Cover Page The handle

Cover Page

The handle http://hdl.handle.net/1887/29985 holds various files of this Leiden University dissertation

Author: Leonhard, Wouter

Title: Recapitulating polycystic kidney disease in mice

Issue Date: 2014-12-10

Irma S. Lantinga-van Leeuwen

Wouter N. Leonhard

Annemieke van de Wal

Martijn H. Breuning

Sjef Verbeek

Emile de Heer

Dorien J.M. Peters

1Leiden University Medical Center,Center for Human and Clinical Genetics, The Netherlands

2Leiden University Medical Center, Dept. of Pathology, Leiden, The Netherlands

Genesis 2006; 44:225–232

CHAPTER 2

Transgenic mice expressing tamoxifen-inducible

Cre for somatic gene modification in renal

epithelial cells.

ABSTRACT

Gene inactivation often leads to an embryonic-lethal phenotype. In focal diseases like renal cell carcinomas and polycystic kidney disease, somatic gene inactivation in subsets of cells is likely to occur at later stages. We generated a transgenic mouse line with an inducible form of Cre recombinase for conditional gene modifications in kidney epithelial cells. To this end a 1.4-kb promoter fragment of the kidney-specific-cadherin gene (KspCad) was cloned upstream of a tamoxifen-inducible Cre recombinase (CreER^T2) encoding sequence.

Expression and activity of Cre was evaluated using RT-PCR analysis and by crossbreeding to Z/EG reporter mice. One KspCad-CreER^T2 line showed kidney-specific Cre expression and mediated recombination upon tamoxifen treatment in Z/EG reporter mice. No reporter gene expression was detected in untreated animals or in extra renal tissues upon treatment.

Within the kidneys, enhanced green fluorescent protein (EGFP) fluorescence was observed in epithelial cells in several nephronic segments. In addition, the system successfully recombined a floxed Pkd1 gene.

2

INTRODUCTION, RESULTS AND DISCUSSION

Transgenic mouse models for renal diseases can provide insight into the mechanisms and pathogenesis that underlie those diseases and can be used for therapeutic testing. Especially the recently developed strategies for conditional knockout mouse lines have great potential.

A major drawback of gene inactivation is that many genes participate in a variety of developmental and physiological processes and that homozygous inactivation often results in embryonic or prenatal lethality. Homozygous polycystic kidney disease 1 (Pkd1)-mutated embryos, for instance, die around embryonic day (E)15.5 and mice carrying homozygous mutations in the gene encoding nephrin, which is involved in glomerular filtration, show perinatal death ^1;2. To overcome these limitations, conditional gene targeting systems have been developed allowing gene inactivation under specific conditions. These spatio- temporally controlled systems are based on site specific recombinases. Cre recombinase (Cre) from bacteriophage P1 is most frequently used ³. Cre is a protein that catalyzes recombination between specific 34-bp-long DNA sequences (loxP sites), resulting in removal of intervening sequences when loxP sites are positioned in the same orientation ³.

By mating mice carrying endogenous genes in which loxP sites have been introduced via homologous recombination to transgenic mice that express Cre, genetic modifications can be established. The tissues or cells that will undergo gene modification will be determined by the promoter that directs expression of Cre. Crossbreeding with aquaporin-2 (AQP2)-Cre mice, for instance, will result in gene modifications in epithelial cells of renal collecting ducts

4 whereas the UMOD promoter can drive Cre expression in another part of the kidney, e.g.

the thick ascending limbs of the loops of Henle ⁵(see also Table 1). Another advantage of the Cre/loxP system is that it is possible to time the moment of gene inactivation, by using inducible variants of Cre recombinase ^6-8. In this way gene inactivation cannot only be delayed until adulthood, but also sequential events following gene inactivation can be followed. In the inducible CreER^T2–system, Cre is fused to a mutated estrogen receptor ligand-binding domain ⁹). This Cre fusion protein is no longer able to bind endogenous estrogen but does bind to the synthetic ligands tamoxifen (tam) and 4-hydroxytamoxifen (4-OHT). If expressed in the absence of its ligands the inducible CreER^T2 remains in the cytoplasm, where it is proposed to be bound to the heat shock protein complex ^9;10. In the presence of its ligands, CreER^T2 is translocated to the nucleus, where it can exert its enzymatic activity (Fig. 1).

