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• W2051209896 abstract "Mouse models for autosomal-dominant polycystic kidney disease (ADPKD), derived from homozygous targeted disruption of Pkd1 gene, generally die in utero or perinatally because of systemic defects. We introduced a loxP site and a loxP-flanked mc1-neo cassette into introns 30 and 34, respectively, of the Pkd1 locus to generate a conditional, targeted mutation. Significantly, before excision of the floxed exons and mc1-neo from the targeted locus by Cre recombinase, mice homozygous for the targeted allele appeared normal at birth but developed polycystic kidney disease with a slower progression than that of Pkd-null mice. Further, the homozygotes continued to produce low levels of full-length Pkd1-encoded protein, suggesting that slight Pkd1 expression is sufficient for renal cyst formation in ADPKD. In this viable model, up-regulation of heparin-binding epidermal growth factor-like growth factor accompanied increased epidermal growth factor receptor signaling, which may be involved in abnormal proliferation of the cyst-lining epithelia. Increased apoptosis in cyst epithelia was only observed in the later period that correlated with the cyst regression. Abnormalities in Na+/K+-ATPase, aquaporin-2, and vasopressin V2 receptor expression were also identified. This mouse model may be suitable for further studies of progression and therapeutic interventions of ADPKD. Mouse models for autosomal-dominant polycystic kidney disease (ADPKD), derived from homozygous targeted disruption of Pkd1 gene, generally die in utero or perinatally because of systemic defects. We introduced a loxP site and a loxP-flanked mc1-neo cassette into introns 30 and 34, respectively, of the Pkd1 locus to generate a conditional, targeted mutation. Significantly, before excision of the floxed exons and mc1-neo from the targeted locus by Cre recombinase, mice homozygous for the targeted allele appeared normal at birth but developed polycystic kidney disease with a slower progression than that of Pkd-null mice. Further, the homozygotes continued to produce low levels of full-length Pkd1-encoded protein, suggesting that slight Pkd1 expression is sufficient for renal cyst formation in ADPKD. In this viable model, up-regulation of heparin-binding epidermal growth factor-like growth factor accompanied increased epidermal growth factor receptor signaling, which may be involved in abnormal proliferation of the cyst-lining epithelia. Increased apoptosis in cyst epithelia was only observed in the later period that correlated with the cyst regression. Abnormalities in Na+/K+-ATPase, aquaporin-2, and vasopressin V2 receptor expression were also identified. This mouse model may be suitable for further studies of progression and therapeutic interventions of ADPKD. Autosomal-dominant polycystic kidney disease (ADPKD) is one of the most common life-threatening inherited diseases, characterized by the development of gradually enlarging renal cysts and a progressive loss of normal kidney tissue that can lead to chronic renal failure. It affects between 1 in 600 and 1 in 1000 live births in all ethnic groups worldwide. Cysts in the liver, pancreas, and spleen as well as a variety of cardiovascular, cerebrovascular, and connective tissue abnormalities are also common.1Torres VE Harris PC Autosomal dominant polycystic kidney disease.Nefrologia. 2003; 23: 14-22PubMed Google Scholar The fluid-filled cysts in an affected kidney are lined by monolayer epithelial cells derived from every segment of the nephron, but predominantly from the collecting duct.2Verani RR Silva FG Histogenesis of the renal cysts in adult (autosomal dominant) polycystic kidney disease. A histochemical study.Mod Pathol. 