Use of the Tetracycline System for Inducible Protein Synthesis in the Kidney
Anna Rachel Gallagher*,
Kai Schönig,
Nelson Brown*,
Hermann Bujard and
Ralph Witzgall
*Institute for Anatomy and Cell Biology I and Center for Molecular Biology, University of Heidelberg, and Institute for Molecular and Cellular Anatomy, University of Regensburg, Germany.
Correspondence to Ralph Witzgall, Institute for Molecular and Cellular Anatomy, University of Regensburg, Universitätsstr. 31, 93053 Regensburg, Germany. Phone: +49-941-9432821; Fax: +49-941-9432868;
ABSTRACT. The great advantage of the tetracycline-induciblesystem lies in its ability to address a large variety of biologicalquestions in a time-dependent and tissue-specific manner. Thisstudy describes a transgenic mouse line, rTALAP-1, which producesthe reverse tetracycline transactivator under control of theliver activator protein (LAP) promoter. Two reporter lines withluciferase and LacZ reporter genes were used to demonstratepredominant expression in the kidney and liver when doxycyclinewas added to the drinking water. In the kidney, transgene expressionwas found primarily in cortical proximal tubules. No luciferaseand -galactosidase activity was detected in mice without doxycyclinein the drinking water, which attests to the tight control ofthis system. One of the advantages of the tet system lies inits reversibility, and indeed, a virtually complete remissionof transgene activity in both the kidney and liver was observedwhen doxycycline was withdrawn. Also examined was transactivatoractivity during development by exposing the mothers producingthe reverse transactivator to doxycycline before mating. Transgeneactivity was detected in newborn kidneys and liver, indicatingthat sufficient amounts of doxycycline had crossed the placentalbarrier. During nephron development, the LAP promoter appearedto be only active in the more mature proximal tubules. Finally,the rTALAP-1 line was used to inducibly express the human PKD2cDNA in proximal tubules of transgenic mice, but no cystic changeswere detected, even after 6 mo of induction. E-mail: ralph.witzgall@vkl.uni-regensburg.de
The understanding of gene function has been greatly aided bya multitude of transgenic mice. Conventional knockout mice,however, have the disadvantage that inactivation of the geneof interest may cause embryonic lethality or so severe a phenotypethat it does not allow its analysis in all organs of adult mice.Therefore, a number of systems have been developed, which caneither inactivate or overexpress genes conditionally in a tissue-specificand/or time-dependent manner. Of the few inducible expressionsystems that have been devised so far, the tetracycline-dependentsystem (tet system) probably has shown the greatest potential.
The tet system consists of two components. The first representsa tetracycline-dependent transactivator (tTA) driven by a specificpromoter. The second contains tet operator sequences next toa minimal promoter, that control the transcription of the cDNAor gene of interest. Two versions of the tet-inducible systemexist. In the original design, tTA binds to the tet operatoronly in the absence of tetracycline (1). In a subsequent version,the reverse tetracycline-inducible system (rtTA), a mutatedtTA will bind to the tet operator only in the presence of tetracycline(2).
Although first established to study the function of genes inmammalian cell lines, the tet system soon also made a successfultransition into animals (3,4). A similar extent of transgeneexpression has been achieved with both the tTA and rtTA systems;induction kinetics, however, were slower with the tTA systembecause withdrawal of doxycycline is necessary before transcriptionof the transgene can be induced. Recent modifications to thetransactivator sequence have led to an improved tetracycline-dependentreverse transactivator (rtTA2S-S2), which reaches the same activationlevels as the original rtTA, while its regulatory range is tenfoldenhanced as a result of its lower background activity (5).
A number of tet-inducible mouse lines have been generated (6),but none has been directed specifically to the kidney. For oneline, in which the reverse transactivator is driven by the humancytomegalovirus promoter (rtTACMV-3), tet-inducible proteinsynthesis was described in many organs, among them the kidney(3). There, the transgene is specifically expressed in corticaland inner medullary collecting ducts (7). In this report, wedescribe another transgenic mouse line, in which an optimizedreverse transactivator is only active in the liver and the kidney.