Presently, a number of genetically modified mice expressing Cre in kidneys have been established; however, the majority of these transgenes are not inducible and often show expression in other organs (Table 1). Depending on the gene expression pattern of floxed target genes, these mice could be useful. To study the kidney specific role of genes with broad expression, however, renal-specific Cre mice are necessary. Therefore, we established a transgenic mouse line expressing the CreER^T2 recombinase under the control of regulatory elements of the Ksp-cadherin gene. Promoter fragments of this gene have been used by others to establish mice that express transgenes in epithelial cells of the kidneys ^11;12.

2

A 1.4 kb fragment of the Ksp-cadherin gene promoter was amplified by PCR and cloned upstream of a CreER^T2 encoding sequence (Fig. 1b). Sequence analysis confirmed the integrity of the promoter fragment.

As an initial experiment to validate the functionality of the DNA construct, a dog renal epithelial cell line (MDCK) was transiently transfected with the plasmid, and stained using anti-Cre antibodies. As expected, cytoplasmic expression of Cre was observed in untreated transfected cells. In the presence of tamoxifen, however, nuclear staining for Cre was observed (Fig. 3a). These results show that the KspCad-CreER^T2 construct is expressed in renal epithelial cells and, moreover, that Cre translocates from the cytoplasm to the nucleus upon induction.

This construct was microinjected into fertilized C57Bl6 mouse oocytes. We generated six founder mouse lines that transmitted the Cre transgene through their germline and one without germline transmission. Copy numbers of the transgene in the different founder lines were estimated by Southern blotting and varied between one and approximately forty.

Reverse transcription PCR revealed that two lines, F427 (carrying one or two transgenic copies) and F577 (carrying 4-5 copies), express Cre mRNA in the kidneys (Fig. 2a).

To determine if recombination occurred in vivo, we crossbred both RT-PCR-positive KspCad- CreER^T2 lines to a Z/EG reporter mouse line. This line carries a ß-geo cassette flanked by two loxP sites and an EGFP gene coding for enhanced green fluorescent protein ¹³ (Fig. 1c).

EGFP cannot be expressed from this transgene unless the β-geo cassette is excised. Thus, these mice only express EGFP after Cre-mediated recombination. Offspring carrying both transgenes (Cre; Z/EG) received 5 mg tamoxifen orally for five consecutive days. One week after tamoxifen-administration mice were sacrificed, and kidneys and several other tissues were collected. Cryosections of the tissues were examined by fluorescence microscopy. Line F577 showed no green fluorescence in the kidneys or in any of the other tissues examined.

Bright green fluorescent signals, however, were observed in renal epithelial cells of mouse line F427, demonstrating successful Cre-mediated recombination in vivo. Fluorescent cells were detected in the cortex and inner medulla and to a lesser extent in the outer medulla of tamoxifen-treated F427 mice (Fig. 3b). Importantly, mice not treated with tamoxifen showed no fluorescence in kidneys (Fig. 3c) and extrarenal tissues collected from treated mice, i.e. liver, heart, pancreas, lungs, spleen, brain, stomach, testis and uterus were also negative except for a very weak staining in a few cells (<0.1%) in the stomach. Concordantly, RT-PCR analysis did not detect Cre mRNA in the tissues analyzed with the exception of lungs that showed a hardly visible RT-PCR product after 35 or more cycles of amplification in the PCR reaction (Fig. 2b). Together, these results indicate that the tamoxifen-dependent Cre activity of this KspCad-CreER^T2 mouse line is highly kidney-specific.

To determine whether Cre activity is widespread or limited to specific parts of the nephron, we performed immunohistochemistry by using antibodies directed against Aquaporin-2 (AQP2), Tamm Horsfall glycoprotein (THP), and megalin. Kidney sections obtained from double transgenic mice carrying both the Cre transgene and Z/EG reporter gene were analyzed one