1988; 1: 457-463PubMed Google Scholar The pathogenesis of the renal cyst formation and progression is currently thought to involve 1) dysregulated epithelial cell proliferation and differentiation, 2) alternations in specific membrane protein polarity, 3) changes in cell-matrix interactions, and 4) abnormality in fluid accumulation.3Wilson PD Polycystic kidney disease.N Engl J Med. 2004; 350: 151-164Crossref PubMed Scopus (604) Google Scholar Since there is currently no effective treatment for ADPKD except for dialysis and renal transplantation, much attention has been focused on understanding the molecular mechanism underlying the pathogenesis of renal cyst expansion. Throughout the past decade, the mutated genes responsible for ADPKD were identified by positional cloning strategies. In most cases, ADPKD is recognized as a monogenic disorder caused by mutation in two genes: PKD1, accounting for ∼85% of cases; and PKD2, accounting for ∼10% of cases.4European Polycystic Kidney Disease Consortium The polycystic kidney disease 1 gene encodes a 14 kb transcript and lies within a duplicated region on chromosome 16.Cell. 1994; 77: 881-894Abstract Full Text PDF PubMed Scopus (743) Google Scholar, 5American Polycystic Kidney Disease Consortium Analysis of the genomic sequence for the autosomal dominant polycystic kidney disease gene (PKD1) predicts the presence of a leucine-rich repeat.Hum Mol Genet. 1995; 4: 575-582Crossref PubMed Scopus (237) Google Scholar, 6International Polycystic Kidney Disease Consortium Polycystic kidney disease: the complete structure of the PKD1 gene its protein.Cell. 1995; 81: 289-298Abstract Full Text PDF PubMed Scopus (621) Google Scholar, 7Mochizuki T Wu G Hayashi T Xenophontos XL Veldhuisen B Saris JJ Reynolds DM Cai Y Gabow PA Pierides A Kimberling WJ Breuning MH Deltas CC Peters DJM Somlo S PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein.Science. 1996; 272: 1339-1342Crossref PubMed Scopus (1149) Google Scholar Most mutations identified in affected families appear to inactivate the PKD genes. However, patients with ADPKD are heterozygotes, having inherited one mutant and one normal allele of the PKD1 or PKD2 genes. Studies of cyst-lining epithelial cells isolated from individual cysts in both disorders have demonstrated loss of heterozygosity at the wild-type PKD1 allele8Qian F Watnick TJ Onuchic LF Germino GG The molecular basis of focal cyst formation in human autosomal dominant polycystic kidney disease type I.Cell. 1996; 87: 979-987Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 9Watnick TJ He N Wang K Liang Y Parfrey P Hefferton D St. George-Hyslop P Germino G Pei Y Somatic mutations of PKD1 in ADPKD2 cystic tissue suggests a possible pathogenic effect of trans-heterozygous mutations.Nat Genet. 2000; 25: 143-144Crossref PubMed Scopus (106) Google Scholar that has led to a two-hit mechanism for cyst formation. This mechanism requires not only a germ-line mutation of PKD1 or PKD2 but also an additional somatic mutation in the wild-type gene to initiate the formation of cysts. Briefly, loss-of-function mutations in both alleles of either PKD1 or PKD2 are necessary and sufficient for renal cyst formation in ADPKD. The results of animal studies also support the two-hit mechanism because mice heterozygous for targeted disruption of either Pkd1 or Pkd2 develop late-onset renal cysts, whereas homozygous animals die in utero or perinatally with severe cystic disease.10Lu 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 targeted Pkd1 mutation.Nat Genet. 1997; 17: 179-181Crossref PubMed Scopus (373) Google Scholar, 11Lu W Shen X Pavlova A Lakkis M Ward CJ Pritchard L Harris PC Genest DR Perex-Atayde AR Zhou J Comparison of Pkd1-targeted mutants reveals that loss of polycystin-1 causes cystogenesis and bone defects.Hum Mol Genet. 2001; 10: 2385-2396Crossref PubMed Scopus (156) Google Scholar, 12Wu G D'Adati V Cai Y Markowitz G Park JH Reynolds DM Maeda Y Le TC Hou H Kucherlapati R Edelmann W Somlo S Somatic inactivation of Pkd2 results in polycystic kidney disease.Cell. 1998; 93: 177-188Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar This mechanism would explain the late age of onset in ADPKD and the focal nature of epithelial cells giving rise to cysts. Although such second hits do indeed occur within individual cysts, the frequencies are low (between 17% and 24% in PKD1).8Qian F Watnick TJ Onuchic LF Germino GG The molecular basis of focal cyst formation in human autosomal dominant polycystic kidney disease type I.Cell. 1996; 87: 979-987Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 13Brasier JL Henske EP Loss of the polycystic kidney disease (PKD1) region of chromosome 16p13 in renal cyst cells supports a loss-of-function model for cyst pathogenesis.J Clin Invest. 