Human Molecular Genetics, 2000, Vol. 9, No. 18 2617-2627
© 2000 Oxford University Press
A human PKD1 transgene generates functional polycystin-1 in mice and is associated with a cystic phenotype
Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK, 1Renal Division, Department of Medicine, Brigham and Women’s Hospital, Boston, MA 02115, USA, 2Department of Pathology, University of Washington Medical Center, Seattle, WA 98195, USA and 3Department of Nephrology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA
Received 15 June 2000; Revised and Accepted 1 September 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Three founder transgenic mice were generated with a 108 kb human genomic fragment containing the entire autosomal dominant polycystic kidney disease (ADPKD) gene, PKD1, plus the tuberous sclerosis gene, TSC2. Two lines were established (TPK1 and TPK3) each with
30 copies of the transgene. Both lines produced full-length PKD1 mRNA and polycystin-1 protein that was developmentally regulated, similar to the endogenous pattern, with expression during renal embryogenesis and neonatal life, markedly reduced at the conclusion of renal development. Tuberin expression was limited to the brain. Transgenic animals from both lines (and the TPK2 founder animal) often displayed a renal cystic phenotype, typically consisting of multiple microcysts, mainly of glomerular origin. Hepatic cysts and bile duct proliferation, characteristic of ADPKD, were also seen. All animals with two copies of the transgenic chromosome developed cysts and, in total, 48 of the 100 transgenic animals displayed a cystic phenotype. To test the functionality of the transgene, animals were bred with the Pkd1del34 knockout mouse. Both transgenic lines rescued the embryonically lethal Pkd1del34/del34 phenotype, demonstrating that human polycystin-1 can complement for loss of the endogenous protein. The rescued animals were viable into adulthood, although more than half developed hepatic cystic disease in later life, similar to the phenotype of older Pkd1del34/+ animals. The TPK mice have defined a minimal area that appropriately expresses human PKD1. Furthermore, this model indicates that over-expression of normal PKD1 can elicit a disease phenotype, suggesting that the level of polycystin-1 expression may be relevant in the human disease. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive renal cyst development and enlargement, typically leading to end stage renal disease in late middle age. As a disease that affects
1 in 1000 individuals, it is one of the commonest autosomal disorders (1). Yet, despite the cloning of PKD1 and PKD2, the major genes responsible for the disease (2,3), understanding the disease pathogenesis is sketchy and therapeutic intervention remains elusive.
Mutation of PKD1 accounts for 85% of ADPKD. Polycystin-1, its protein product, is a large membrane-bound molecule with a predicted unglycosylated molecular mass of 460 kDa (4,5). Its function remains unknown, although a role in cell–cell and/or cell–matrix interactions is predicted (4,6); a concept supported by recent localization data (7,8) and evidence of involvement in signalling (9,10). Further insight into possible function comes from homologies between polycystin-1, polycystin-2, other PKD-like molecules (polycystin-L, polycystin-L2 and PKDREJ) and voltage-activated and transient receptor potential ion channel proteins (3,11–15). These structural predictions are strengthened by recent functional evidence that polycystin-L can act as a Ca2+-regulated cation channel, showing a direct role for polycystin molecules in ion transport (16).
ADPKD is dominant; inheritance of one mutant allele is sufficient to cause disease. Analysis of PKD1 mutations has revealed a range of mainly stop and frameshifting changes, suggesting that the mutant allele is inactivated (17). Analysis of rare cases with severe PKD and tuberous sclerosis (TSC), which have large deletions disrupting the adjacent PKD1 and TSC2 genes (and in some cases completely removing PKD1), also indicate that a null PKD1 allele is associated with cyst development (18). Knockout models of Pkd1 (Pkd1del34 and Pkd1L) further support the notion that cyst development is caused by reduction or loss of protein. In the Pkd1del34 model older heterozygotes have occasional cysts, whereas homozygotes die in the perinatal period with enlarged and cystic kidneys (19,20). Cystic kidneys are also found in Pkd1L homozygotes, but they have a more severe vascular phenotype, with earlier embryonic lethality (21). In the human disease, evidence is accumulating that at the cellular level cysts develop in a recessive manner due to somatic second hits to the normal PKD1 allele, therefore explaining the focal nature of cyst development (22,23). This two-hit model is supported by a mouse model of Pkd2 (WS25) in which an additional exon 1 is targeted adjacent to the normal exon resulting in a hypermutable locus (24). Somatic mutation of PKD2 in cystic epithelia (25,26) and recently examples of trans-heterozygous cysts, with PKD1 somatic mutations in PKD2 cysts and vice versa (27,28), have also been described.