FIG. 1. Kidney-specific CreER^T2-mediated recombination. (A) Scheme to achieve kidney-specific deletions using the CreER^T2 system. A kidney-specific promoter (Ksp-prom) drives the expression of a tamoxifen-inducible Cre recombinase (CreER^T2). Transgenic mice carrying this CreER^T2 transgene are crossbred to mice carrying floxed DNA sequences. In the absence of tamoxifen Cre is expressed in the kidneys but Cre protein is not active. Upon tamoxifen-administration, however, Cre can mediate recombination of DNA sequences flanked by loxP sites (floxed) resulting in deletion of the intervening sequence (u; loxP site). In extrarenal tissues Cre protein is not expressed, leaving floxed transgenes intact. (B) Schematic representation of the KspCad-CreER^T2 construct. The tamoxifen- inducible CreER^T2 is placed under the control of regulatory elements of the Ksp-cadherin gene. Restriction sites used to linearize the DNA construct and for Southern blot analysis are shown: S, SalI and E, EcoRI. The probe used for genotyping by Southern blotting is shown. (C) The Z/EG reporter gene (Novak et al., 2000). In the absence of active Cre recombinase there is expression from the β-geo-cassette resulting in β-galactosidase expression.

Deletion of the floxed β-geo cassette by Cre-mediated recombination results in expression of enhanced green fluorescent protein (EGFP). pCAGGS, modified chicken β-actin promoter; β-geo, β-geo cassette (that is lacZ/

neomycin-resistance fusion gene); pA, polyadenylation sequence; EGFP, enhanced green fluorescence protein; u, loxP site. Adapted from (Novak et al., 2000).

2

week after tamoxifen-treatment. EGFP-positive cells were observed particularly in megalin- positive cells (proximal tubules) but green fluorescence was also seen in AQP2-positive cells (collecting ducts) and in a number of THP-positive cells (loops of Henle and distal tubules) (Fig.

3d). Most of the tubules contain only one or two green fluorescent cells per cross-section.

Thus, renal tubular epithelial cells and collecting duct cells show Cre activity, although in a mosaic pattern. Green fluorescence was not observed in glomeruli (data not shown). Shao et al. used a similar fragment of the Ksp-cadherin promoter to establish non-inducible Cre mouse lines and, in contrast to our findings, observed expression mainly in thick ascending limbs of loops of Henle and collecting ducts ^11;12. There are several explanations for these findings, for instance the site of integration of the transgene. Furthermore, expression of the Z/EG reporter gene seems to be mosaic, therefore, 100% recombination was not expected

13). We have tested several protocols for tamoxifen administration. Our preferred protocol is oral feeding (5 x 5 mg/day). However, in our experience, using a small number of mice and the Z/EG reporter mouse as indicator for Cre recombinase efficiency, there were no obvious differences between oral feeding (5 x 5 mg/day) and intraperitoneal injections (5 x 2 mg/

day), nor did we observe sex-dependent differences in recombination efficiency. Mice that received only a single feeding with tamoxifen showed less recombination efficiency while no major differences were seen between three and five days feeding (not shown).

FIG. 2. (A) Cre expression in kidneys of transgenic mice. RT-PCR analysis revealed Cre mRNA in kidneys from founder lines F427 and F577. The RT-PCR reaction, using 1 µg total RNA, was performed in the absence (-) and presence (+) of reverse transcriptase to control for DNA contamination. M, Marker. (B) Analysis of Cre mRNA expression in extrarenal tissues of mouse line F427. RT-PCR analysis of Cre mRNA and β-actin mRNA was performed without (-) and with (+) reverse transcriptase. Amplification of β-actin mRNA served as control for the presence of RNA.

Recombination was further analyzed by crossing the Cre mice to mice containing a floxed polycystic kidney disease 1 (Pkd1) gene (Lantinga-van Leeuwen et al., 2004). We performed PCR and Southern blot analysis for the detection of deletion in kidneys and liver before and after administration of tamoxifen. PCR analysis using primers flanking the floxed fragment, exons 2-11 of the Pkd1 gene, amplified a 142-bp PCR product only in DNA extracted from kidneys of tamoxifen-treated double transgenic animals (Cre,wt; Pkd1^lox,wt; Fig 4A). Southern blot analysis confirmed that floxed DNA excision had occurred in kidneys of tamoxifen- treated double transgenic animals (Fig. 4B). The recombination efficiency was quantified by real-time PCR on DNA isolated from mice carrying one floxed allele and one wild-type Pkd1 allele (n=3). DNA isolated from heterozygous del,wt mice (n=4) in which 50% of the Pkd1 alleles contain a deletion served as reference DNA. After tamoxifen-treatment of Cre,wt;

Pkd1^lox,wt mice approximately 20-25% of the Pkd1 alleles contain a deletion, indicating a recombination efficiency of 40-50% (Fig. 4C). No recombination was detected in double transgenic mice not treated with tamoxifen (n=3, data not shown). A five-times dilution of del,wt DNA with DNA isolated from wild-type mice (n=4) nicely showed the expected decrease in percentage of deletion alleles. Tamoxifen-administration to newborn mice, via the mother (3x 5 mg/day) resulted in similar recombination efficiencies (data not shown).