1997; 99: 194-199Crossref PubMed Scopus (224) Google Scholar In addition, the germ line wild-type PKD1 is still expressed continuously in most cyst epithelial cells of kidneys from ADPKD patients having inherited one mutant PKD1 allele.14Ong AC Harris PC Davies DR Pritchard L Rossetti S Biddolph S Vaux DJ Migone N Ward CJ Polycystin-1 expression in PKD1, early-onset PKD1, and TSC2/PKD1 cystic tissue.Kidney Int. 1999; 56: 1324-1333Crossref PubMed Scopus (91) Google Scholar The evidence suggests that the majority of somatic mutations in PKD1 are likely to be missense changes if the two-hit mechanism is operational. In the present study, we found that only partial inhibition of Pkd1 expression was sufficient for renal cyst formation in mice, suggesting the other possible mechanism that any actions hampering the expression and/or biological function of PKD1 may initiate the renal cyst formation in ADPKD. Complete loss-of-function in either PKD1 or PKD2 may not be strictly required for development of the common ADPKD. Polycystin-1, the novel protein encoded by PKD1, is a large (>460 kd) membrane protein of ∼4300 amino acids with 11 transmembrane domains. Its extensive extracellular amino terminus contains a number of adhesive domains that implicate polycystin-1 in cell-cell and cell-matrix interactions.3Wilson PD Polycystic kidney disease.N Engl J Med. 2004; 350: 151-164Crossref PubMed Scopus (604) Google Scholar, 5American Polycystic Kidney Disease Consortium Analysis of the genomic sequence for the autosomal dominant polycystic kidney disease gene (PKD1) predicts the presence of a leucine-rich repeat.Hum Mol Genet. 1995; 4: 575-582Crossref PubMed Scopus (237) Google Scholar, 6International Polycystic Kidney Disease Consortium Polycystic kidney disease: the complete structure of the PKD1 gene its protein.Cell. 1995; 81: 289-298Abstract Full Text PDF PubMed Scopus (621) Google Scholar The short intracellular carboxyl terminus of the protein has a G protein binding site and many sites for phosphorylation that could respond to regulators of signal transduction, as well as a coiled-coil domain that is in physical interaction with PKD2-encoded polycystin-2.3Wilson PD Polycystic kidney disease.N Engl J Med. 2004; 350: 151-164Crossref PubMed Scopus (604) Google Scholar, 15Delmas P Nomura H Li X Lakkis M Luo Y Segal Y Fernández-Fernández JM Harris P Frischauf A Brown DA Zhou J Constitutive activation of G-proteins by polycystin-1 is antagonized by polycystin-2.J Biol Chem. 2002; 277: 11276-11283Crossref PubMed Scopus (156) Google Scholar Polycystin-2 is also a membrane protein, with six transmembrane domains, that functions as a Ca2+-permeant cation channel and has significant homology with the transient receptor potential family (TRP).16Delmas P Padilla F Osorio N Coste B Raoux M Crest M Polycystins, calcium signaling, and human diseases.Biochem Biophys Res Commun. 2004; 322: 1374-1383Crossref PubMed Scopus (86) Google Scholar Recent studies have shown that polycystin-1 and polycystin-2 are interacting partners within a receptor-ion channel complex in which polycystin-1 acts as a receptor that gates Ca2+-permeant polycystin-2 channels.16Delmas P Padilla F Osorio N Coste B Raoux M Crest M Polycystins, calcium signaling, and human diseases.Biochem Biophys Res Commun. 2004; 322: 1374-1383Crossref PubMed Scopus (86) Google Scholar This polycystin complex has been found in primary cilia and basolateral membrane of renal epithelial cells, where it may participate in sensing fluid shear stress and cell-cell/matrix interaction, respectively.17Wilson PD Polycystin new aspects of structure, function, and regulation.J Am Soc Nephrol. 2001; 12: 834-845Crossref PubMed Google Scholar, 18Nauli SM Alenghat FJ Luo Y Williams E Vassilev P Li X Elia AEH Lu W Brown EM Quinn SJ Ingber DE Zhou J Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells.Nat Genet. 2003; 33: 129-137Crossref PubMed Scopus (1585) Google Scholar However, the diverse functions of the polycystin complex are not fully characterized, and its roles in the process of renal cystic transformation are still under extensive investigation. Polycystin-1 and −2 are widely expressed in fetal and adult tissues,7Mochizuki T Wu G Hayashi T Xenophontos XL Veldhuisen B Saris JJ Reynolds DM Cai Y Gabow PA Pierides A Kimberling WJ Breuning MH Deltas CC Peters DJM Somlo S PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein.Science. 