The PKD1 mRNA is widely expressed in fetal and adult tissues (29). Analysis with various polycystin-1 antibodies indicates expression in tubular epithelia of the developing kidney and in ductal structures in many other organs, plus in the vasculature and cardiac and skeletal muscle (29–32). The widespread expression reflects the systemic nature of the cystic and non-cystic abnormalities associated with ADPKD (33) and disruption of the murine locus (21). Expression in the adult appears more limited, with renal expression mainly restricted to distal tubules and collecting ducts (30,32) and murine polycystin-1 markedly down-regulated 2–4 weeks postnatally (34). Interestingly, analysis of ADPKD tissue has revealed persistent, or even enhanced, polycystin-1 immunoreactivity of cystic epithelia in the majority of cysts (29,30,35). This finding appears at odds with the requirement for an inactivating PKD1 second hit to initiate cystogenesis (unless there is a very high level of trans-heterozygotes) and questions whether these somatic events are required prior to cyst development (36).
In vitro evidence suggests that polycystin-1 and -2 interact (37,38) and analysis of polycystin-2 expression indicates a similar cellular pattern to that of polycystin-1 (32,39), although some differences have been noted (40). Other possible binding partners have been identified (7,8,41,42) suggesting that polycystin-1 may form part of a protein complex and indicating precise regulation of the protein level, as well as the temporal and spatial pattern of expression. Efforts to identify sequences required for regulating expression and generating possible splicing variants of the gene, have been hampered by the size of the transcript (encoded by 46 exons) and the complexity of the reiterated genomic region containing the 5' three-quarters of the human gene (2,43). Attempts to address these questions by analysis of PKD1 in vitro have been complicated by problems expressing the full-length transcript (44). Furthermore, these in vitro systems do not characterize the PKD1 promoter or elements required to control spatial or developmental expression of polycystin-1. No clearly defined cellular phenotype is associated with altered expression of the protein.
In an attempt to define a genomic region required for appropriate expression of PKD1 and to develop a model system where functionality of polycystin-1 can be tested, we have generated transgenic mice expressing human PKD1. This system allows the expression and role of human polycystin-1 to be tested in the mouse and the phenotypic consequences of altering the level of expression of the protein to be assessed.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation of PKD1 transgenic mice
To isolate a genomic clone containing the entire PKD1 gene a P1-derived artificial chromosome (PAC) library was screened with probes flanking the gene at the 5' (N54) and 3' (1A-1) ends (Fig. 1). A positive clone, 136-M1, was identified and the insert of
135 kb mapped in detail using PKD1 and TSC2 probes to ensure that it matched the genomic structure. A PvuI restriction fragment of 108 kb was identified that contained the PKD1 gene plus 20 kb upstream and 43 kb downstream, including the entire adjacent TSC2 gene (Fig. 1). 136-M1 was digested with PvuI and micro-injected into the pronuclei of fertilized C57BL/6 x CBA mouse eggs. Three transgenic progeny were identified by Southern blotting of tail DNA with the human-specific probe JH1. Transgenic lines were established from two of these animals, TPK1 and TPK3, but the TPK2 founder remained too small to breed and died at 5 months. The approximate copy number of the transgenic insert in each line was assessed by quantitative Southern blotting using a probe for the human transgene and mouse control (Fig. 2). The copy number was highest in TPK2, with 90 copies of the transgene, with 30 copies and 28 copies estimated for TPK1 and TPK3, respectively. Fluorescence in situ hybridization (FISH) in the TPK1 and TPK3 lines showed a single integration site in chromosomal regions 7E3-F1 and 18A2, respectively (mouse Pkd1 maps to 17A–B). Analysis of the transgenic lines with probes lying close to the ends of the PvuI fragment, N54 and CW24 (Fig. 1), revealed a strong common BamHI fragment consistent with the PKD1 transgenes being organized in a head-to-tail array. However, further complexity of multiple minor bands was detected with both probes indicating that the structure at the integration site may be complex with some copies separated by other DNA fragments (data not shown).
