Podocyte Cell–Specific Expression of Doxycycline Inducible Cre Recombinase in Mice
Juuso Juhila*,
Ramon Roozendaal,
Markus Lassila*,
Sjef J. Verbeek and
Harry Holthofer*
* Research Program in Molecular Medicine, Biomedicum, University of Helsinki, Helsinki, Finland; and Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
Address correspondence to: Dr. Harry Holthofer, Biomedicum Helsinki, Department of Molecular Medicine, University of Helsinki, Helsinki, FI-00014 Finland. Phone: +358-9-19125500; Fax: +358-9-19125501; E-mail: harry.holthofer{at}helsinki.fi
Received for publication June 14, 2005.
Accepted for publication December 13, 2005.
Conventional silencing of many podocyte-specific genes in miceis associated with embryonic or perinatal lethality. Therefore,it would be of great importance to generate mouse models thatallow the modification of genes that are expressed in podocytesat later stages of age. Herein is described a transgenic mousewith doxycycline-inducible podocyte-specific expression of Crerecombinase. For the generation of this binary system, a singletransgenic construct that contained two separate genes was used:One encoding the optimized M2 version of the doxycycline-dependenttranscription transactivator reverse tetracycline-controlledtranscriptional activator (rtTA) under control of the humanpodocin (NPHS2) promoter and the other encoding the recombinaseCre under control of the rtTA/doxycycline-responsive minimalcytomegalovirus (CMV) Tet operator sequence 7 promotor. Microinjectionof the JRC-CRE construct in fertilized oocytes from FVB/N miceresulted in 16 transgenic founders. Double-transgenic offspringfrom breeding of a selected founder with the Z/AP reporter mouseshowed alkaline phosphatase staining only upon doxycycline administrationand exclusively in podocytes. These data indicate that thisnew inducible Cre recombinase mouse line is an excellent toolin conditional, kidney glomerular podocyte-specific gene deletionin adult mice.
The kidney glomerulus produces the primary urine. The essentialstructural element that is responsible for this function, theglomerular filtration barrier, consists of a fenestrated capillaryendothelium, a layered glomerular basement membrane (GBM), andglomerular visceral epithelial cells called podocytes. Podocytescover the GBM from the outside and present with highly orderedcellular extensions, foot processes, linked to each other bythe slit diaphragm (1). This structure seems to be crucial inregulating the passage of circulating plasma proteins into primaryurine. Glomerular diseases with proteinuria manifest with characteristicmorphologic changes of the podocytes, including loss of theslit diaphragm and foot process effacement. The degree of thesechanges is associated with the severity of proteinuria (2).Children with congenital nephrotic syndrome of the Finnish type(CNF) have severely impaired podocyte function and, before currenttransplantation therapy, used to die within a few months afterbirth as a result of massive proteinuria. After a meticuloussearch for the responsible genetic defect in CNF, a nonfunctionalNPHS1 gene was found (3). NPHS1 encodes the slit diaphragm–specificprotein nephrin. Consistent with the human CNF disease phenotype,nephrin knockout (KO) mice die within a few days after birth,and the podocytes show closely similar morphologic abnormalitiesas the glomeruli of patients with CNF (4,5). Furthermore, otherdistinct single-gene mutations of genes that are expressed preferentiallyin the podocytes are associated with severe glomerular phenotypesin humans and mice. These include Wilms tumor gene (WT-1) (6),podocin (7,8), -actinin-4 (9,10), and CD2AP (11). Conventionalgene KO are not always useful tools to study podocyte-specificgenes because deficiency of these genes results in perinataldeath, thus preventing their detailed analysis within the fullyfunctional kidney of the adult mouse.
