. Fig. 2E shows that there is roughly twice as much light production from ¯ies homozygous for the tTA Fig. 2. Analysis of the actin-tTA system in adults. (A) Dose curve of repression. Double transgenic ¯ies were maintained on regular ¯y food without dox (0), or food supplemented with 1, 10 or 100 mg/ml of drug for 48 h. Whole ¯y extracts were analyzed on Western blots using a luciferase monoclonal antibody. There is signi®cant repression when ¯ies are fed at 1 mg/ml, and nearly total repression at 100 mg/ml. (B) Kinetics of repression. Double transgenic ¯ies were transferred from regular food to vials containing food supplemented with 100 mg/ml of dox and collected immediately 0, 12, 24, 36 or 48 h later. Control ¯ies contain only the luciferase target transgene (2), or a transgene where luciferase is expressed under the control of a CREB-responsive promoter (1) (18). There is no detectable signal from the luciferase antibody on Western blots performed on wild type extracts (not shown). Extracts were prepared and analyzed on Western blots. Twelve hours of feeding at 100 mg/ml is suf®cient to repress transgene expression. (C) Induction kinetics upon dox withdrawal. Flies were maintained on regular ¯y food supplemented with 100 mg/ml of dox for 48 h, then transferred to food without drug. They were collected immediately 0, 3, 6, 12 or 24 h later. Whole ¯y extracts were made and analyzed on Western blots. Detectable induction occurs after 3±6 h of feeding, but continues to increase for 24 h. (D) Kinetics of in vitro luciferase activity upon dox withdrawal. In parallel with (C), ¯ies were fed dox for 48 h before being transferred to drug-free food. Extracts were made immediately 0, 3, 6, 12 or 24 h later and in vitro luciferase activity was measured. About ten ¯ies were used for each time point, and the values (with SEM) are shown. There is approximately a 100-fold induction when ¯ies are maintained on drugfree food for 24 h (relative to the t 0 time point). (E) Effect of transactivator gene dosage on luciferase activity. Flies containing one (tTA/CyO) or two (tTA/tTA) copies of the transactivator transgene and two copies of the luciferase transgene were used to measure in vivo luciferase activity using a luminometer. Approximately 20 ¯ies of each genotype were fed luciferin for 24 h before they were transferred to individual wells of a 96-well microtiter dish. Light output was measured directly from individual ¯ies and the SEM is shown. There is about twice as much light output when the transactivator gene dosage is doubled.
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Fig. 3. Analysis of the actin-tTA system in embryos. (A) Genotype of the embryos analyzed. (B) Embryo whose mother was fed dox. A typical embryo from mothers who were maintained on yeast paste supplemented with 1 mg/ml dox for 48 h. The embryo was ®xed and incubated with luciferase polyclonal sera. Detection and staining were completed with Ni-DAB in conjunction with an HRP-conjugated secondary antibody. There is no detectable luciferase expression when mothers are fed dox. (C) Embryo whose mother was not fed dox. A typical embryo from mothers treated in parallel to (B) except with no dox in the yeast paste. There is strong, ubiquitous expression of the transgene when mothers are not fed the drug.