Transgenic Animals
Three strains of transgenic mice were used in our study. Thereverse transactivator line rTALAP-1, in which the reverse transactivatoris driven by the liver activator protein (LAP) promoter, hasbeen previously described (8,9). It contains an expression cassettethat consists of 2.8 kbp of the LAP promoter and a cDNA encodingthe S2 version of the reverse transactivator (5). The firstreporter line, LC1, contains a bidirectional expression cassetteof the luciferase gene and Cre cDNAs under control of tet operatorsequences (9). A second reporter line again contains a bidirectionalexpression cassette, but this time with the nLacZ gene (encoding-galactosidase with a nuclear localization signal) and the humanPKD2 cDNA under control of tet operator sequences. The latterwas generated by cloning the human PKD2 cDNA into the PstI andSalI sites of the pBI3 plasmid (10). After the resulting constructwas linearized with AseI, it was isolated by gel electrophoresis,passed twice through an Elutip D column (Schleicher & Schuell,Dassel, Germany), and injected into fertilized oocytes of C57Bl/6x DBA mice. All transgenic lines were maintained on a C57Bl/6background. Founders were identified by screening tail biopsyspecimens for the presence of nLacZ and PKD2 cDNA by PCR. Thefollowing oligonucleotide pairs were used: 5'-CAAACCATCGAAGTGACCAG-3'and 5'-CAATTTAACCGCCACTCAGG-3' to amplify a 388-bp fragmentof the nLacZ gene; and 5'-ATTTGCAGATCTGTTCTCACATATCGG-3' and5'-CTCTCAATCCTGGGGGAA-3' to amplify a 407-bp fragment of thePKD2 cDNA. The reaction conditions were 94°C for 4 min,followed by 25 cycles of 94°C for 30 s, 52°C for 30s, and 72°C for 1 min. Thirteen out of 49 founders werepositive for both nLacZ and PKD2. Of the 13 founders, only fourwere inducible for -galactosidase activity.
Mice of either gender were used for our studies. For the inductionkinetics (both for luciferase and -galactosidase), mice werebetween 35 and 42 d old. Mice used in the withdrawal study were6 mo old. For the dose-response experiments and the tissue survey,mice were 12 mo (no doxycycline) and 4 mo old (0.2 and 2 mg/mlof doxycycline).
Genotype Analysis
Genomic DNA was isolated from tail biopsy specimens of miceand subjected to Southern blot analysis according to standardprotocols (11). To detect the presence of the nLacZ and luciferasetransgenes, 10 µg of genomic DNA was digested with BamHIand hybridized with the respective fragments; to detect thereverse transactivator transgene, genomic DNA was digested withEcoRI.
Doxycycline Administration
Doxycycline hydrochloride (Sigma, Deisenhofen, Germany) andsucrose (5% final concentration) were dissolved in water. Thedoxycycline-sucrose solution was prepared fresh every 3 to 4d in a brown drinking bottle.
In Vivo Bioluminescence Imaging
Mice were anesthetized, injected intraperitoneally with 100µg D-luciferin (Promega, Madison, WI) per gram of bodyweight, and immediately placed in a dark chamber. Luminescencewas captured by a photon-counting camera (two-stage ICCD C2400-47;Hamamatsu Photonics Deutschland, Herrsching, Germany) fittedwith a Nikon lens (35 mm/fl.2) for a period of 1 to 2 min. Theimages were then digitized with the Argus 20 image processor(Hamamatsu Photonics Deutschland) and later processed by AdobePhotoshop software (Adobe Systems, San Jose, CA).
Luciferase Assay
Tissues from adult mice were homogenized for 10 s in 500 µlof lysis buffer (20 mM DTT, 25 mM Tris pH 7.8, 2 mM EDTA, 10%glycerol, 1% Triton X-100) immediately after removal; in thecase of newborn mice tissues were sonicated for 10 s in 100µl of lysis buffer. After homogenization, the tissueswere snap-frozen in liquid nitrogen and stored at -80°C.When luciferase activity was measured, the samples were thawedand centrifuged for 15 min at 14,000 rpm and 4°C. Ten microlitersof the supernatant were combined with 250 µl of 25 mMglycylglycine, 15 mM MgSO4, 5 mM ATP, 0.5 mM D-luciferin andassayed for 10 s in a Lumat LB9501 (Berthold, Wildbad, Germany).All measurements were performed in duplicate. An aliquot ofthe lysate was used to determine the protein concentration bymeans of an improved Bradford assay (12).
-Galactosidase Staining
Adult mice were perfused through the distal abdominal aortawith 4% paraformaldehyde,1x PBS for 3 min at a pressure of 180mmHg. The relevant organs were removed, sliced, and immersedin 18% sucrose,1x PBS until they sank to the bottom. Organsfrom newborn mice were removed without prior perfusion, cutinto slices, fixed in 1.25% paraformaldehyde, 0.2% glutaraldehyde,and 1x PBS for 15 min and then immersed twice in 30% sucrose,1xPBS for 15 min each. All tissues were subsequently frozen inliquid nitrogen-cooled isopentane. -Galactosidase staining wasperformed on 7-µm cryosections. The sections were equilibratedin staining solution (5 mM potassium ferricyanide, 5 mM potassiumferrocyanide, 2 mM MgCl2, 20 mM NaCl, 10 mM EGTA pH 8.0, 10mM sodium phosphate pH 8.0) and then incubated overnight at30°C in the presence of 1 mg/ml of X-gal. The sections werewashed twice in PBS and then either counterstained with eosinand mounted, or used further for immunohistochemistry.