To circumvent this, a conditional KO strategy was designed onthe basis of the use of transgenic mice with doxycycline-induciblepodocyte-specific Cre recombinase expression. The binary tetracycline-controlledtranscriptional activation system is a powerful tool in achievingtemporal control of transgene expression in mammals (12). Forthis purpose, two different tetracycline-controlled activatorsare widely used in transgenic mice. The original transcriptionalactivator (tTA) activates transcription of the target gene inthe absence of tetracycline, after binding to the Tet operatorsequence located in the 5' region of the target gene (Tet-offsystem) (13). The reverse tetracycline-controlled transcriptionalactivator (rtTA) binds to Tet operator sequence 7 and activatestranscription of the target gene in the presence of tetracycline(Tet-on system) (12). Recently, improved rtTA versions thatrequire far lower tetracycline concentration for the inductiontogether with minimal background expression of Cre recombinasein the absence of tetracycline were developed (14).
Here we present a podocyte-specific doxycycline-inducible Cretransgenic mouse generated by a single oocyte injection stepbased on a modified approach described by Utomo et al. (15).Within one transgene construct, all required DNA elements werecombined: The Cre recombinase gene under control of the rtTA-M2(14) transcription factor together with the rtTA-M2 encodinggene under control of a 2.5-kb fragment of the mouse NPHS2 (podocin)promoter. The use of this single-transgene construct obviatesthe need for multiple rounds of transgenesis and elaborate crossings.This mouse will enable studies of the phenotypic consequencesof loss of defined proteins in the podocytes in the adult mouseby direct crossing with mice with appropriate floxed alleles.
Cloning of Doxycycline-Inducible Cre Recombinase Construct
The p2.5PodocinpnlacF plasmid, which contains 2.5 kb of genomicsequence of human NPHS2 gene located 5' to the translation initiationcodon, was provided by Dr. Lawrence Holzman (16,17). The "core"construct was provided by W.H. Lee (15). An EcoRI-HindIII fragmentfrom the original rtTA of the core construct was replaced withthe optimized version, rtTA-M2 (14). In our hands, several attemptsto clone promoters into the designated cloning site failed asa result of instability of the construct. Therefore, the twofunctional EcoRI-ScaI fragments from the core construct werereconstituted in a pBR322-based, low-copy backbone after modificationof the polylinker, generating NotI restriction sites insteadof ScaI on both sides of the transgene. Finally, unique SalIand SmiI sites were introduced at the position of the EcoRIsite. The resulting construct was stable and designated RRC-M2(Figure 1).
Figure 1. In a binary reverse doxycycline-regulated vector, an NPHS2 (podocin) promoter drives the expression of the recombinant inducible transcription factor reverse tetracycline-controlled transcriptional activator (rtTA; M2). In the presence of doxycycline, rtTA binds to the Tet operator sequence element linked to a minimal cytomegalovirus (CMV) promoter and drives the expression of Cre recombinase.
The unique XbaI site in p2.5PodocinpnlacF was replaced by aSalI using a linker (sense 5'-ctagcagatctaagcagtcgaca-3' andantisense 5'-ctagtgtcgactgcttagatctg-3'). The resulting SalI-NcoIfragment of the podocin promoter was cloned into the SalI-SmiI–digestedRRC-M2 construct as a SalI-blunt fragment. The NcoI end wasblunted using T4 polymerase (New England Biolabs, Ipswich, MA).Identity of the clones was verified by restriction mapping andsequencing. The final construct is referred to as JRC-CRE (Figure 1).
Generation and Identification of JRC-CRE Transgenic Mice
The podocin-rtTA construct was released from the plasmid vectorbackbone by digestion with NotI and purified by gel electrophoresis,and DNA was extracted using a QIAEX II Gel Extraction Kit (QiagenGmbH, Hilden, Germany). The purified construct DNA was introducedinto the pronuclei of fertilized oocytes from the FVB/N mouseby microinjection using standard techniques. All experimentshad approval of the local committee for laboratory animal welfareof the University of Helsinki.