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Expression of the PKD1 transgene
To determine whether the PKD1 transgene was transcriptionally active, transgenic mouse mRNA was assayed by northern blotting and RNAase protection. Widespread expression was found in newborn and adult tissues (kidney, liver, brain, heart, skeletal muscle, thymus, spleen, skin, lung and testis), as well as in fetal tissues from both lines. Figure 3a illustrates human PKD1 expression, as a 14 kb transcript, in adult brain and kidney tissues from TPK1 and TPK3, but not the control mouse tissues, with a similar expression pattern seen for murine Pkd1 (Fig. 3c). The RNAase protection assay was used to compare the level of expression of the transgene between all three lines and with the endogenous mRNA. Comparison of the level of human PKD1 in adult tissue showed a slightly higher level in TPK1 than in TPK3 (1.25–2x) and that expression in TPK2 was approximately twice that in TPK1. The transgene was expressed at a significantly higher level than the endogenous gene (7.5–14.5x, TPK1; and 2.5–7x, TPK3). This over-expression of the human transcript did not, however, affect the level of endogenous mouse Pkd1, which was not significantly different in the transgenic lines compared with normal adult animals. In addition to PKD1, the transgenic fragment contains the entire TSC2 gene (Fig. 1). Analysis of the TSC2 mRNA revealed expression of a 5.5 kb transcript in adult brain and kidney tissue (Fig. 3b) from both transgenic lines.
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Expression of transgenic human polycystin-1
To test whether the transgene was appropriately translated we analysed expression of the protein by western blotting. We have previously described a monoclonal antibody to the leucine-rich repeat motif, close to the N-terminus of human polycystin-1, which consistently detects polycystin-1 as an
400 kDa protein in human kidney tissue (35). Although this antibody can detect mouse polycystin-1, it is with a lower affinity and we developed methods using western blotting with 10 µg of membrane protein to achieve specific detection of the human protein. A large polycystin-1 product (400 kDa) was detected in western blots of membrane protein samples from TPK1 and TPK3 neonates, but not in the wild-type littermate controls (Figs 4a and 5). These and related experiments showed that the transgenic polycystin-1 was expressed widely during fetal and neonatal life. Analysis of kidney membrane fractions from both transgenic lines revealed similar developmental regulation of the transgene, with a marked down-regulation of the transgene between 2 weeks and 1 month postnatally, continuing at a low or absent level in the adult (Fig. 4b), similar to that previously described for murine polycystin-1 (34). The intensity of the polycystin-1 signal detected from a low level of membrane protein suggests that the human transgene is over-expressed compared with the endogenous protein, but quantification has not been possible due to the absence of a mouse polycystin-1-specific antibody. Analysis of tuberin expression in neonatal tissue showed a more restricted pattern with signal mainly limited to brain (Fig. 5).
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We have been unable to establish conditions for immunohistochemical studies with the polycystin-1 antibodies, 7e12 and PKS-A, to specifically detect the transgenic human protein, without cross-reactivity with murine polycystin-1. Consequently, we have been unable to localize just the transgenic protein. Nevertheless, if we assume that the antibodies are detecting the transgenic and endogenous protein, we can conclude that the transgenic protein is localized similarly to the murine product, with no aggregation observed. Renal localization is mainly in the cytoplasm of the maturing tubules, as previously shown in human tissue (29,32).