A steadily increasing number of genes involved in kidney-related diseases or renal development are now placed between loxP sites allowing conditional gene modification.

The fraction of cells undergoing Cre-mediated recombination after tamoxifen-treatment, in kidneys of mice carrying this transgene and a floxed endogenous target gene, will likely depend on the locus of the floxed genes, the moment of induction, and possibly on the induction protocols used ^14;15. An absolute knockout phenotype, however, is not always required. For example, tumorigenesis is a focal disease and also in polycystic kidney disease cysts develop in approximately <1% of the nephrons. Therefore, to study the molecular basis of those diseases an incomplete knockout may actually be advantageous.

In summary, we have generated a mouse line, F427, called KspCad-CreER^T2, with tamoxifen- inducible expression of Cre in different cell types of the renal epithelium. Importantly, other tissues and non-induced mice showed virtually no Cre activity. These mice can be used to mutate target genes in the kidneys at different timepoints during development or in adulthood.

2

FIG. 3. In vitro and in vivo analysis of KspCad- CreER^T2 (A) In vitro analysis of KspCad-CreER^T2. MDCK cells were transiently transfected with the KspCad-CreER^T2 construct, treated without (A-C) or with (D-F) tamoxifen, and stained using an antibody against Cre recombinase (shown in red). Cell nuclei were visualized with DAPI (shown in blue). (A,D) DAPI-staining of cells. (B,E) Anti-Cre staining of cells without (B) or with (E) tamoxifen. (C,F) Merged images of DAPI and anti-Cre-staining. Transfected cells showed cytoplasmic Cre localization in the absence of tamoxifen. Cre translocates to the nucleus after tamoxifen-treatment.

(B) Induced Cre-activity in kidneys of mouse line F427. Cre-mediated recombination was monitored in double transgenic mice (Cre;Z/EG). Mice received tamoxifen (5 mg) orally for five consecutive days and were sacrificed one week later. Green fluorescence cells indicate that recombination had occurred in the reporter gene. Nuclei were stained with DAPI (blue). Representative 7 µm cryosections of tissues are shown. Green fluorescent cells were present in the cortex and inner medulla of the kidneys and to a lesser extent in the outer medulla. (A,C,E) Boundary of the inner (left) and outer (right) medulla. (B,D,F) Boundary of the outer medulla (left) and cortex (right). (A,B) DAPI-staining of cells. (C,D) EGFP fluorescence. (E,F) Merged images of DAPI and EGFP-signals.

(C) Absence of Cre-activity in kidneys of untreated F427 mice. (A,B) Kidney sections of untreated double transgenic mice (Cre; Z/EG), showing that Cre is not active in kidneys of mice that were not administered with tamoxifen. (A) DAPI-staining of cells. (B) EGFP (no signal).

(D) Kidney-segment-specific activity of Cre.

Localization of Cre-mediated recombination in kidney sections from double transgenic (Cre;Z/

EG) mice after oral administration of tamoxifen for 5 consecutive days. Cryosections were stained using antibodies against megalin, a proximal tubule marker, Tamm Horsfall protein (THP, i.e.

uromodulin), a marker for the loops of Henle and distal tubules and aquaporin-2 (AQP2), a collecting duct marker. (A) anti-megalin, (D) anti-THF, (G) anti-AQP2, (B,E,H) EGFP and (C,F,I) merged images.

Green fluorescence was observed in the different cell types.

FIG. 4. KspCad-CreER^T2-mediated recombination of a floxed Pkd1 allele. (A) Agarose gel analysis of PCR products amplified from genomic DNA isolated from Cre,wt; Pkd1^lox,wt mice treated (+) or not treated (-) with tamoxifen.