Transgenic mice were identified by PCR on tail genomic DNA (50to 100 ng), using CreForward (5'-gaccaggttcgttcactca-3') andCreReverse (5'-tagcgccgtaaatcaat-3') primers and 30 cycles of95°C (30 s), 54°C (30 s), and 72°C (30 s) and afinal extension of 72°C for 5 min. The reaction was performedwith HotStar Taq DNase polymerase (Qiagen) in a total reactionvolume of 20 µl, including 400 nM of both primers. Sampleswere analyzed by standard agarose gel electrophoresis (1.5%gel).
Phenotype characterization and behavioral screening was madeusing modification of the Irwin procedure (18). Observed parametersare listed in Table 1.
Table 1. Phenotype characterization of JRC-CRE micea
Doxycycline Administration in JRC-CRE Mice
F1 littermates were used to analyze Cre recombinase proteinexpression in mouse tissues. Doxycycline was administered indrinking water (0.2 mg/ml in 5% sucrose), and administrationwas started at the age of 8 to 10 wk. Water was changed twicea week, and the bottles were covered with aluminum foil to preventdegradation by light.
Reverse Transcription–PCR of Cre-Specific mRNA of JRC-CRE Mouse Kidneys
After cervical dislocation, mouse tissues were dissected immediately,snap-frozen in liquid nitrogen, and stored at –78°Cuntil used. Total RNA was extracted from homogenized lysatesof the cortex of frozen adult kidneys using the RNeasy MiniKit (Qiagen) according to the manufacturer’s instructions.
cDNA was prepared from 1 µg of RNA with the M-MLV reversetranscriptase (Promega, Madison, WI) using random primers (RocheDiagnostics GmbH, Mannheim, Germany; RT+). To confirm the RNAorigin of the PCR signals, we also analyzed each sample withoutthe reverse transcriptase reaction (RT–). Cre-specificcDNA was amplified by using the Cre thermocycling protocol (25cycles; see above), and NPHS1 primers were used as housekeepinggene controls: NPHS1Forward 5'-cctggagctaccctgcata-3 and NPHS1Reverse5'-ggacttggtaaggcagcaaa-3'.
Generation of Double-Transgenic JRC-CRE x Z/AP Reporter Line
Generation of double-transgenic JRC-CRE x Z/AP reporter lineJRC-CRE transgenic founder was bred with Z/AP (lacZ/human placentalalkaline phosphatase) reporter mice (19) to obtain double-transgenicJRC-CRE x Z/AP reporter mice. The Z/AP reporter mouse line carriesa -geo cassette flanked by two loxP sites and an alkaline phosphatase(AP) gene (19). AP cannot be expressed in this transgene unlessthe -geo cassette is excised. Thus, these mice express AP afterCre-mediated recombination. Double-transgenic mice were identifiedby PCR on tail genomic DNA for Z/AP using Z/APForward (5'-ctgctaaccatgttcatgcc-3')and Z/APReverse (5'-ggcctcttcgctattacg-3') primers and Cre primers(see above), using the same amplification protocol. Offspringthat carried both transgenes (JRC-Cre x Z/AP) were administered0.2 mg/ml doxycycline in the drinking water for 14 consecutivedays.
AP Substrate Staining in JRC-CRE x Z/AP Mouse Kidney
For testing the presence of AP, the JRC-CRE x Z/AP and wild-typecontrol mice either received (n = 3) or did not receive (n =3) doxycycline, and tissues were collected as described above.The list of the tissues that were tested is provided in Table 2.
Table 2. List of tissues of JRC-CRE x Z/AP mice tested by AP substrate staininga
Before staining, slides were refixed in cold PBS that contained0.2% glutaraldehyde for 10 min. After fixation, slides werewashed in PBS for 5 min and endogenous AP was inactivated byincubating slides in PBS at 75°C for 30 min. Slides thenwere rinsed with PBS, washed in AP buffer (100 mM Tris-HCl [pH9.5], 100 mM NaCl, and 10 mM MgCl2) for 5 min, followed by incubationwith BM purple (Roche) for 1 h at 4°C to detect AP activity.Gurr Aquamount (BDH, Poole, UK) was used for mounting.