Phenotypic consequences of expressing human polycystin-1
Analysis of the transgenic lines indicated that human PKD1 was significantly over-expressed compared with the endogenous gene. To determine whether this over-expression and/or expression of the human gene in the mouse had any phenotypic consequences, transgenic animals were sacrificed at various ages and analysed histologically. Interestingly, kidneys from animals of both transgenic lines displayed bilateral cystic abnormalities (Table 1). Figure 6A shows a severely affected kidney from a 6-month-old TPK3 animal which appears pale with numerous cystic lesions visible on the surface. A section through this kidney (Fig. 6B) shows focal clusters of microcysts of various sizes throughout the cortex. Closer examination of a TPK1 kidney shows that the majority of the cysts are of glomerular origin, with the glomerular tuft still visible in many, although dilated tubules were also present (Fig. 6C). The renal abnormalities observed in the different transgenic lines were similar (Fig. 6D–F) with the characteristic pattern of glomerulocystic injury seen in each. In four moderately to severely affected animals marked cystic dilation of the renal pelvis was also detected. Accompanying the cystic lesions were varying degrees of tubulointerstitial fibrosis, mild patchy tubular atrophy and, occasionally, glomerulosclerosis. The tubulointerstitial changes were associated with marked interstitial inflammation which was most heavily concentrated at the cortico-medullary junction. There were no identifiable changes within the vasculature.
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The transgenic phenotype was highly variable between animals, although similar, bilateral presentation was usually seen. Thirty-eight per cent of transgenic animals showed a cystic phenotype (35% TPK1 and 40% TPK3) with disease more common in older animals (35% younger; 43% older; see Table 1 for details). None of the 20 C57BL/6 x CBA control cohort sacrificed at 18–20 months of age displayed a cystic renal phenotype. Animals from both lines bred to contain two copies of the transgenic chromosome were universally small and all displayed moderate to severe renal cystic lesions when analysed before 11 months of age (Table 1), suggesting that the level of transgenic expression may be important. The founder TPK2 animal, which had a higher level of human PKD1 mRNA, also had a moderate to severe phenotype. The cystic phenotype, including severe disease, was seen in males and females from both lines.
Animals from both the TPK1 and TPK3 lines displayed liver abnormalities characteristic of human polycystic kidney disease (Table 1). These abnormalities ranged from gross cystic changes (Fig. 6G and H) which were more common in older animals, to distinctive abnormalities of the portal tracts, such as ductal plate abnormalities showing chronic inflammatory infiltrates and bile ductular proliferation (Fig. 6I). It is interesting to note that most animals with liver disease also displayed renal cysts (Table 1).
The PKD1 transgenic product is functionally active in mouse
Based on analysis of the TPK1 and TPK3 transgenic lines, it appeared that over-expression of human PKD1 in mice could result in a cystic phenotype. One possible explanation for these observations was that the human protein was functionally inactive, owing to mutation, inappropriate expression or incompatibility between the human and mouse proteins. If this were the case the observed phenotype may be due to a dominant negative effect of the transgenic product, creating a high level of functionless polycystin-1-containing complexes. To test whether the transgenic product was functional in mouse, experiments were performed to determine whether the transgene would rescue the perinatal lethal phenotype of the Pkd1 knockout homozygote, Pkd1del34/del34 (19).