1996; 272: 1339-1342Crossref PubMed Scopus (1149) Google Scholar, 19Ward CJ Turley H Ong AC Comley M Biddolph S Chetty R Ratcliffe PJ Gattner K Harris PC Polycystin, the polycystic kidney disease 1 protein, is expressed by epithelial cells in fetal, adult, and polycystic kidney.Proc Natl Acad Sci USA. 1996; 93: 1524-1528Crossref PubMed Scopus (213) Google Scholar and their expressions are developmentally regulated.20Geng L Segal Y Peissel B Deng N Pei Y Carone F Rennke HG Glucksmann-Kuis AM Schneider MC Ericsson M Reeders ST Zhou J Identification and localization of polycystin, the PKD1 gene product.J Clin Invest. 1996; 98: 2674-2682Crossref PubMed Scopus (171) Google Scholar, 21van Adelsberg J Chamberlain S D'Agati V Polycystin expression is temporally and spatially regulated during renal development.Am J Physiol. 1997; 272: F602-F609PubMed Google Scholar Homozygous mutant mice with targeted disruption of either Pkd gene generally die in utero or perinatally with cardiac septal defects, bone abnormalities, and severe cystic manifestations in nephrons and pancreatic ducts.10Lu 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 targeted Pkd1 mutation.Nat Genet. 1997; 17: 179-181Crossref PubMed Scopus (373) Google Scholar, 11Lu W Shen X Pavlova A Lakkis M Ward CJ Pritchard L Harris PC Genest DR Perex-Atayde AR Zhou J Comparison of Pkd1-targeted mutants reveals that loss of polycystin-1 causes cystogenesis and bone defects.Hum Mol Genet. 2001; 10: 2385-2396Crossref PubMed Scopus (156) Google Scholar, 22Kim K Drummond I Ibraghimov-Beskrovnaya O Klinger K Arnaout A Polycystin 1 is required for the structural integrity of blood vessels.Proc Natl Acad Sci USA. 2000; 97: 1731-1736Crossref PubMed Scopus (263) Google Scholar These previous studies confirm the requirement of either Pkd gene during embryonic development. Animal models are important tools in experimental medical science to understand better the pathogenesis of human diseases and to test therapeutic approaches. Currently, no mouse model for the most common and severe form of ADPKD derived from targeted disruption of Pkd1 is viable and amenable for further investigation. In the experiments described herein, we used homologous recombination to introduce a Neo cassette into intron 34 of mouse Pkd1 to partially inhibit its expression. The mouse homozygotes appear normal at birth but still have small renal cysts. Cyst enlargement and interstitial fibrosis are developed later, recapitulating the phenomena in the human ADPKD. The resulting mouse models for ADPKD are viable and suitable for advanced studies in the future. A targeting plasmid was constructed using genomic DNA fragments derived from 129/Ola mouse strain. A loxP site, which has a neomycin cassette flanked by two loxP sites, and a thymidine kinase cassette were introduced into the Pkd1 locus (Figure 1A). Embryonic stem (ES) cell (line E14) electroporation, selection, and screening were performed using standard gene targeting techniques. Briefly, genomic DNA was isolated from neomycin and gancyclovir double-resistant ES cell clones and screened for a specific targeting event by polymerase chain reaction (PCR) using a forward primer outside the targeting vector and downstream of exon 36 (5′-CAAAGCCCTGACTATCCATTGG-3′) and a reverse primer in the neo sequence (5′-CCTCTTGAAAACCACACTGCTCG-3′). The correct targeting of PCR-positive clones was confirmed by Southern blot analysis. For this analysis, 10 μg of DNA from selected clones was digested with XbaI, and DNA was electrophoresed and transferred onto nylon membranes using capillary transfer. Blots were hybridized with P32-labeled probe external to the targeting vector (Figure 1A). Blots were also hybridized with neo-specific probes to verify the absence of additional random integration of the targeting vector. To demonstrate the presence of the loxP in intron 30, one set of primers located upstream from the loxP site (5′-GGTAGGTCCTGTGAGGTTTGCTG-3′) and neo (5′-GCCTTCTATCGCCTTCTTGACG-3′) were used. All of the PCR products amplified from positive