Immunofluorescence Staining in Mouse Tissues
Immunofluorescence staining was performed on frozen tissue sections(6 µm). The sections were fixed in acetone (–20°C)for 10 min on ice, washed with PBS, and blocked with CAS-blocksolution (Zymed Laboratories Inc., San Francisco, CA) for 10min. Sections were incubated overnight with the primary antibodyin ChemMate Antibody Diluent (DakoCytomation, Glostrup, Denmark).The used primary antibodies were anti-CRE polyclonal antibody(Stratagene, La Jolla, CA; 1:1000), anti-human placental APpolyclonal and monoclonal antibodies (Biomeda, Foster City,CA; 1:50), anti–WT-1 polyclonal antibody (Santa Cruz Biotechnology,Santa Cruz, CA; 1:50), and anti-synaptopodin mAb (Progen BiotechnikGmbH, Heidelberg, Germany; 1:200). After washing, the sectionswere incubated with affinity-purified TRITC-conjugated goatanti-rabbit IgG (Jackson ImmunoResearch Laboratories Inc., WestGrove, PA; 1:200) and/or affinity-purified FITC-conjugated rabbitanti-mouse IgG (DakoCytomation; 1:200) in ChemMate AntibodyDiluent for 30 min and mounted with Vectashield Mounting Mediumfor fluorescence (Vector Laboratories Inc., Burlingame, CA).
JRC-CRE Mice
Before pronuclear injection, the transgene was tested in vitroin A293 cell culture, and Cre recombinase expression was localizedwithin the nuclei and was tightly regulated by doxycycline administration(data provided as supplemental data; Supplemental Figures 1and 2). Sixteen of 122 offspring were identified to carry thetransgene after oocyte injections. Founders were bred with C57Bl/6wild-type mice. All founders transmitted the transgene to theiroffspring. All founders and their F1 offspring were fertile,and the phenotype was normal by gross morphology and by lightmicroscopy of the kidneys (data not shown). After a 2-wk administrationperiod of doxycycline, the phenotype of JRC-CRE litters remainednormal, and no difference in the body weight of the mice comparedwith wild-type controls was observed (Table 1). The behavioralobservations were a modification of the Irwin procedure (18).
Cre recombinase expression was tested in tissues from all founderlines by using AP substrate staining (Table 2). Three of 16founder lines showed Cre recombinase expression in podocytesalso without doxycycline administration. Two of 16 founder linesexpressed Cre recombinase also in heart tissue after 2 wk ofdoxycycline administration. For the further experiments, onefounder line was selected on the basis of the high Cre recombinaseexpression level in podocytes and lack of leakiness withoutdoxycycline and in other tissues in preliminary experiments(data not shown).
Doxycycline-Inducible Expression of Cre Recombinase mRNA
Cre recombinase mRNA expression was studied in the kidneys ofJRC-CRE–positive mice with or without doxycycline administrationby reverse transcription–PCR. Doxycycline administrationwas started at the age of 8 to 10 wk. Expression of Cre recombinasemRNA was observed only after 2 wk of doxycycline treatment (Figure 2).
Figure 2. Reverse transcription–PCR of Cre-specific mRNA of JRC-CRE mouse kidneys. RNA was isolated after 2 wk of doxycycline (0.2 mg/ml) administration from JRC-CRE–positive mouse kidneys. Genomic DNA that was isolated from JRC-CRE mouse tails was used as positive control. Expected fragment size for Cre recombinase genomic and cDNA was 420 bp for both. For NPHS1, genomic DNA fragment size was 570 bp and for cDNA was 317 bp.
Expression of Cre Recombinase Protein in Adult Mice
To determine the spatial localization of Cre recombinase proteinexpression in kidney, we examined renal tissues by Cre antibodyimmunofluorescence staining of cryosections in JRC-CRE mice.Expression of Cre recombinase was prominent in the peripheryof glomeruli, in localization consistent with podocytes (Figure 3, D and F).Cre recombinase protein expression was absent inwild-type mice (Figure 3, A and B) and in JRC-CRE–positivemice that were not treated with doxycycline (Figure 3, C and E).Cre recombinase protein expression was not found in otherobserved tissues (heart, liver, and pancreas) in JRC-CRE micewith or without doxycycline administration (Figure 4).