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embryonic staining detected in older embryos is due to zygotic tTA-TetO gene expression. We were not able to detect any adverse effects on embryonic development when mothers were fed dox at 1 mg/ml. These embryos developed normally through larval and pupal stages, and enclosed to give normal sized adults. In contrast, larvae suffer clear effects on developmental morphology and behavior when fed concentrations of 100 mg/ml or greater (data not shown). The larvae generally appear smaller and spend less time burrowing in the food that contains the drug, consistent with previous ®ndings (Bello et al., 1998; Bieschke et al., 1998). However, we were able to fully repress expression of a TetO-LacZ transgene when larvae were raised on 10 mg/ml dox-containing food (Fig. 4), consistent with previous results using the TetOff system with a spatially-delimited promoter (Bello et al., 1998). There were no obvious effects on larvae when they were fed at concentrations of 10 mg/ml, or lower. At this concentration, repression of transgene expression is effective in a variety of tissues including larval brains (Fig. 4D), wing discs (Fig. 4C), leg discs, (Fig. 4B), gut, and salivary glands (data not shown). 3.3. Spatially-restricted induction in adults We combined the well-characterized Gal4-UAS (Brand and Perrimon, 1993) and Tet-Off expression systems (Gossen and Bujard, 1992) in a tripartite system (Fig. 5)
gene relative to ¯ies that carry only one copy of this gene. These measurements were made in living ¯ies using a luminometer, a technique with proven sensitivity and reliability (Belvin et al., 1999). This data indicates that in this range of transactivator expression, the amount of the transactivator protein is still rate-limiting for induction. 3.2. The actin system in embryos and larvae Embryonic reporter expression can be induced strongly if the mothers of the embryos are fed drug-free food. Embryos (6±15 h) were collected from mothers carrying two copies of the actin-tTA and TetO-luciferase transgenes. If the mothers were maintained on food supplemented with 1 mg/ml dox for 48 h prior to embryo collection, there is no detectable antibody staining (Fig. 3B). Apparently the antibiotic consumed by the mother is deposited into the embryo, repressing transgene expression. Very robust and ubiquitous staining occurs if the mothers were fed drug-free food (Fig. 3C). Early embryos (0±3 h) from the double transgenic mothers, embryos from wild-type mothers, or embryos lacking a tTA transgene did not produce any detectable staining (data not shown). These comparisons indicate that the doxcontaining embryos are totally repressed. This result is consistent with the expression data in adults (Fig. 2B, lane 2). The lack of early embryonic staining indicates that
Fig. 4. Analysis of the actin-tTA system in larvae. (A) Genotype of the larvae analyzed. (B±D) Leg discs, wing discs, and brains from wandering third instar larvae raised on dox free food (left) or food containing 10 mg/ml dox (right). Larvae were dissected, ®xed in 4% paraformaldehyde, and stained in X-gal solution. Staining is undetectable in larvae raised on dox food even when staining times are extended overnight. Robust staining was observed in all larval tissues tested when raised on dox-free food.
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Fig. 5. The spatially-restricted tTA system. This tripartite system relies upon three separate transgenes that are crossed into a single ¯y. The ®rst transgene (A) expresses the Gal4 protein under the spatially restricted control of any genomic enhancer element. The second transgene (B) expresses the transactivator (tTA) under the control of Gal4 binding sites (UAS). The ®nal transgene expresses a target gene under the control of tetracycline operators (TetO). Withdrawal of dox from the food results in temporal induction of the target gene. The availability of Gal4 drivers with the desired expression pattern is the only limitation on the adaptability of this system.
to achieve tissue-speci®c control of tTA expression. Gal4UAS transgenes are used to control tTA expression in a spatially-restricted manner. Any of the widely available Gal4 driver lines (transgene A in Fig. 5) is combined with an UAS-tTA transgenic line (B in Fig. 5) to limit tTA to a particular expression pattern. Both of these transgenes are combined with a third transgene (C in Fig. 5) which contains the tetO-target cassette. Again, target gene expression is achieved upon withdrawal of dox from the food. The boundary elements SCS and SCS 0 were used to ¯ank the UAS-tTA and TetO-luciferase transgenes. Using an in vivo assay for luciferase activity, we detect greater than a 100-fold induction that is spatially and temporally regulated (Fig. 6B). Flies homozygous for the UAStTA and TetO-luciferase transgenes were crossed to ¯ies carrying the Gal4 gene under the control of the sevenless gene promoter (sev-Gal4). Flies were fed luciferin for 12 h then loaded into a 96-well microtiter dish containing agar. The drug-fed ¯ies were maintained on agar with 100 mg/ml dox and luciferin for 24 h, while the drug-free ¯ies were maintained on agar and luciferin. Activity measurements were made in the live animal using a luminometer. There is about a 115-fold (219/1.9) difference in activity between drug-free and drug-fed ¯ies that contain all three transgenes. There is no induction if ¯ies are missing the sev-Gal4 driver transgene, and a 1.9-fold increase when the Gal4 driver is present and the ¯ies are maintained on dox-containing food. Similar results were obtained with three other combinations of UAS-tTA and TetO-luciferase lines when they were combined with the sev-Gal4 driver. Therefore, the induction that is seen is not a function of speci®c lines that might