Immunohistochemistry and Alkaline Phosphatase Staining
Immunoperoxidase staining was carried out according to the VectastainABC kit instructions (Vector Laboratories, Burlingham, CA).Primary antibodies were a rabbit polyclonal anti–Tamm-Horsfallglycoprotein antibody (Biotrend, Cologne, Germany; diluted 1:100)and a rabbit polyclonal anti-HA antibody (Sigma; diluted 1:2,000).-Galactosidase staining was carried out overnight before immunohistochemistry.Alkaline phosphatase histochemistry was performed by exposingcryosections to the appropriate reaction solution (0.3 mM nitroblue tetrazolium chloride, 0.3 mM 5-bromo-4-chloro-3-indolyl-phosphate,4-toluidine salt in 0.2 M Tris·HCl, pH 9.5) for 25 min.The sections were subsequently washed twice with bidistilledwater for 10 min and then mounted in bicarbonate-buffered glycerolpH 8.6. All sections were examined with a Leica Polyvar 2 microscope;pictures were taken with Nikon digital camera DXM1200. Finally,all images were processed with Adobe Photoshop software (AdobeSystems).
Cell Culture and Transfection Protocols
HtTA-1 cells, which are HeLa cells producing tTA, were stablytransfected with the AseI-linearized pBI3 plasmid containingthe full-length PKD2 cDNA and the nLacZ gene by using a polyornithineprotocol (13). Forty-eight hours after transfection, cells wereplated onto 10-cm petri dishes and selected with puromycin (0.5µg/ml; Calbiochem, Darmstadt, Germany). Approximately2 wk later, resistant colonies were isolated and tested for-galactosidase and polycystin-2 synthesis.
Protein Preparation and Western Blot Analysis
Proteins were prepared from cell lines by lysing the cells ina buffer containing 1% Triton X-100, 0.05% SDS, 150 mM NaCl,10 mM Tris HCl pH 7.5, 2 mM EDTA pH 8.0, 1 µg/ml of leupeptin,1 mM PMSF. After the protein concentration was determined, aliquotscontaining 30 µg of protein were analyzed by Western blot.To prepare protein from organs, 6-wk-old mice were placed ondoxycycline for 2 wk; mice not receiving doxycycline were usedas negative controls. Upon removal, kidneys and livers wereadded to 5 ml of a homogenization buffer containing 250 mM sucrose,25 mM Tris pH 7.4, 5 mM EDTA, 2 µg/ml of leupeptin, 2µg/ml of aprotinin, 1 mM benzamidine, 1 mM PMSF, 20 µg/mlTAME (p-tosyl-L-arginine methyl ester). After the tissues werehomogenized with a motor-driven Teflon pestle, they were centrifugedfor 15 min at 500 x g and 4°C to remove debris. An aliquotof the supernatant containing 50 µg of protein was analyzedby Western blot.
Proteins were run on polyacrylamide gels under denaturing andreducing conditions and then transferred onto a polyvinylidenedifluoride (PVDF) membrane. The membrane was incubated withthe 12CA5 mouse monoclonal anti-HA epitope antibody (cell culturesupernatant diluted 1:30). Final detection of the HA-epitope–taggedpolycystin-2 protein was done with horseradish peroxidase–conjugatedgoat anti-mouse IgG Fab (diluted 1:10,000; Sigma) and the chemiluminescencereagents from NEN (Bad Homburg, Germany).
Immunocytochemistry and -Galactosidase Staining of Cells
Cells were plated on glass coverslips and allowed to grow foran additional 3 d in the absence of doxycycline. After fixationwith 2% PFA, 1x PBS, the cells were washed with 1x PBS and thenstained for -galactosidase activity in a solution containing5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 1mM MgCl2, 1x PBS, and 1 mg/ml of X-gal. The next morning, thecells were washed with 1x PBS, permeabilized with 0.2% TritonX-100, 1x PBS, 2% BSA for 45 min and then incubated with theanti-HA–epitope antibody 12CA5 (diluted 1:30) for 2 hat room temperature. Subsequently, the cells were washed threetimes with 1x PBS and incubated for 60 min with Cy3-conjugatedrat anti-mouse IgG antibody (Dianova, Germany; diluted 1:300).The coverslips were mounted and examined with a Leica Polyvar2 microscope. Pictures were taken with Nikon digital cameraDXM1200.