clones were confirmed by sequencing. Positive clones were injected into C57BL/6 blastocysts to generate chimeras. The chimeras that were derived from C57BL/6 blastocysts were bred with C57BL/6 or FVB inbred strain mice. Homozygotes were generated by a cross between F1 heterozygotes, and heterozygotes and homozygotes were determined by Southern blot or PCR of tail genomic DNA. RT-PCR analysis was performed with 4 μg of total RNA extracted from various organs. First-strand cDNA was synthesized by Super Script II RT (Gibco BRL, Eggenstein, Germany) with 25 ng of oligo(dT)12-18 primer and 200 U of Super Script II per reaction. Samples (5 μl) of the RT reaction were used in a 100-μl PCR reaction. Two sets of primers were used for analysis of the Pkd1 transcript. Set 1, F6 in exon 6 (5′-AAGCACAGGAGCAATGTCGGAC-3′) and B11 in exon 11 (AATGAGGTCACCAGGGAGCATAGG-3′) amplified an 839-bp fragment from the wild-type Pkd1 transcript. Set 2, F33 in exon 33 (5′-GGAAGATGGTGCCTCTCTGGTTAG-3′) and B37 in exon 37 (5′-CACACGCTCACTTACAGGGGTTAC-3′) amplified a 583-bp fragment from the wild-type Pkd1 transcript. A set of primers was used for analysis of the EGF transcript; 5′-TCCGTCCGTCTTATCAGGCATC-3′ and 5′-GGCACATTCATTGACATCTTCGC-3′ amplified a 375-bp fragment. Another set of primers was used for analysis of the heparin-binding epidermal growth factor (HB-EGF) transcript; 5′-AAGTTGCTTTCTCCTCCAAGCC-3′ and 5′-CCACGATGACAAGAAGACAGACG-3′ amplified a 321-bp fragment. Each transcript from various genotyped mice was cloned and sequenced by the dideoxy method. We generated rabbit polyclonal antibodies specific for mouse polycystin-1 using a segment of the first extracellular PKD domain polypeptide (containing amino acids 866 to 882 of murine polycystin-1), as described in a previous study.18Nauli SM Alenghat FJ Luo Y Williams E Vassilev P Li X Elia AEH Lu W Brown EM Quinn SJ Ingber DE Zhou J Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells.Nat Genet. 2003; 33: 129-137Crossref PubMed Scopus (1585) Google Scholar The peptide was also conjugated to NHS-activated Sepharose 4B (Amersham Biosciences, Uppsala, Sweden) for affinity purification of these polyclonal antibodies. A rabbit antiserum to Tamm-Horsfall glycoprotein was purchased from Biomedical Tech-nologies Inc. (Stoughton, MA). A purified rabbit antibody to lysozyme was purchased from DakoCytomation (Glostrup, Denmark). Mouse monoclonal antibodies against erb-B2, Na+/K+-ATPase α1-chain, and proliferating cell nuclear antigen were purchased from Upstate Biotechnology (Lake Placid, NY). Mouse monoclonal antibodies against α-smooth muscle actin (α-SMA), Na+/K+-ATPase β2-chain, β-actin, Grb2, and Shc were purchased from BD Biosciences (Franklin Lakes, NY). Affinity-purified rabbit antibodies against EGF receptor and phospho-EGF receptor were purchased from Cell Signaling Technology (Beverly, MA). Affinity-purified goat polyclonal antibodies to aquaporin-2 were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). A rabbit antiserum to Na+/K+-ATPase β1-chain was purchased from United States Biological (Swampscott, MA). For Western blot analysis, total protein was measured by Bio-Rad protein assay (Hercules, CA), for which 50 μg/lane of membrane and cytosol fractions from various tissue extracts were separated on sodium dodecyl sulfate-polyacrylamide gels at 80 V at room temperature. Protein was transferred onto polyvinylidene difluoride membrane at 35 V for 16 hours at 4°C in Tris-glycine transfer buffer. The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween-20 and incubated with primary antibodies. The immunoreactive protein was captured by horseradish peroxidase-conjugated secondary antibodies and was detected by enhanced chemiluminescence. For histological analysis, specimens were fixed in formalin, embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin (H&E) as well as Masson's trichrome for light microscopic examination. For immunostaining, a standard immunoperoxidase protocol (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) was used. After blocking with goat serum, sections were incubated with primary antibodies for 1 hour at room temperature, rinsed in phosphate-buffered saline, incubated with biotinylated goat anti-rabbit or anti-mouse secondary