Figure 3. Immunofluorescence staining in JRC-CRE mouse kidneys. (A) Nuclear staining by DAPI in wild-type control mouse. Cre recombinase protein expression in wild-type control mouse (B), JRC-CRE mouse without doxycycline treatment (C), doxycycline-treated JRC-CRE mouse (D), JRC-CRE mouse without doxycycline treatment (E), and doxycycline-treated JRC-CRE mouse (F). (G through J) Alkaline phosphatase substrate staining in JRC-CRE x Z/AP without doxycycline treatment (G and I) and in doxycycline-treated JRC-CRE x Z/AP mouse kidneys (H and J). Magnification, x200 in A to D; x40 in G and H; and x600 in E, F, I, and J.
Figure 4. Immunofluorescence staining in JRC-CRE mouse tissues. Cre recombinase protein expression in pancreas without doxycycline treatment (A), pancreas after doxycycline (B), liver without doxycycline treatment (C), liver after doxycycline treatment (D), heart without doxycycline treatment (E), and heart after doxycycline treatment (F). Magnification, x200.
Functional Cre Recombinase Activity
To test Cre recombinase functionality (capability to catalyzerecombination between two loxP sites), we performed cross-breedingwith Z/AP reporter mouse line. Kidney sections from double-transgenicmice with or without 2 wk of doxycycline administration wereanalyzed by AP substrate staining. AP expression was found inthe podocytes only after the doxycycline treatment (Figure 3, G through J).Wild-type control specimen did not show any staining.Other tissues of the double transgenic mice that were studieddid not show any staining measured by AP substrate (Table 2;Supplemental Figure 3) or immunofluorescence assay (SupplementalFigure 4).
Localization of AP Protein in the Glomeruli
To determine the localization of human placental AP proteinexpression, we examined the kidney tissues with several antibodiesand immunofluorescence microscopy in JRC-CRE x Z/AP mice thatwere treated with doxycycline. Staining of AP co-localized withsynaptopodin protein but not with WT-1 staining (Figure 5).A total of 100 glomeruli from several sections were counted.All glomeruli that were positive for synaptopodin also showedhigh expression of AP in doxycycline-treated double-transgenicmice. AP protein expression was not observed in double-transgenicmice without doxycycline (Figure 5) or in wild-type controls(data not shown). Nevertheless, it cannot be claimed that allpodocytes express Cre recombinase upon induction, but our datasuggest that the percentage is very substantial.
Figure 5. Immunofluorescence stainings in JRC-CRE x Z/AP mouse kidneys. (A, C, and E) Double stainings in mice without doxycycline treatment. (B, D, and F) Double stainings in mice after doxycycline (0.2 mg/ml) treatment. Magnification, x400.
Here we describe a transgenic mouse line with tightly regulatedinducible expression of the Cre recombinase exclusively in thekidney podocytes in the adult mouse. This viable and fertiletransgenic mouse line was generated by injection of oocyteswith a single DNA construct that contained a podocyte-specificpromoter (NPHS2) that drives the expression of rtTA M2 and aCre recombinase gene whose expression is initiated by rtTA inthe presence of doxycycline.
There are several advantages to using a single inducible transgenevector that confers site-specific expression. First, the celltype specificity can be selected easily by a single cloningstep that exchanges the one desired cell type–specificpromoter for the other. Moreover, any gene of interest can bemade inducible and cell type specific by putting it in the placeof the Cre recombinase gene. Second, the single construct givessuperior speed and economy, because no multiple rounds of oocyteinjections and further breeding steps are needed. Third, thetransgene expression can be turned off as desired, giving invaluabletargeting, e.g., to a particular stage of embryonic developmentor in adult life, thus overcoming the limitations of embryoniclethality or redundancy.