In Vivo Imaging and Dose Dependency
The transactivator mouse line, rTALAP-1 (8,9), was created byusing the optimized S2 version of the reverse transactivator(5) under control of the LAP promoter (14). Mice double-transgenicfor rtTALAP-1 and the luciferase reporter gene were administereddoxycycline at a concentration of 2 mg/ml in their drinkingwater. Using a noninvasive imaging system that can detect luminescence(8,15), it appeared that the LAP promoter was liver specific(Figure 1, a). However, after removing a number of organs andexamining them for luciferase activity, pronounced luminescencealso originated from the kidney (Figure 1, b), which could probablynot be seen by noninvasive imaging because of the retroperitoneallocation of the kidneys. The comparison of lysates from thekidneys with those from a number of other organs (i.e., adrenalgland, brain, heart, intestine, lung, pancreas, skeletal muscle,spleen, testis, and thymus) showed at least 100-fold higherluciferase values in the kidneys (Table 1). A lower dose of0.2 mg/ml of doxycycline in the drinking water resulted in amore than 90% reduction of luciferase activity in both the kidneysand the liver (Figure 1, c; note the logarithmic scale).
Figure 1. Imaging and dose dependency of luciferase activity. (a) Double-transgenic mice, which contained one expression cassette consisting of the reverse transactivator under control of the liver activator protein (LAP) promoter and a second expression cassette consisting of a luciferase cDNA under control of tetracycline-dependent system (tet) operator sequences, were administered doxycycline at a concentration of 2 mg/ml in the drinking water for 7 d. Then they were injected with D-luciferin and immediately placed into a dark chamber to collect photons. It can be seen by projecting the signal onto the mouse that most photons originated from the upper abdomen. (b) Upon removal of the liver and both kidneys, this luminescence could be attributed to the liver, but very strong luciferase activity was also present in the kidneys. (c) A dose-response experiment showed that administering doxycycline for 14 d at a concentration of only 0.2 mg/ml in the drinking water resulted in a pronounced decrease of luciferase activity. Data are presented as mean + SD; the number of mice is given above the bars.
Table 1. Luciferase activities in various tissues after 14 days of administration of doxycycline (2 mg/ml) in drinking watera
Localization of the Tet Activator in the Kidney
To determine in which cells of the kidney the LAP promoter isactive, mice double-transgenic for the reverse transactivatorand the nLacZ gene were administered doxycycline at a concentrationof 2 mg/ml in the drinking water for 14 d. Cryosections of thekidneys were first stained for -galactosidase activity overnight,and then either subjected to alkaline phosphatase histochemistryor stained with antibodies against markers for the various nephronsegments. -Galactosidase activity was most prominent in thecortical portion of the proximal tubule (Figure 2, a), whichbecame evident when costained for the brush border enzyme alkalinephosphatase, thus strongly suggesting that the LAP promoterwas only active in the S1 and S2 but not in the S3 portion ofthe proximal tubule (Figure 2, b). It is noteworthy that quiteoften not all cells in the same tubular profile showed -galactosidaseactivity; this mosaic pattern was seen in all animals examined.Only in approximately 20% of alkaline phosphatase–positiveprofiles all nuclei of a given profile produced -galactosidase,whereas in approximately the same percentage of profiles no-galactosidase-positive nuclei were detected (Figure 2, e).In addition to the cortex, there was some -galactosidase activityin the inner stripe, suggesting transgene induction also inthick ascending limbs. Indeed, by costaining with an antibodyagainst the Tamm-Horsfall glycoprotein, thick ascending limbprofiles in the inner stripe and the cortex were identifiedthat contained blue nuclei (Figure 2, c and d). We found noprofile in the inner stripe in which all nuclei produced -galactosidase,whereas in approximately 90% of the profiles, none of the nucleiwas positive for -galactosidase, which is in stark contrastto the situation in proximal tubules (Figure 2, e). Using antibodiesagainst the thiazide-sensitive NaCl cotransporter (a markerof distal convoluted tubules) and against aquaporin-2 (a markerof collecting ducts), blue nuclei could very rarely be detectedin cortical distal convoluted tubules and collecting ducts (datanot shown).