antibodies, rinsed, then incubated with streptavidin-conjugated peroxidase, rinsed, then incubated with 3-amino-9-ethyl-carbazole or diaminobenzidine as a chromogen, counterstained with hematoxylin, and examined by light microscopy. For specific labeling of the collecting duct, sections were directly incubated with biotinylated Dolichos biflorus lectin (purchased from Vector Laboratories), rinsed, and then incubated with streptavidin-conjugated peroxidase. For morphometric analysis of the interstitial fibrosis in the kidney, a standard point-counting method was used to quantitate the matrix score for α-SMA expression in the renal interstitium. Briefly, under high magnification (×200), 12 nonoverlapping fields from each renal section were immunostained with α-SMA and photographed. A grid containing 100 (10 × 10) sampling points was superimposed on each photograph. Points falling on cystic cavities, glomerular structures, or large vessels were excluded from the total count. The staining scores of interstitial α-SMA expression were assessed by the number of points overlying positive α-SMA expression and then converted to a percentage. For each genotype, 4-μm-thick sections of formalin-fixed renal tissue from 3-month-old mice were deparaffinized and hydrated in graded alcohols. After digestion in proteinase K and immersion in 2% H2O2, sections were incubated with 10 μmol/L biotin-16-dUTP and 0.2 U/μl TdT in TdT buffer [30 mmol/L Tris-HCl (pH 7.2), 140 mmol/L sodium cacodylate, 1 mmol/L cobalt chloride]. The enzymatic reaction was stopped by immersion in TB buffer (300 mmol/L sodium chloride, 30 mmol/L sodium citrate). After incubation with 2% bovine serum albumin, labeled nucleotides were reacted with avidin/biotin complex (Vectastain; Vector Elite, Burlingame, CA), followed by visualization with 3,3′-diaminobenzidine and counterstained with periodic acid-Schiff. Apoptosis was quantitated by averaging the number of stained nuclei per cystic and noncystic tubule in a minimum of 500 tubular epithelial cells/kidney. Consistent with the broad expression of Pkd1 during early organogenesis, mice homozygous for targeted disruption of Pkd1 exhibit developmental abnormalities causing embryonic or perinatal lethality.10Lu 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 targeted Pkd1 mutation.Nat Genet. 1997; 17: 179-181Crossref PubMed Scopus (373) Google Scholar, 11Lu W Shen X Pavlova A Lakkis M Ward CJ Pritchard L Harris PC Genest DR Perex-Atayde AR Zhou J Comparison of Pkd1-targeted mutants reveals that loss of polycystin-1 causes cystogenesis and bone defects.Hum Mol Genet. 2001; 10: 2385-2396Crossref PubMed Scopus (156) Google Scholar, 22Kim K Drummond I Ibraghimov-Beskrovnaya O Klinger K Arnaout A Polycystin 1 is required for the structural integrity of blood vessels.Proc Natl Acad Sci USA. 2000; 97: 1731-1736Crossref PubMed Scopus (263) Google Scholar, 23Boulter C Mulroy S Webb S Fleming S Brindle K Sandford R Cardiovascular, skeletal, and renal defects in mice with a targeted disruption of the Pkd1 gene.Proc Natl Acad Sci USA. 2001; 98: 12174-12179Crossref PubMed Scopus (247) Google Scholar The complex renal and extra-renal phenotypes of the Pkd1-targeted mutants obstruct further investigation at later stages of development and mask direct from indirect consequences of Pkd1 inactivation. To circumvent this limitation, we first created a floxed Pkd1 mouse for conditional inactivation of polycystin-1 by Cre/loxP-mediated gene targeting. The targeted strategy and genotype analysis of the ES clones and mice are summarized in Figure 1, A and B. A single loxP site was inserted into intron 30, and a loxP-flanked mc1-neomycine (mc1-neo) cassette was inserted into intron 34 in reverse orientation relative to the targeted Pkd1 gene. The linearized targeting vector was introduced into E14 ES cells by electroporation, and 279 colonies resistant to G418 and gancyclovir were selected. Sequencing of PCR products formed by primers outside the 3′ end of the short homologous arm and in the neo gene identified 21 independent ES cell clones with Pkd1 targeted by homologous recombination. All these positive clones were double-checked by Southern blot analysis with a 3′-external probe. The presence of the loxP site in