The Pkd1del34/+ allele was crossed into the mixed background used for the transgenic experiments. The phenotype of the C57BL/6 x CBA Pkd1del34/del34 fetuses was similar to those previously described in C57BL/6 or BALB/c backgrounds (19), with the majority of animals dying in late fetal life and none surviving beyond the immediate postnatal period. Pkd1del34/+ + TPK1 and Pkd1del34/+ + TPK3 animals were crossed with Pkd1del34/+ heterozygotes and the offspring typed using a Southern blot assay (Fig. 7). Table 2 shows that Mendelian ratios were observed for the TPK1- and TPK3-positive offspring of these crosses demonstrating that the transgenic human polycystin-1 could rescue the lethal Pkd1del34/del34 phenotype and therefore that the transgene generated a functional product. The rescued animals appeared healthy and remained viable into adulthood (the oldest being sacrificed at 11 months); inter-breeding of rescued animals also generated viable offspring. The results from histological analysis of rescued animals and of del34 heterozygotes in the C57BL/6 x CBA background (without the transgene) are summarized in Table 3. The phenotypes in both sets of animals were similar, with only very occasional renal abnormalities detected, but hepatic cystic disease seen in more than half of the animals. It therefore appears that although the transgene effectively rescues the lethal phenotype of the del34 homozygotes, hepatic cystic disease is common; mirroring the phenotype of del34 heterozygotes (Table 3) (20). Interestingly, the number of rescued animals with renal cysts appears lower than in the corresponding transgenics (Tables 1 and 3). This could be due to the lower overall level of polycystin-1 in the rescues than in the transgenics, due to loss of the endogenous protein.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have generated three independent transgenic founders (and established two lines) that express PKD1 from a large genomic DNA fragment. Analysis of mRNA and protein has demonstrated expression of human polycystin-1 in mice that appears to mirror the endogenous pattern, with widespread PKD1 expression and down-regulation of the protein at the conclusion of murine renal development. Functional complementation with the transgene rescues the lethal Pkd1del34/del34 phenotype demonstrating that the transgenic protein is active and appropriately expressed. The viability of the rescued animals indicates that human polycystin-1 can replace the endogenous protein, interacting with murine binding partners. However, the appearance of multiple liver cysts and occasional renal cysts in some older rescued animals indicates that all of these interactions may not be optimal and/or the level of expression may be critical to maintain normal liver and renal morphology.
A significant proportion of animals from the TPK1 and TPK3 lines and the TPK2 founder animal displayed a distinctive renal cystic phenotype with the appearance of multiple focal cysts, primarily of glomerular origin. The similarity of the phenotype in three independently derived transgenic lines indicates that it is expression of the transgene and not disruption at the site of integration that causes the phenotype. Glomerular cysts are not usually a characteristic feature of adult ADPKD. However, in severe early onset cases and on the rare occasions that fetal ADPKD kidneys have been examined, glomerular cysts are the major manifestation of disease (45–47). In common with ADPKD and other animal models of PKD, the transgenic cysts are usually associated with interstitial inflammation and sometimes fibrosis. The observed liver changes, such as ductal plate proliferation and biliary cysts, are also characteristic of ADPKD. Interestingly, the renal cystic phenotype is similar to that described for the Pkd1 and Pkd2 knockouts (19,21,48) with a predominance of glomerular cysts and is strikingly similar to the older Pkd1del34/+ heterozygotes, with marked phenotypic variability and the appearance of focal glomerular and tubular cysts, plus ductal plate malformations and cysts in the liver (20). Therefore, although the TPK mice are not an exact model of adult ADPKD, they manifest a phenotype similar to that associated with mutation to human or murine PKD1.
The TPK animals and cell lines derived from them will be useful for studying the PKD1 gene without the homologous gene (HG) loci and characterizing the expression and localization of the human protein at any period of development, providing pointers for the analysis of the endogenous protein. Detection of the transgenic protein with 7e12 has further verified the specificity of this antibody for polycystin-1. A complication of this model is the presence of a transcribed and translated copy of TSC2, which could contribute to the phenotype seen in the transgenics. However, the similarity of the cysts to ADPKD, without the strongly eosinophilic-staining plump epithelium associated with disruption of TSC2 (18) and the restricted expression pattern of the transgenic tuberin (Fig. 5), suggests that the cystic phenotype is associated with PKD1 over-expression. Indeed, given the close proximity of PKD1 and TSC2 [whose primary transcripts are likely to overlap on opposite strands (49)], and the similar expression pattern of the two genes (but not proteins) in the transgenics, the presence of a functional TSC2 may be necessary for appropriate regulation of PKD1. Further development of transgenic systems employing smaller constructs will help in defining the size of upstream and downstream regions (including TSC2) required for normal PKD1 expression.