K, kidney; L, liver. Del: a 142-bp deletion-specific PCR product, which was only observed in DNA extracted from kidneys of tamoxifen-treated animals. Ref: a 93-bp PCR product of Pkd1 exon 36 served as control for the presence of DNA. Positive control (+) is Pkd1 del,wt DNA; negative control (-) is water. (B) Southern blot analysis of transgenic mouse DNA digested with XbaI and hybridized with a Pkd1 intron 1 probe. The recombined floxed Pkd1 allele was detected in kidney DNA of tamoxifen-treated mice, but not in liver DNA. K, kidney; L, liver; C, control DNA indicating the floxed (2.0 kb) and recombined (7.5 kb) fragments. (C) Real-time PCR analysis of genomic DNA isolated from kidneys of Cre,wt; Pkd1^lox,wt mice treated with tamoxifen (n=3). Tamoxifen was orally administrated to adult mice in three independent experiments (5x 5 mg/day). DNA isolated from heterozygous Pkd1^del,wt mice (n=4) with a deletion in 50% of Pkd1 alleles served as reference. After tamoxifen-treatment approximately 20-25% of the Pkd1 alleles contain a deletion, showing that recombination had occurred in 40-50% of the floxed Pkd1 alleles. Pkd1 del,wt DNA 1:5-diluted with wild-type DNA (n=4) showed the expected 10% deletion allele; 50%:5. Floxed Pkd1 mice not treated with tamoxifen showed no recombination (n=3, not shown). Measurements were performed twice and in duplicate.

2

METHODS

Plasmid constructs

A 3606 bp promoter fragment of the murine Ksp-cadherin gene was amplified by PCR using PfuTurbo DNA polymerase (Stratagene) and the primers KspF1 5’-CCACACACATAAAGGGAAACTGAGG-3’ and KspR1 5’-CAAGAGCTCTCAGGCACTCACCTT-3’. In a nested PCR reaction, a 1681 bp promoter fragment was generated with the primers KspF3 5’-GGGAGCCCTTGAGACCTAGT-3’ and KspR2 5’-ACAACTGCAGTCTCCCTTGGTCCAGTTTCCAG-3’ (with attached PstI site (bold)).

The 1681 bp PCR product was digested with PstI and HindIII and the resulting 1.4 kb HindIII- PstI fragment (containing bp 2430-3825 of Genbank accession no. AF118228,) was cloned into the PstI and HindIII of pBS(KS-), and subsequently inserted into the multiple cloning site of pSE280 after Pst1 and Kpn1 digestions, generating pSE-KspCad. The DNA sequence of the promoter fragment was verified by sequencing. In parallel a 2866 bp StuI-SalI fragment of pCreER^T2 (a kind gift of P. Chambon, IGBMC, Illkirch-Cedex, France ⁹ was cloned into the SalI and StuI sites of pSE280 resulting in pSE-CreER^T2. Finally, the BssHII-PstI fragment of pSE-KspCad was cloned into the NsiI and MluI sites of pSE-CreER^T2, generating the targeting vector pSE-KspCad-CreER^T2.

Cell culture and transfection

Madin-Darby canine kidney (MDCK) cells (ATCC, Rockville, USA) were grown to 60%

confluency on coverslips in a 24-wells plate. The cells were transfected with 250 ng of pSE-KspCad-CreER^T2 per well using Transfast^TM transfection reagent (Promega), and treated overnight with or without 100 nM of 4-OHT-tamoxifen (Sigma-Aldrich Chemie). Cells were fixed with cold acetone/methanol (2:1) for 10 minutes, washed with PBS, and blocked with PBS/5% nonfat dry milk for 1 hour. The cells were washed with PBS and incubated overnight with a polyclonal antibody against Cre (1:3000; Novagen) in PBS/2% BSA. Upon washing with PBS the cells were incubated with Alexa 594-conjugated goat-anti-rabbit (1:2000;

Molecular Probes) for 1 hour, washed with PBS and rinsed with distilled water. Coverslips were embedded in gelvatol with 1 μg/ml 4’,6-diamidino-2-phenylindole.2HCl (DAPI) and analyzed with a Leica fluorescence microscope.

Generation of transgenic mice

The targeting construct was linearized by digestion with SalI and purified transgene DNA was microinjected into pronuclei of fertilized oocytes of C57Bl6/jico mice (Charles River Lab).