Figure 2. Localization of -galactosidase activity in the kidney. Double-transgenic mice, which contained liver activator protein (LAP) promoter/rtTA2S-S2 and tetracycline-dependent (tet) operator/nLacZ expression cassettes, were fed doxycycline at a concentration of 2 mg/ml in the drinking water for 14 d. (a) Histochemical staining for -galactosidase activity and subsequent counterstaining with eosin demonstrates many blue nuclei in the cortex of the kidney. (b) Staining for -galactosidase and alkaline phosphatase, a brush border enzyme, reveals that most -galactosidase–positive nuclei are present in proximal tubules. Note that there are also some nuclei in proximal tubules, which did not turn blue. (c, d) When histochemical staining for -galactosidase was combined with immunostaining against Tamm-Horsfall protein, a marker of thick ascending limbs, some blue nuclei were also detected in thick ascending limb cells in the cortex (arrows in c) and the inner stripe (d). Asterisks in c mark proximal tubules with many blue nuclei. (e) A careful quantitation substantiates that -galactosidase–positive nuclei were much more frequent in proximal tubules than in thick ascending limbs, but it also demonstrates the considerable degree of mosaicism. Data are presented as mean values.
Induction Kinetics of Gene Expression
To test the induction kinetics of the rtTALAP-1 transgene inthe kidney, female mice containing the LAP/rtTA2S-S2 cassettewere mated with males containing the luciferase and nLacZ reportergenes under the control of the tet operator. When the offspringwere approximately 5 wk old, they were exposed to doxycyclinefor 1 to 14 d. Luciferase activity was then measured in thelysates from the right and left kidney, liver, and spleen ofdouble- and single-transgenic mice (Figure 3).
Figure 3. Kinetics of luciferase induction in adult mice. Mice containing only the liver activator protein (LAP) promoter/rtTA2S-S2 expression cassette (rtTA), only the tetracycline-dependent (tet) operator/luciferase expression cassette (luc), or both expression cassettes (rtTA/luc) were fed doxycycline in the drinking water for 1, 4, 7, and 14 d before both kidneys, the liver, and spleen were removed. Already after 24 h on doxycycline, pronounced luciferase activities were observed in the kidneys (a) and the liver (b), whereas only a very modest luciferase activity was measured in the spleen, even after 14 d of doxycycline administration (c). The tightness of the system is evident from the very low luciferase activity of rtTA/luc mice without exposure to doxycycline and from equally low luciferase activity of luc mice exposed to doxycycline for 14 d. RK and LK, right and left kidney, respectively. Data are presented as mean + SD; the number of mice is given above the bars.
As expected, instrument background was detected for the transgenicanimals containing only the reverse transactivator. The tightnessof the system became evident by the fact that the organs containingonly the luciferase gene, and the organs from double-transgenicanimals with no exposure to doxycycline also only showed backgroundactivity. After 1 d of doxycycline administration, however,a high luciferase activity of remarkably similar levels wasseen in the right and left kidney of double-transgenic mice,thus demonstrating a uniform induction and validating our approach.As expected, luciferase values rose with increasing exposureto doxycycline (Figure 3, a). The same was seen for the liver,where induction was at least 10,000-fold higher than backgroundafter 14 d of doxycycline administration (Figure 3, b). Thespleen, which was used as a control tissue, showed less than1% of the hepatic and renal luciferase values (Figure 3, c).
The same time course was used to investigate the induction andlocation of -galactosidase activity. -Galactosidase positivenuclei were already seen after 1 d of doxycycline administration,although we could not detect more than five blue nuclei persection in the cortex of the kidney. The longer the exposureto doxycycline, the more cells became induced, which correlatedwell with the luciferase data. Apparently tubular cells in thecortex reacted first, whereas -galactosidase positive cellswere fewer in the inner stripe and took longer to be induced(data not shown).
Developmental Kinetics of Gene Expression
Because we were able to control gene expression with doxycyclinein adult kidneys, we also wanted to see if this was possibleduring development. For this part of the study, the motherscarrying the LAP/rtTA2S-S2 transgene were placed on doxycycline1 wk before mating. The offspring were analyzed on the day ofbirth as well as 7, 14, and 35 d after birth. A high level ofluciferase activity was detected in newborn mice, indicatingthat sufficient amounts of doxycycline had crossed the placentalbarrier. Luciferase activity in the kidneys of newborn micewas similar to that in the kidneys of adult mice after 1 d ofexposure to doxycycline. However, 1 and 2 wk postpartum, luciferaseactivity was markedly reduced, suggesting that doxycycline wasnot transduced at sufficiently high concentrations through themothers’ milk (Figure 4, a). In livers of newborn mice,luciferase activity was equivalent to that of livers from adultmice after being induced for 14 d. A pronounced decline in activitywas also seen in the liver over the first couple of weeks, similarto that in the kidney (Figure 4, b). However, in both hepaticand renal tissues, a subsequent increase can be seen in 35-d-oldmice similar to the levels seen in tissues from adult mice after14 d of doxycycline administration (Figure 4, a and b). A similarpattern, although at much lower levels, was also observed inthe spleen (Figure 4, c). -Galactosidase histochemistry revealedthat the induced cells in newborn kidney were limited to themore mature tubular profiles because no blue nuclei could bedetected in the nephrogenic zone below the capsule (Figure 4, d).No blue nuclei could be detected in the kidneys of 14-d-oldmice (data not shown).