It is interesting that the rescued animals often have hepatic cystic disease, but only rarely exhibit renal cysts. Yoder et al. (50) previously described a transgenic rescue experiment of the orpk model of PKD in which the renal but not the hepatic defect was rescued. A difference in renal and hepatic cyst development is also evident in the Pkd1 and Pkd2 homozygous knockouts, as they develop renal cysts but do not have liver abnormalities (19,21,48). Because of the lethality of these models it is unknown if (and when) liver cysts would develop, but the corresponding heterozygotes do develop cystic liver disease in older animals (20,48) (Table 1). It appears that a lack (or reduction) of the polycystins has an effect later in the liver than in the kidneys, possibly reflecting later expression in that organ. It is not clear why the hepatic phenotype is not as readily rescued as the renal disease, but it is interesting that the changes mirror those seen in the del34 heterozygotes (Table 3).
There is growing evidence that cyst formation in ADPKD is a two-hit process, in which somatic inactivation of the normal polycystin allele is required before a cyst can develop. However, the primary importance for cyst initiation of the observed somatic events has been questioned because of the polycystin immunoreactivity of cystic epithelia (see Introduction). In the case of the PKD1 transgenics, loss of all polycystin-1 seems an unlikely scenario for disease causation given the multiple copies of the transgene and the two endogenous alleles. RNA expression studies indicate that transgenic expression does not down-regulate the endogenous gene and the rescue experiments show that the transgene is functional. It is possible that the tandem array of transgenes is unstable, but we have not seen clear germline changes in copy number and somatic deletion of the whole locus would still leave the endogenous genes. Although truncated products, which could have a dominant negative effect, may be expressed from the transgenic array after somatic mutation, there is no evidence of a dominant negative effect in ADPKD. The Pkd1 knockout models may generate some aberrant products (21; W. Lu et al., submitted), but as the disease is much milder in heterozygotes than homozygotes it is unlikely that such products can exert a dominant negative effect.
It seems likely that the level of PKD1 expression explains cyst development in the transgenics. There are examples of dominant genetic disorders, such as Darier disease (51) and Pelizaeus–Merzbacher disease (PMD) (52), in which dosage reduction (haplo-insufficiency) or dosage increase (by gene duplication) lead to a disease phenotype. Duplication, missense mutation and inactivation of the disease gene in PMD, PLP, results in a similar phenotype, suggesting that the level of the protein is critical. Indeed, transgenic mice that over-express PLP have a similar disease phenotype to mouse models with mutations in the Plp gene. It has been suggested that abnormal levels of PLP disrupt complex formation during transport through a secretory pathway (53). A similar explanation could be applied to the PKD1 transgenic mice if polycystin-1 forms part of a complex in which strict molar ratios of the components are required for efficient assembly and in which imbalance leads to a high level of functionless or aberrantly functioning complexes. This could be possible if polycystin-1 is involved in cell–cell interactions (7), cell–matrix (8) interactions and/or ion transport. A critical imbalance of subunits in a polycystin-1-containing complex during branching, or in maintenance of the tubular network in the kidney, may lend to cyst initiation. The observed focal nature of cyst formation in the transgenic mice is less readily explained without secondary genetic events, but it is possible that stochastic factors and threshold levels determine whether a cell tips into a cystic cascade. It is worth noting that focal cysts are found in many other transgenic models that overexpress factors associated with proliferation (54–58). It is unlikely that somatic mutational events are important for cyst development in these transgenic models, and therefore, it seems that focal cysts can often result from global aberrant gene expression.