express at particularly high levels, or are particularly easy to induce due to their surrounding chromatin structure. The use of insulators may have contributed to the equivalent induction that we see with different combinations of the UAS-tTA and TetO-luciferase transgenes. The adaptability of this system was tested using other Gal4 drivers. The glass enhancer is known to express exclusively in the adult eye, while the elav enhancer is ubiquitously expressed in the nervous system (Kidd et al., 1998). These drivers produced a range of activation from approximately 30-fold (glassGal4) to 250-fold (elav-Gal4)). Therefore, the induction is also independent of the particular driver that is used and the particular combinations of driver, UAS-tTA, or TetO-luciferase lines. In situ hybridization of adult head sections shows that luciferase expression is spatially-restricted to the correct pattern for the sevenless enhancer. Luciferase mRNA is detected in the adult eye in a driver-dependent and doxdependent manner (Fig. 6C, middle panel). This pattern of in situ expression matches the known pattern of expression of the sev-Gal4 driver. There is undetectable background staining when triple transgenic ¯ies are fed dox for 24 h (Fig. 6C, left panel), or when ¯ies missing the Gal4 driver (-Gal4 driver) are not fed the drug (Fig. 6C, right panel). Since the induction occurs in the adult eye, is driver-dependent, and is dependent upon drug-free food, it is spatially and temporally regulated. 3.4. Spatially-restricted induction in embryos We used an elav-Gal4 driver to spatially limit tTA expression in embryos. The elav regulatory sequence drives expression in the central nervous system of embryos beginning at approximately stage13 eggs. Flies homozygous for UAS-tTA and TetO-luciferase were mated to homozygous elav-Gal4 ¯ies (Fig. 7A), and embryos were stained with an anti-luciferase antibody. We detect strong Gal4 driver-dependent and dox-responsive luciferase staining in embryos. In the presence of the elav-Gal4 driver, there is strong luciferase staining in the central nervous system of embryos from approximately stage 13 on (Fig. 7C) if their mothers were fed regular food. Embryos that do not have the elav-Gal4 driver, but contain the other two transgenes, do not show any staining (Fig. 7D). If the mothers were fed dox at 1 mg/ml for 48 h, there is no detectable staining in their triple transgenic embryos (Fig. 7B). Similar results were obtained when two other combinations of UAS-tTA and TetO-luciferase reporter transgenes were crossed to the elav-Gal4 driver. A second driver that uses the regulatory regions from the scabrous gene was used to con®rm the adaptability of the system (Kidd et al., 1998). The sca-Gal4 driver, in the presence of UAS-tTA and TetO-luciferase transgenes, produces luciferase expression in the early central nervous system, consistent with the known expression pattern of this driver. Dox feeding of the mothers abolishes this staining
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(data not shown). Therefore, in the embryo, as in the adult, induction occurs in the appropriate spatial pattern for the drivers that are used, is driver-dependent and requires a drug-free mother. 4. Discussion We describe two improved inducible systems for Drosophila, one resulting in ubiquitous expression, the other allowing spatially- and temporally-regulated expression. The second version is adaptable to currently available Gal4 driver lines. Combined with the induction properties of our systems, these changes represent major improvements to inducible technology in Drosophila. Four parameters characterize an inducible system: leakiness, the induction ratio, the kinetics of induction, and the pleiotropic effects of the inducer. Our data is consistent with the general consensus from tissue culture that Tet-Off