Because of perinatal lethality, conventional KO mouse modelsoften fail to reveal the detailed biologic function and dynamicsof specific proteins within the adult, fully functional kidney.In case of nephrin, the structure–function relationshipis relatively well described, whereas several key questionsconcerning gene regulation in vivo still remain unresolved.Site-specific recombinases (e.g., Cre-loxP system) are excellenttools to establish tissue-specific gene deletions in the adultmouse. The cell type specificity depends on the expression characteristicsof the promoters that drive the expression of the recombinasegene. Recently, two podocyte-specific Cre recombinase mouselines were generated. In the first line, Cre recombinase expressionis under the control of the NPHS1 (nephrin) promoter (20) andin the other line under NPHS2 promoter (17). Moeller et al.(21) used the Cre-loxP system to tag podocytes in a murine modelof crescentic glomerulonephritis and was able to show that cellswithin the glomerular crescents are derived from the podocytecell lineage. One drawback with these two models is that Crerecombinase is already expressed at early developmental stagesof the embryo. Subsequently, the phenotype after gene silencingduring embryogenesis is often lethal.
Thus, inducible cell type–specific Cre recombinase mouselines will be needed to silence genes that are expressed inpodocytes in the adult animal. Inducible gene deletion has beenused for many mouse tissues, including the liver (22,23), heart(24,25), and, e.g., for molecules such as prostaglandin receptors(26). Previously, inducible Cre recombinase mouse lines havebeen generated, particularly one specific for the kidney collectingducts cells (27). Bugeon et al. (28) developed a mouse linein which a transgene encoding the mutated estrogen receptor–Crerecombinase fusion protein was introduced into the mouse genome.Animals were crossed with Z/AP reporter mice, and after injectionof the inducer drug tamoxifen, Cre fusion protein translocatesto the nuclei of podocytes, where it becomes active and mediatesrecombination of DNA carrying loxP target sequences. These animalsprovide for the first time a tool for silencing genes in podocytesof adult animals, but complete tissue-specific activity wasnot achieved (28). Furthermore, doxycycline-controlled podocin-specific-galactosidase expression has been reported under control ofNPHS2 promoter (29). Still, no kidney-specific inducible genedeletion has been published.
This novel doxycycline-inducible Cre recombinase mouse linewill be useful for conditional deletion of essential podocyteproteins and study of their functions in detail in adult mice.This is important for further diagnostic and pharmacologic developmentalplatforms. Furthermore, the binary construct can serve as aninvaluable tool to overexpress particular podocyte-specificproteins and to tightly control inducible gene deletion in othertissues by changing cell type–specific promoters, as aconcept already shown by Utomo et al. (15) with the originalconstruct.
Acknowledgments
This study was supported by grants from the Academy of Finland(106906), Alfred Kordelin Foundation, Orion Pharma Foundation,Paavo Nurmi Foundation, European Union (QLGI-CT-2000-00619),the Sigrid Juselius Foundation, and Finnish Diabetes Foundation.
Part of this work was presented at the American Society of NephrologyMeeting, St. Louis, MO, October 27 to November 1, 2004.
We thank Dr. Lawrence Holzman for providing the podocin promoterand When Hwa Lee for the core construct. Special acknowledgmentsare also due to Ms. Kristiina Nokelainen, Mr. Jarmo Koponen,and Mr. Marcel Messing for the excellent technical support.
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Published online ahead of print. Publication date availableat www.jasn.org.
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Abstract
Introduction
-actinin-4 (9,10), and CD2AP (11). Conventional gene KO are not always useful tools to study podocyte-specific genes because deficiency of these genes results in perinatal death, thus preventing their detailed analysis within the fully functional kidney of the adult mouse. 
-geo cassette flanked by two loxP sites and an alkaline phosphatase (AP) gene (19). AP cannot be expressed in this transgene unless the 