intron 30 of the targeted allele was confirmed in 15 clones by sequencing of the PCR products formed by primers 5′ upstream from the loxP site and in the neo gene. The remaining six clones contained only the loxP-flanked mc1-neo cassette in intron 34 of the targeted Pkd1 allele (Lneo). Two independent clones (no. 175 and no. 186) with the neo gene and all three loxP sites in the targeted allele (L3) were injected into C57BL/6 blastocysts and implanted into pseudopregnant females. The resulting chimeric mice, with >80% of the agouti coat, were crossed with C57BL/6 or FVB mice to produce Pkd1L3/+ F1 progeny. The phenotypes described below were not significantly influenced by genetic background. Most of the phenotypic analyses were performed with C57BL/6–129 background. Pkd1L3/+ F1 progeny derived from clone no. 175 were intercrossed and the F2 progeny were genotyped at 3 weeks of age (Figure 1B): 58 wild-type (Pkd1+/+), 114 Pkd1L3/+, and 54 Pkd1L3/L3 animals are shown. Genotyping of the clone no. 186 F2 progeny showed 11 Pkd1+/+, 25 Pkd1L3/+, and 13 Pkd1L3/L3 animals, similar to clone no. 175 F2 progeny. The ratio of Pkd1+/+:Pkd1L3/+:Pkd1L3/L3 in either of the mouse lines did not differ significantly from the 1:2:1 ratio expected for a nonlethal mutation. Most of the phenotypic analyses were performed with the no. 175-derived mouse line, but identical results also were obtained with the no. 186-derived line. RT-PCR of total RNA from 30-day-old Pkd1+/+, Pkd1L3/+, and Pkd1L3/L3 mice, using 5′-exon 33 primer (F33) and 3′-exon 37 primer (B37), revealed only a single DNA band of predicted size for wild-type transcript in mRNA from all three genotyped mice (Figure 1C). DNA sequences of these RT-PCR products were identical, suggesting that the mc1-neo cassette in reverse orientation within intron 34 is not spliced into the Pkd1 transcript of the Pkd1 L3 allele. Interestingly, the transcript was expressed at a lower level in homozygotes after normalization with β-actin expression. RT-PCR analysis using the other set of primers located in exons 6 (F6) and 11 (B11) also showed a similar result (Figure 1C). To determine whether the protein level of polycystin-1 was also decreased in homozygous mutants, we performed Western blot analysis with a polyclonal antibody directed against the extracellular first PKD domain of mouse polycystin-1. A strong immunoreactive band of ∼460 kd was detected in wild-type and heterozygous mice, and once again the intensity of this band showed a fourfold to fivefold decrease in homozygous mutants (Figure 1D). These results indicate that the insertion of the mc1-neo into intron 34 causes a significant disturbance but does not completely abrogate in Pkd1 expression. Unlike the previously established homozygous mutants with targeted disruption of Pkd1, the Pkd1L3/L3 mice expressing a low level of polycystin-1 appeared normal at birth with no gross anatomical abnormality (Figure 2A). The short stature, distended abdomens, and massively enlarged cystic kidney were not exhibited until several days after birth (Figure 2). A total of 104 Pkd1L3/L3 and 46 Pkd1L3/+ mice (age range, 1 to 360 days) were examined for kidney size and cystic lesions in their pancreas and liver, as well as the size of their heart and lungs. Grossly, the kidneys of the Pkd1L3/L3 mice enlarged rapidly during the first 30 days and gradually shrunk in volume by more than 50% thereafter, resulting in a bumpy and distorted appearance (Figure 2, E–H). We also successively measured the abdominal girth, an indirect indicator of kidney size, of the Pkd1L3/L3 mice for 6 months. Coinciding with the finding of kidney size variations, the abdominal girths also rapidly increased during the first 30 days after birth and subsequently decreased. The massive translucent cysts in bilateral kidneys were grossly identifiable under general illumination in all examined Pkd1L3/L3 mice older than" @default.
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• W2051209896 date "2006-01-01" @default.
• W2051209896 modified "2023-10-09" @default.
• W2051209896 title "Defining a Link with Autosomal-Dominant Polycystic Kidney Disease in Mice with Congenitally Low Expression of Pkd1" @default.
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