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systems are less leaky than Tet-On systems. In adults, total repression, or minimal leakiness, can be achieved with 12 h of feeding on drug-free food. If mothers feed for 48 h on drug-free food, their embryos are totally repressed. In both cases, the level of repressed expression is similar to the level of expression seen in the absence of the transactivator gene. Similar results are seen with the spatially-restricted system. In adults there is only a 1.9fold difference between ¯ies with- and without a Gal4 driver. These results suggest that almost all of the transactivator protein is inactive in the presence of drug. In spite of the strong repression, both systems exhibit high induction ratios, exceeding 100-fold in adults. Because of the instability of a luciferin-luciferase complex, luciferase accumulation is unlikely to contribute to an overestimation of the amount of induction. The actin system can be induced with 6±24 h of drug withdrawal. It is likely that the spatially-restricted system will have similar kinetics, since the fold-induction at 24 h is similar in both systems. However, the detailed pharmacokinetics of drug withdrawal may vary in different tissues, and needs to be empirically determined. We would like to stress that the magnitude and kinetics of induced expression for both the Actin5c and Gal4 based systems must be determined empirically for each target transgene. We fully expect that there will be differences based upon transgene insertion site, feeding protocol, the pharmacokinetics of drug delivery to the tissue of interest, and the mRNA and protein stability of the target gene. There are no obvious harmful effects of feeding dox up to 1±2 mg/ml in adult ¯ies, nor does this empirically appear to
Fig. 6. Analysis of the spatially-restricted tTA system in adults. (A) Schematic of the cross used to generate all of the experimental ¯ies. They contained one copy of the eye-restricted Gal4 driver (sev-Gal4), one copy of the transactivator transgene (UAS-tTA), and one copy of the target transgene (TetO-Luc). The control ¯ies, which lacked the driver transgene (-Gal4 driver), were the same genotype as the mothers. (B) In vivo luciferase activity. Flies containing one (UAS-tTA/CyO) or two transgenes (UAStTA/sev-Gal4) on the second chromosome, and one copy of the reporter transgene on the third chromosome were individually placed into the wells of a microtiter dish. They were kept on regular agar with 25 mM luciferin (2Dox) or on agar supplemented (1Dox) with 100 mg/ml of dox and 25 mM luciferin for 24 h. Light production was measured using a luminometer. N , 20 and the SEM is shown. There is signi®cant induction of light output when ¯ies containing all three transgenes are fed drug-free food. Flies without the Gal4 driver had identical activity in the absence or presence of antibiotic. If this value is set to one, there is a 1.9-fold increase when a Gal4 driver is added and the ¯ies are fed the drug (leakiness) and a 219-fold increase when the ¯ies are drug-free. This results in a 115-fold increase in the absence versus presence of dox. (C) In situ hybridization of luciferase expression. Flies containing either two (2Gal4 driver) or three transgenes were maintained on regular food (2Dox) or food supplemented with 100 mg/ml of dox for 48 h before adult head cryostat sections were cut and probed with digoxygenin-labeled RNA probes. All ¯ies contained one copy of the TetO-luciferase target, and the UAS-tTA transgene. The Gal4 driver was a sev-Gal4 line that expresses in the adult eye. Eye-speci®c expression of luciferase is detected in ¯ies that were either not fed dox or lacked a Gal4 driver.
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Fig. 7. Analysis of the spatially-restricted tTA system in embryos. (A) Genotype of the cross used to generate ¯ies with three transgenes. Progeny from this cross contain a single copy of the Gal4 driver (elav-Gal4), the transactivator (UAS-tTA), and the target transgene (TetO-Luc). Flies missing the Gal4 driver (2Gal4 driver) had the mother's genotype. (B) A typical embryo from mother's fed dox (1Dox) at 1 mg/ml in yeast paste for 48 h. (C) A typical embryo from mothers fed regular yeast paste (2Dox). (D) A typical embryo from uncrossed ¯ies (2Gal4 driver) with the mother's genotype. All embryos were ®xed, treated with the luciferase monoclonal antibody, and the immunohistochemical signal was detected using an HRPconjugated secondary antibody in conjunction with DAB. A strong signal is detected in the central nerve cord of stage 15 embryos, consistent with the elav-Gal4 expression pattern.
affect egg laying and embryonic development. However, there are noticeable effects of feeding antibiotic to larvae, even at concentrations as low as 100 mg/ml. However, we were able shut off expression of a TetO-lacZ transgene with 10 mg/ml dox and observed no developmental abnormalities when larvae were fed at this lower concentration. We have heard anecdotally of a number of failures in implementing the tetracycline system in ¯ies using similar component parts to those that we have used here. Even in the reports where the Tet-Off system has been successfully used, its utility and induction capabilities are limited due to the spatial and temporal limitations of the promoters being used to drive tTA expression. We suspect that our use of the SCS and SCS 0 boundary elements has made a signi®cant contribution to the effectiveness of our systems. When