Figure 4. Activity of the reverse transactivator during development. (a through c) Female mice carrying a liver activator protein (LAP) promoter/rtTA2S-S2 expression cassette (rtTA) were fed doxycycline in the drinking water from 7 d before being mated with male mice carrying a tetracycline-dependent (tet) operator/luciferase expression cassette (luc). Luciferase activity was determined in both kidneys, liver, and spleen of newborn mice and 7, 14, and 35 d after birth. High luciferase activity was observed in the kidneys and liver of newborn double-transgenic (rtTA/luc) mice and 35 d after birth, whereas it was considerably lower in the intermediate time points, suggesting that sufficient levels of doxycycline crossed the placenta, but that not enough doxycycline was provided with the mothers’ milk. After weaning, when the offspring began to drink water with doxycycline, the reverse transactivator obviously became activated again. (d) Histological section of a kidney from a newborn mouse with -galactosidase instead of luciferase as a reporter protein. Double staining for -galactosidase and alkaline phosphatase, which marks differentiated proximal tubules, demonstrates that the LAP promoter is not active in the nephrogenic zone immediately below the capsule but only in mature proximal tubules. RK and LK, right and left kidney, respectively. Data are presented as mean + SD; the number of mice is given above the bars.
Persistence of Transgene Expression
One of the attractive features of the tet system lies in itsreversibility, which depends on the decrease of doxycyclinelevels after removal as well as on the half-life of the mRNAand protein encoded by the transgene. We therefore analyzedthe decline in luciferase levels of double-transgenic mice,which were treated with doxycycline for 14 d. When doxycyclinewas removed from the drinking water, luciferase activity inthe liver decreased to background levels within 14 d after discontinuationof doxycycline (Figure 5). Luciferase activity in the kidneyshad fallen 100-fold after 14 d and to just above backgroundlevels after 35 d. Again, -galactosidase activity mimicked theresults of the luciferase assays because blue nuclei were stillpresent in the kidneys 14 d after removal of doxycycline; noblue nuclei were detected in the kidney after 35 d of withdrawal(data not shown).
Figure 5. Persistence of reverse transactivator activity. Adult double-transgenic mice containing liver activator protein (LAP) promoter/rtTA2S-S2 and tetracycline-dependent (tet) operator/luciferase expression cassettes were fed doxycycline in the drinking water for 14 d and then transferred to drinking water without doxycycline. It becomes evident that the activation of the reverse transactivator is reversible, but that it takes longer in the kidney than in the liver before the transactivator returns to its uninduced state. Data are given as mean + SD; the number of mice is given above the bars.
Inducible Expression of PKD2
To test the inducibility of the bidirectional expression cassettecontaining the human PKD2 cDNA and the nLacZ gene in the tetsystem, stably transfected HeLa cell lines were generated thatinducibly produce full-length HA-tagged polycystin-2 and -galactosidasein the absence of doxycycline. The clones were tested firstfor -galactosidase activity and then for polycystin-2 immunoreactivity.All -galactosidase-positive cells also stained positive forHA-tagged polycystin-2 protein (Figure 6, a and b). These immunocytochemicalfindings were subsequently confirmed by Western blot analysis.It can be clearly seen that the PKD2 cDNA is switched off inthe presence of doxycycline and induced upon withdrawal of doxycycline(Figure 6, c).
Figure 6. Simultaneous induction of polycystin-2 and -galactosidase in stably transfected cells. (a through c) A bidirectional expression cassette consisting of the human PKD2 cDNA and a nLacZ gene under control of tetracycline-dependent (tet) operator sequences was stably transfected into HeLa cells producing a tTA. Upon induction, the nuclei of transfected cells stained positive for -galactosidase (a), whereas the HA-tagged polycystin-2 protein was detected in the cytoplasm (b). In the case of polycystin-2, this immunocytochemical finding was subsequently confirmed by Western blot analysis (c; molecular weights are given in kDa).