Genotypes of transgenic mice were determined by PCR analysis of tail DNA. The primers used for amplification of the Cre recombinase gene were: CreI 5’-GCCTGCATTACCGGTCGATGCAAC-3’

and CreII 5’-GTGGCAGATGGCGCGGCAACACCATT-3’. The presence of the transgene was verified by Southern blot analysis using this 725-bp Cre PCR product as probe and digesting

genomic DNA with EcoRI, resulting in a 2.1 kb hybridization band. All experiments using mice were approved by the local animal experimental committee of the Leiden University Medical Center and by the Commission Biotechnology in Animals of the Dutch Ministry of Agriculture.

Tamoxifen-treatment of mice

An aliquot of 600 mg tamoxifen-free base (Sigma, Cat no. T5648) was dissolved in 900 µl ethanol and suspended in 5.1 ml sunflower oil. This 100 mg/ml tamoxifen solution was sonicated for 2 min (2x 1 min using 6-seconds pulses) and then stored at -20ºC. For oral administration the solution was thawed at 55ºC and administered to mice (50 µl) by a feeding needle. For intraperitoneal (i.p.) injections of mice (50 µl), the 100 mg/ml tamoxifen solution was diluted 2.5 times in ethanol/oil. Mice were analyzed 5 days after the last tamoxifen administration. For five days 5 mg tamoxifen/day was administrated.

RT-PCR analysis

Tissues were frozen in liquid nitrogen and stored at -80ºC. RNA was isolated using the GenElute mammalian Total RNA kit (Sigma), and cDNA was synthesized with SuperScript II reverse transcriptase (GibcoBRL). One µg total RNA was transcribed into cDNA and amplified by PCR using the Cre primers 5’-GACCAGGTTCGTTCACTCA-3’ and 5’-TAGCGCCGTAAATCAAT-3’.

As a cDNA synthesis control, a 75-bp fragment of the β-actin mRNA was amplified using the primers 5’-GCTCTGGCTCCTAGCACCAT-3’ and 5’-GCCACCGATCCACACAGAGT-3’.

Detection of Enhanced Green Fluorescent Protein

To analyze Cre activity in vivo, KspCad-CreER^T2 mice were crossed with Z/EG reporter mice ¹³. Offspring carrying both transgenes were treated with tamoxifen and sacrificed one week later. Tissues were fixed for 1 hour in 10% buffered formalin with 10% sucrose, embedded in OCT (Tissue Tek, Sakura), frozen in liquid nitrogen and stored at -80ºC. Tissue sections (7 µM thick) were cut with a cryostat, air-dried for 20 min, examined with a Leica CW4000 microscope, and photographed using a Leica DC350FX digital camera. As controls, samples from single transgenic littermates and untreated double transgenic animals were photographed under identical exposure conditions.

Real-time PCR and Southern blot analysis

To determine recombination efficiency at a floxed Pkd1 gene, real-time PCR analysis of DNA samples was performed using 1x Evagreen (Gentaurer), 1x ROX (Invitrogen) and 1 unit AmpliTaq DNA polymerase (Applera Europe), and the 7900HT Fast Real time PCR System (Applied Biosystems). All measurements were performed twice and in duplicate.

Amplification cycles were 95ºC for 2 min, followed by 40 cycles at 95ºC for 20 s, at 60ºC for 20 s and at 72ºC for 20 s, and a final extension at 72ºC for 1 min. To detect the deletion the

2

following primers were used: mPkd11-DelF3 5’-TTACGGGCTGCAGGAATTCGAT-3’, in intron 1, and mPkd1-DelR2 5’-ATGCCCAAGATGAGGACAATGC-3’, in intron 11, which yield a 142-bp PCR product after Cre-mediated recombination at the Pkd1 locus. As reference PCR, we amplified a 93-bp PCR product from exon 36 of the Pkd1 gene using the primers DNARefFor2 5’-TGCTCTTGGTAGCTGTAGCTGT-3 and DNARefRev2 5’-AGCTGCTTGAGAGAAGCCACAT-3’.

For Southern blot analysis, tissues were frozen in liquid nitrogen and stored at -80ºC.

Genomic DNA was isolated using Qiagen genomic-tip 100/G columns (Qiagen). DNA was digested with XbaI, separated by 0.8% agarose gel eletrophoresis, transferred to Hybond N+

(Amersham) and hybridized with an intron 1 Pkd1 probe, detecting a 1.9 kb fragment in a wild-type Pkd1 allele, a 2.0 kb fragment in a floxed Pkd1 allele and an approximately 7.5 kb fragment in a recombined (deletion) allele.