multiple transgenes are involved, the cumulative effects of random insertion sites can be quite signi®cant. By insulating our transgenes from position effect, we may have put tighter control on expression levels and patterns. Thus the use of boundary elements may have increased expression levels from both the tTA and TetO transgenes. In addition, the use of insulators has been shown to cause independent insertions of each transgene to have nearly identical properties (Chung et al., 1993; Felsenfeld et al., 1996). This may decrease the number of combinations of transgenes that need to be screened, and simpli®es the genetics when combining three transgenes into a single ¯y. In spite of the use of insulators, it is still possible that random insertion sites may affect the leakiness and magnitude of induction. Similarly, it is possible, if enough independent lines are screened, that insertions can be found that have no leaky expression, whether the target transgene is insulated or not (Baron and Bujard, 2000). Although we used insulators, the Tet-Off systems does show some leakiness. For applications involving expression of potentially toxic proteins we recommend that users consider an uninsulated vector and screen many independent target lines. For certain biological questions, the Tet-On system may be more practical than Tet-Off. The Tet-On system utilizes a mutant transactivator that binds to TetO sequences only in the presence of dox (Gossen et al., 1995). The currently available Tet-On system in Drosophila uses the Actin5c promotor to achieve ubiquitous expression of the rtTA (Tet-On) transactivator (Bieschke et al., 1998). Overall induction in this system is slow, taking approximately 5 days to achieve 10-fold induction of bgal, rendering it of limited use for most applications. Since bgal activity is very stable, accumulation of lacZ protein probably contributes to an overestimation of the actual induction. Recently, second generation Tet-On transactivators have been published (Urlinger et al., 2000). Experiments to evaluate whether these transactivators signi®cantly improve the currently available system are underway, and the preliminary results are promising (M. Stebbins et al., manuscript in preparation). Our improvements to the tTA expression system should open the door for a wide array of applications in Drosophila. The actin-based tTA system is now a viable alternative to the heat-shock induction system, thereby by-passing possible pleiotropic effects of heat-shock itself. The availability of tTA and heat-shock also allows the combinatorial manipulation of multiple transgenes. The tTA systems may be particularly useful for rescue of developmentally lethal genes, where the rescuing genes are expressed throughout development, or for the study of heat-shock proteins. The spatially-restricted system will facilitate detailed investigations of tissue and temporal requirements for gene function. Until now, this type of experiment has been technically dif®cult to perform due to the ubiquitous nature of the heat-shock inducible system and the temporal restrictions of the Gal4-UAS system. This will be particularly useful for
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studying complex behavioral phenotypes such as learning, memory, courtship and circadian rhythms. Finally, the graded response to dox suggests that it should be possible to sustain intermediate levels of induction simply by controlling drug dosage in the food, providing a controllable ªrheostatº for inducible gene expression in Drosophila. Acknowledgements We thank Jim DeZazzo for ADF-1 antibody, reagents, advice and comments on early versions of the manuscript. We also thank Grisha Enikolopov, Maurice Kernan, Paul Schedl and Sid Strickland for valuable discussions and Hong Zhou for technical assistance. This work was supported by grants from NIH (R01 NS35575), the McKnight Foundation, and start-up funds from Cold Spring Harbor Laboratory (to J.C.P.Y.). References Baron, U., Bujard, H., 2000. Tet repressor-based system for regulated gene expression in eukaryotic cells: principles and advances [in process citation]. Methods Enzymol. 327, 401±421. Bello, B., Resendez-Perez, D., Gehring, W.J., 1998. Spatial and temporal targeting of gene expression in Drosophila by means of a tetracyclinedependent transactivator system. Development 125, 2193±2202. Belvin, M.P., Zhou, H., Yin, J.C., 1999. The Drosophila dCREB2 gene affects the circadian clock. Neuron 22, 777±787. Bieschke, E.T., Wheeler, J.C., Tower, J., 1998. Doxycycline-induced transgene expression during Drosophila development and aging. Mol. Gen. Genet. 258, 571±579. Brand, A.H., Perrimon, N., 1993. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401±415. Chung, J.H., Whiteley, M., Felsenfeld, G., 1993. A 5 0 element of the chicken beta-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila. Cell 74, 505± 514. DeZazzo, J., Sandstrom, D., de Belle, S., Velinzon, K., Smith, P., Grady, L., DelVecchio, M., Ramaswami, M., Tully, T., 2000. Nalyot, a mutation of the Drosophila myb-related Adf1 transcription factor, disrupts synapse formation and olfactory memory. Neuron 27, 145±158. Felsenfeld, G., Boyes, J., Chung, J., Clark, D., Studitsky, V., 1996. Chromatin structure and gene expression. Proc. Natl. Acad. Sci. USA. 93, 9384±9388. Furth, P.A., St Onge, L., Boger, H., Gruss, P., Gossen, M., Kistner, A., Bujard, H., Hennighausen, L., 1994. Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proc. Natl. Acad. Sci. USA 91, 9302±9306. Girard, F., Bello, B., Laemmli, U.K., Gehring, W.J., 1998. In vivo analysis
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