Mice double-transgenic for the reverse transactivator and thetet operator/PKD2/nLacZ expression cassette were administereddoxycycline in the drinking water for 14 d, animals withoutdoxycycline in the drinking water served as a negative control.When protein lysates from the kidney and liver were subjectedto Western blot analysis, the epitope-tagged polycystin-2 proteinwas only detected in animals receiving doxycycline (Figure 7, a).Analysis of cryosections revealed that the expression ofPKD2 was most pronounced in the proximal tubule, therefore mirroringthe -galactosidase staining (Figure 7, b and c). To investigatewhether the overexpression of the human PKD2 cDNA would leadto alterations in the kidneys, we administered doxycycline long-termstarting at 8 wk after birth. Even after 6 mo, however, no morphologicchanges were visible (Figure 7, d and e).
Figure 7. Inducible expression of the human PKD2 cDNA in the rTALAP-1 mouse line. The bidirectional PKD2/nLacZ expression cassette was used to establish transgenic mice, which were then mated with mice containing the reverse transactivator under control of the liver activator protein (LAP) promoter. (a) Western blot analysis was performed with 50 µg of protein prepared from kidney and liver. Only in those mice receiving doxycycline in the drinking water ("on"), polycystin-2 (arrow) can be detected in both the kidney (K) and the liver (L); without doxycycline ("off"), neither organ produces polycystin-2 (the numbers on the left indicate the molecular weight in kDa). (b, c) When kidney sections from double-transgenic mice that had been fed doxycycline in the drinking water were subjected to immunohistochemistry with the anti-HA epitope antibody, polycystin-2 was detected in cortical tubular profiles. Nomarski optics were used to identify these profiles as proximal tubules through the presence of their brush border (asterisks in b and c). (d, e) PAS staining of kidney sections from double-transgenic mice reveals no morphologic changes to nontreated mice (d) even after exposure to doxycycline for 6 mo (e).
The aim of our study was to characterize the transgenic mouseline rTALAP-1, which allows the inducible and reversible expressionof transgenes predominantly in proximal tubules. In this linethe tetracycline-inducible transactivator is driven by the LAPpromoter. This promoter based on the analysis of other transgeniclines, was previously thought to be liver-specific (3). It wastherefore somewhat unexpected to detect transactivator activityin the kidney, which, however, was a stable effect because weobserved renal expression in many crosses and over six generations.Whether this particular expression pattern of the rtTA2S-S2transgene is due to a positional effect of its integration siteremains a matter of speculation.
Strongest transgene expression was detected in the corticalproximal tubules, although transactivator activity in otherparts of the nephron was also observed. Expression in thickascending limb cells was variable and seemed to depend on thelength of induction and on the tet operator line used, whilethere was consistently strong expression in proximal tubulesindependent of the tet operator mouse lines. Such a patternof tet operator activity has already been seen in the brain,where a reverse transactivator line under the control of theCaMKII- promoter showed expression in the striatum and septumwhen placed only shortly on doxycycline; with a longer inductionperiod, expression could also be detected in the cortex andhippocampus (16). We also noticed that even though there wasstrong expression in proximal tubules, in many cases, not everynucleus in a given tubular profile was blue. This mosaic patternof LacZ expression has been noted previously (17), but the underlyingmechanisms are poorly understood, although it is thought todepend on chromosomal context and transgene copy number (18).
Depending on the kind of study to be conducted, mosaicism representsa more or less serious problem. For example, if one wants toinvestigate tumor induction, the mosaic inactivation of a tumorsuppressor gene upon the induction of Cre recombinase wouldstill permit the development of tumors and might actually beadvantageous because it would be possible to perform a genotype-phenotypecorrelation of normal and malignant tissue in the same organ(the prediction being that in tumors both copies of a tumorsuppressor gene are inactivated, whereas in nontransformed tissueone or both alleles are still wild-type). On the other hand,physiologic studies may be severely hindered by mosaicism becausewild-type cells would be able to compensate for the cells inwhich the inactivation event has taken place. Those investigationswould therefore only be feasible at a low level of mosaicism.