We have described a novel model of PKD caused by over-expression of PKD1. Although this is not an entirely faithful model of human ADPKD, as extra copies of the gene are present rather than a germline mutation to one allele, it does suggest that the level of polycystin-1 may be important in preventing cyst formation in the kidney and elsewhere. Excess polycystin-1, as in the transgenic model, can lead to cyst formation. These findings indicate that it is worth reassessing whether the reduction in the level of polycystin-1 in Pkd1 knockout heterozygotes (which have a phenotype similar to that of the TPK mice and rescued animals) and in ADPKD patients may be sufficient to trigger focal cyst formation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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DNA methods and reagents
The human DNA and cDNA probes used in this study have been described previously (2,59), except LRR (471 bp) which consists of nucleotides 281–751 of the PKD1 transcript (GenBank accession no. L33243). Newly described mouse cDNA probes are ex5–9 (1061 bp), ex23–26 (784 bp) and ex36–41 (795 bp) from nucleotides 1026–2088, nucleotides 8790–9573 and nucleotides 11 102–11 896 of the mouse transcript, respectively (60) (GenBank accession no. U70209). Southern blotting and hybridization was performed as previously described (61) and band intensities were quantified using the ImageQuant software and a STORM 860 phosphoimager (Molecular Dynamics, Sunnyvale, CA). FISH analysis employed metaphase spreads prepared from freshly harvested transgenic spleen. Cosmids corresponding to parts of the transgenic insert (JH2A and CW9D) (2,59) were hybridized and detected as previously described (62).
Generation of transgenic mice
The PAC clone 136-M1 was isolated from a library in the vector pCYPAC2N (63) by hybridization to gridded filters (HGMP, MRC). PAC DNA was prepared using a standard alkaline lysis protocol and 20 µg digested with PvuI in a total volume of 100 µl. The digested DNA was diluted to 1 ml with 10 mM Tris pH 7.5, 0.1 mM EDTA and purified using a G-50 Sepharose resin column. A 0.5 ng/µl solution of the eluted DNA was micro-injected into the pronuclei of fertilized (C57BL/6 x CBA) F1 mouse eggs (64). Transgenic progeny were identified by analysis of tail DNA and were mated to (C57BL/6 x CBA) F1 mice to establish lines.
RNA preparation, northern blotting and RNAase protection
Tissue samples were taken from sacrificed mice and immediately snap frozen in liquid nitrogen. RNA was isolated using the method of Chomczynski and Sacchi (65) and northern blots run in denaturing formaldehyde gels using standard procedures. For the RNAase protection assay, 32P-labelled riboprobes to the single-copy area of PKD1 (nucleotides 12 122–12 381) (29) and to Pkd1 (nucleotides 12 634–12 841) were generated by SP6 polymerase using an in vitro transcription kit (Roche Diagnostics, Indianapolis, IN) following the manufacturer’s instructions. A volume equivalent to 1 x 106 c.p.m. of each precipitated riboprobe was hybridized to precipitated total cellular RNA (5 µg of PKD1 probe and ß-actin, or 50 µg of Pkd1 probe) as previously described (66). Different quantities of RNA were employed for the different probes so that similar signal intensities were obtained on the final gel, to aid quantification. Following RNAase digestion and electrophoresis on 8% acrylamide gels, protected bands of the predicted size were quantified using a STORM 860 phosphoimager and ImageQuant software (Molecular Dynamics).