Immunostaining

Kidney-segment identity was characterized using antibodies against Aquaporin-2 (Calbiochem), against Tamm Horsfall glycoprotein (Uromodulin) (Cappel-Organon Teknika) and against megalin ¹⁶. The antibodies were used at dilutions of 1: 300, 1: 5000 and 1:500, respectively. Cryosections were incubated with the primary antibody in PBS/2%

BSA for 1 hour. After repeated washing with PBS the sections were incubated with Alexa 594-conjugated goat-anti-rabbit (1:2000; Molecular Probes) for 1 hour, washed with PBS and rinsed with distilled water.

ACKNOWLEDGEMENTS

We thank Jill Claassen and Jos van der Kaa for technical assistance, P. Chambon for the pCreER^T2 plasmid and Stefan White for reading the article.

REFERENCES

1. Lu W, Peissel B, Babakhanlou H, Pavlova A, Geng L, Fan X, Larson C, Brent G, Zhou J: Perinatal lethality with kidney and pancreas defects in mice with a targetted Pkd1 mutation. Nat Genet 17:179-181, 1997

2. Donoviel DB, Freed DD, Vogel H, Potter DG, Hawkins E, Barrish JP, Mathur BN, Turner CA, Geske R, Montgomery CA, Starbuck M, Brandt M, Gupta A, Ramirez-Solis R, Zambrowicz BP, Powell DR: Proteinuria and perinatal lethality in mice lacking NEPH1, a novel protein with homology to NEPHRIN. Mol Cell Biol 21:4829-4836, 2001

3. Branda CS, Dymecki SM: Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice. Dev Cell 6:7-28, 2004

4. Ge Y, Ahn D, Stricklett PK, Hughes AK, Yanagisawa M, Verbalis JG, Kohan DE: Collecting duct- specific knockout of endothelin-1 alters vasopressin regulation of urine osmolality. Am J Physiol Renal Physiol 288:F912-F920, 2005

5. Stricklett PK, Taylor D, Nelson RD, Kohan DE: Thick ascending limb-specific expression of Cre recombinase. Am J Physiol Renal Physiol 285:F33-F39, 2003

6. Kuhn R, Schwenk F, Aguet M, Rajewsky K: Inducible gene targeting in mice. Science 269:1427- 1429, 1995

7. Lewandoski M: Conditional control of gene expression in the mouse. Nat Rev Genet 2:743-755, 2001

8. Stricklett PK, Nelson RD, Kohan DE: The Cre/loxP system and gene targeting in the kidney. Am J Physiol 276:F651-F657, 1999

9. Indra AK, Warot X, Brocard J, Bornert JM, Xiao JH, Chambon P, Metzger D: Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases. Nucleic Acids Res 27:4324-4327, 1999

10. Brocard J, Warot X, Wendling O, Messaddeq N, Vonesch JL, Chambon P, Metzger D: Spatio- temporally controlled site-specific somatic mutagenesis in the mouse. Proc Natl Acad Sci U S A 94:14559-14563, 1997

11. Shao X, Johnson JE, Richardson JA, Hiesberger T, Igarashi P: A minimal Ksp-cadherin promoter linked to a green fluorescent protein reporter gene exhibits tissue-specific expression in the developing kidney and genitourinary tract. J Am Soc Nephrol 13:1824-1836, 2002

12. Shao X, Somlo S, Igarashi P: Epithelial-specific Cre/lox recombination in the developing kidney and genitourinary tract. J Am Soc Nephrol 13:1837-1846, 2002

13. Novak A, Guo C, Yang W, Nagy A, Lobe CG: Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision. Genesis 28:147-155, 2000 14. Vooijs M, Jonkers J, Berns A: A highly efficient ligand-regulated Cre recombinase mouse line shows

that LoxP recombination is position dependent. EMBO Rep 2:292-297, 2001

15. Feil R, Wagner J, Metzger D, Chambon P: Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem Biophys Res Commun 237:752-757, 1997 16. Verroust PJ, Birn H, Nielsen R, Kozyraki R, Christensen EI: The tandem endocytic receptors megalin

and cubilin are important proteins in renal pathology. Kidney Int 62:745-756, 2002