The rate of induction in the kidney and liver was very fast:we could detect strong reporter gene expression already 1 dafter administering doxycycline in the drinking water, whichobviously is a convenient method of supplying the chemical.Upon discontinuation of doxycycline, the activity in the liverwas reduced to background levels after 14 d of withdrawal. However,transgene expression in the kidney decreased more slowly, reachingbackground levels after 5-wk withdrawal. The difference betweenthe kidney and liver is most likely the result of the pharmacokineticsof doxycycline in these tissues. Studies with other tetracyclineshave demonstrated that higher levels of tetracyclines accumulatein the kidney than in the liver (19,20), which would accountfor the slower reduction of transgene expression in the kidneycompared with the liver. A lower concentration of doxycyclinein the drinking water may help to accelerate the return to backgroundlevels, although it would entail lower induction levels. Thepronounced difference of induction levels between 0.2 and 2mg/ml of doxycycline in the drinking water (approximately tenfold)was somewhat surprising because in vitro the rtTA2S-S2 reversetransactivator shows full activation already at approximately1 µg/ml of doxycycline (5). It is likely that the concentrationof doxycycline in the serum and extracellular fluid is muchlower than that in the drinking water, possibly in the nanogramper milliliter range, but direct measurements would be necessaryto make precise statements. If this explanation is correct,then the intraperitoneal administration of doxycycline may proveadvantageous because the issue of uptake via the intestine iseliminated. Clearly, however, data obtained in vitro have tobe transferred very cautiously to experiments with animals.On the other hand, our withdrawal study illustrates the advantageof the tet-on system in animals. If we had used the originaltet-off system, a rapid induction after withdrawal of tetracyclinewould have been virtually impossible.
Consistent with previous observations that doxycycline can passreadily through the placental barrier (21), we noticed transgeneactivation in kidneys and livers of newborn mice. It is obvious,however, that sufficient levels of doxycycline do not pass throughthe mothers’ milk, an interpretation based on the onlyvery moderate reporter gene activity at 7 and 14 d postpartum.Therefore, a sustained induction in the postnatal period hasto be obtained by intraperitoneal administration of doxycycline.
Finally, we made use of the rTALAP-1 mouse line to induciblyproduce human polycystin-2 in the kidney. Similar to what hadbeen described for a human PKD1 transgene (22), we had hopedthat the expression of the human PKD2 cDNA would lead to thedevelopment of cysts. Even after 6 mo, however, no cysts wereobserved. There may be several explanations for such a result.First, we did not reach high enough levels of polycystin-2 toinduce cystogenesis. Second, there is no critical level abovewhich wild-type polycystin-2 would lead to negative effects.Third, expression of the PKD2 cDNA in other nephron segmentsand not only in the S1 and S2 portions of the proximal tubulewould have resulted in cysts. And fourth, as a result of thespecies difference (we used the human PKD2 cDNA), the cascadetoward cyst formation was not turned on. At this point, it isnot possible to determine what explanation is the correct one,but it could very well be a combination of these factors.
Acknowledgments
We thank Frank Zimmermann and Sascha Dlugosz for performingthe microinjections to establish the transgenic mice. We thankRainer Beck for his assistance in genotyping the mice and Dr.Mazahir Hasan for his scientific input. We acknowledge the technicalexpertise of Hiltraud Hosser, Ingrid Hartmann, and BrunhildeHähnel, and the graphic work of Ton Maurer. The PKD2 cDNAwas a kind gift of Stefan Somlo. Financial support was providedby the European Union (QLRT-2000-01104). Part of this work waspresented at the 2002 meeting of the American Society of Nephrologyin Philadelphia, Pennsylvania.
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Abstract
Introduction
-galactosidase activity was detected in mice without doxycycline in the drinking water, which attests to the tight control of this system. One of the advantages of the tet system lies in its reversibility, and indeed, a virtually complete remission of transgene activity in both the kidney and liver was observed when doxycycline was withdrawn. Also examined was transactivator activity during development by exposing the mothers producing the reverse transactivator to doxycycline before mating. Transgene activity was detected in newborn kidneys and liver, indicating that sufficient amounts of doxycycline had crossed the placental barrier. During nephron development, the LAP promoter appeared to be only active in the more mature proximal tubules. Finally, the rTALAP-1 line was used to inducibly express the human PKD2 cDNA in proximal tubules of transgenic mice, but no cystic changes were detected, even after 6 mo of induction. E-mail: ralph.witzgall@vkl.uni-regensburg.de 
promoter showed expression in the striatum and septum when placed only shortly on doxycycline; with a longer induction period, expression could also be detected in the cortex and hippocampus (16). We also noticed that even though there was strong expression in proximal tubules, in many cases, not every nucleus in a given tubular profile was blue. This mosaic pattern of LacZ expression has been noted previously (17), but the underlying mechanisms are poorly understood, although it is thought to depend on chromosomal context and transgene copy number (18). 