Plasma membrane preparation from tissues and western blot analysis
Tissue samples were taken from sacrificed mice and immediately snap frozen in liquid nitrogen. Samples were homogenized in 1 ml of buffer 1 (0.01 M Tris–HCl pH 7.4, 0.25 M sucrose, 0.2 mM CaCl2). The homogenate was diluted to 5 ml with ice-cold buffer 2 (0.01 M Tris–HCl pH 7.4, 0.025 M sucrose, 1 mM EDTA) and centifuged at 2000 g for 5 min. The resulting supernatant was loaded onto a 35% (w/v) sucrose cushion in SW40 tubes (Beckmann Coutler, Fullerton, CA) and centrifuged at 30 000 g at 4°C for 30 min. The interface was collected, diluted with buffer 3 (0.01 M Tris–HCl pH 7.4, 0.25 M sucrose) and centrifuged finally at 100 000 g at 4°C for 45 min. The final pellet was resuspended in 0.2–0.5 ml of 1% SDS, 10 mM Tris–HCl pH 7.4 and frozen at –70°C in aliquots. All buffers contained protease inhibitor tablets (Complete; Boehringer, Mannheim, Germany). The protein content of each sample was measured using a microtitre Lowry assay (Bio-Rad, Hercules, CA). Ten micrograms of each sample was resuspended in an equal volume of 2x Laemmli sample buffer (100 mM Tris–HCl pH 6.8, 4% SDS, 20% glycerol, 0.1% bromophenol blue, 5% ß-mercaptoethanol), denatured for 10 min at 94°C and loaded on 8% SDS–PAGE gels using a Mini Protean II apparatus (Bio-Rad). Separated proteins were transferred at 30 V overnight onto PVDF membrane (Millipore, Bedford, MA). Filters were blocked for 1 h in 10% non-fat milk in Tris-buffered saline (TBS) pH 7.4, probed with the primary antibody for 1 h and a horseradish peroxidase-conjugated goat anti-mouse IgG1 (Southern Biotechnology Associates, Birmingham, AL) secondary antibody for 1 h. Filters were then washed extensively with TBS pH 7.4 (with added 0.05% Tween). Bound secondary antibody was detected by enhanced chemiluminescence (Boehringer). Antibodies employed in this study were 7e12 and PKS-A, monoclonal antibodies to the LRR and C-terminal regions of human polycystin-1, respectively (29,35), a polyclonal antibody to human tuberin (1895) (67) and a monoclonal antibody to rabbit Na/K-ATPase 
1 subunit (Upstate Biotechnology, Lake Placid, NY).
Histology
Freshly taken transgenic tissues were fixed in formal saline and embedded in paraffin, and 5 µm sections were stained with haematoxylin and eosin by standard methods. The number of cysts in each kidney was documented by scoring a single representative longtitudinal section. Livers were scored by gross analysis and observation of a section including bile duct epithelia.
Immunohistochemistry
Paraffin sections (5 µm) of embryonic day 18.5 transgenic and normal kidneys were cut onto slides, deparaffinized in xylene, rehydrated in a graded series of ethanol and rinsed in distilled water. Endogenous peroxidase activity was blocked by a 30 min incubation in 1% hydrogen peroxide/50% methanol. After rinsing in phosphate-buffered saline (PBS), non-specific binding was blocked with 3% bovine serum albumin (Sigma, St Louis, MO) for 60 min. Sections were incubated at room temperature with the polycystin-1 antibodies, 7e12 or PKS-A, for 30 min, rinsed with PBS and treated with goat anti-mouse immunoglobulin horseradish peroxidase conjugate (Dako, Carpinteria, CA) for 30 min. Bound secondary antibody was detected with diaminobenzidine (Sigma). Counterstaining was carried out with haematoxylin and coverslips were attached using aqueous mounting medium.
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ACKNOWLEDGEMENTS |
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We would like to thank M. Nellist for the tuberin antibody; R. Bengal and S. Butler for technical help; HGMP, MRC, for the PAC filters and clone; and V.E. Torres and Sir D.J. Weatherall for helpful discussions and support. This work was supported by the Medical Research Council, the Polycystic Kidney Research Foundation, the Mayo Foundation and the Wellcome Trust.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 507 266 0541; Fax: +1 507 266 4710; Email: harris.peter@mayo.edu
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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