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Laboratory of Immunobiology, Center for Cancer Research [W. M., S-B. H., N. S., G. W. M., B. Z.], Mouse Cancer Genetics Program [L. T., S. S.], Laboratory of Animal Sciences [E. S.], and Basic Research Program [T. C. B., L. S. S.], Science Applications International Corporation-Frederick, Inc., National Cancer Institute Frederick, Frederick, Maryland 21702; Department of Urology, Yokohama City School of Medicine, Yokohama, 236 Japan [M. B.]; Molecular and Cellular Biology Research, Sunnybrook and Women’s College Health Centre, Toronto, Ontario, Canada M4N 3M5 [C. G. L.]; Surgical Neurology Branch, National Institute of Neurologic Disease, Blindness and Stroke, Bethesda, Maryland 20894 [S. P., A. O. V.]; and Department of Epidemiology and Preventive Medicine, University of Maryland at Baltimore, Baltimore, Maryland 21210 [C. G.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Extensive experimental evidence supports the role of pVHL as the substrate receptor of a ubiquitin ligase (E3) multiprotein complex (15, 16, 17, 18, 19)
. Under normoxic conditions, the E3 ligase complex targets HIF-
subunits for ubiquitin-mediated degradation (20, 21, 22, 23)
. In the absence of functional pVHL, HIF1- and HIF2- accumulate, even in the presence of oxygen, resulting in elevated transcription of a wide variety of HIF-controlled genes (24, 25, 26, 27)
. Overproduction of these angiogenic factors is thought to contribute to the highly vascular tumors that develop in VHL patients. However, a novel VHL mutation that dysregulates HIF1- and its downstream target genes without causing tumors to develop in patients (28)
suggests the possibility that another target gene(s), alone or in combination with HIF, may participate in tumorigenesis.
Most germ-line VHL mutations compromise the binding of the elongins through the -domain of pVHL or the binding of HIF through the ß-domain (15
, 19
, 21)
. In the kidney parenchyma from VHL patients, HIF activation was shown to be an early event occurring in morphologically normal single cells within the renal tubules after VHL inactivation (29)
, leading to progressive up-regulation of the HIF pathway and tumorigenesis. The VHL protein has been shown recently to bind to and stabilize microtubules, protecting them from depolymerization in vivo (30)
. Additionally, pVHL may play a role in the formation of a mature extracellular fibronectin matrix (31
, 32)
.
Several groups have attempted to develop a mouse model that mimics the phenotypic features of VHL disease. Mice homozygous for the VHL null allele died in utero because of vascular abnormalities of the placenta (33) , but heterozygous VHL null mice on a C57BL/6 background were phenotypically normal. Haase et al. (34) used Cre/lox site-specific recombination to avoid embryonic lethality, using an albumin promoter-driven Cre recombinase to conditionally inactivate the VHL gene in the livers of BALB/c mice. Homozygous deletion of VHL in the liver resulted in enlarged blood-filled vascular cavities, severe steatosis, and foci of increased vascularization in the liver parenchyma causing death at 6–12 weeks. Hepatic hemangiomas developed in the livers of heterozygous VHL null mice on a BALB/c background; however, no phenotype was observed in other organs.
The hallmark of human VHL disease is the development of multifocal, highly vascular tumors in VHL target organs. These tumors are thought to arise in cells carrying a germ-line VHL mutation as a consequence of independent somatic mutations or deletions that inactivate wild-type VHL. Indeed, loss of heterozygosity studies of the kidney and CNS of VHL patients have revealed numerous microscopic foci of dysplastic cells, which exhibit inactivation of the wild-type VHL allele (35) . In an effort to produce a VHL conditional knockout mouse model that more closely mimics human VHL disease, we have used a human ß-actin promoter-driven cre transgenic mouse that expresses cre in a mosaic pattern in multiple organs. Homozygous inactivation of VHL in numerous cells of multiple target organs in the mouse mimics the numerous, independent somatic mutations, which occur in human VHL disease. We present the phenotype of the VHL conditional knockout mouse model generated by this approach, including hepatic hemangiomas, angiectasis, and angiogenesis in multiple organs, and defective spermatogenesis, which represents a novel role for the VHL gene in reproductive biology.
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MATERIALS AND METHODS |
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phage library (Stratagene, La Jolla, CA) and subcloned into pBluescript vector. A loxP-neo -loxP cassette was inserted in an EcoRI site upstream of exon 2 for G418 selection, and a second loxP site was inserted in a unique HindIII site, 5.8 kb downstream, destroying the HindIII site. A thymidine kinase gene was inserted into the multiple cloning site of the VHL target vector for negative selection against random insertion. The vector was linearized with NotI and electroporated into strain129-derived mouse ES cells. ES cell clones, which had undergone homologous recombination with the VHL target vector, were selected on G418 (250 µg/ml) and gancyclovir (2 µM) -containing medium, using standard procedures (36)
. Subsequently, the neo cassette was deleted from the correctly targeted ES cells by transient transfection with a cytomegalovirus promoter-driven Cre recombinase expression vector. HindIII-digested DNA from ES cell clones was analyzed by Southern blot analysis using a PCR-generated 250-bp external probe, located upstream of the 5' loxP site, which detected a 7.7 kb HindIII fragment for the VHL wild-type allele, an 18.2 kb HindIII fragment for the VHL floxed allele (without neo gene), and a 12.4 kb HindIII fragment for the VHL deleted allele (Fig. 1, A and B)
.
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Evaluation of Cre Expression in ß-Actin cre-transgenic Mice.
To generate the VHL conditional knockout mice, we selected a transgenic mouse line on a C3H background, in which Cre recombinase expression was under the control of a human ß-actin promoter (38)
.4
Animal care was provided in accordance with the procedures outlined in the "Guide for the Care and Use of Laboratory Animals" (NIH Publication No. 86-23, 1985). Tissue expression of ß-actin promoter-driven Cre recombinase was evaluated in offspring from crosses of the ß-actin cre transgenic mice with ROSA26 reporter mice (39)
. Cre recombinase expression in tissues of ROSA 26 reporter mice will delete the floxed neo expression cassette upstream of the LacZ gene, permitting LacZ gene expression and detection by standard ß-galactosidase staining techniques.
Delivery of Cre Recombinase by Tamoxifen Induction of CreERTM Mice.
Cre ERTM mice carry a transgene encoding a fusion protein between Cre recombinase and the ligand-binding domain of a mutant mouse estrogen receptor, which binds the synthetic ligand tamoxifen, but not endogenous17ß-estradiol (40)
. The CreERTM fusion protein is sequestered in the cytoplasm until administration of tamoxifen, which allows nuclear localization of CreERTM and deletion of the floxed VHL allele. Offspring produced from a cross between female VHLf/f and male VHLd/+/CreERTM mice were injected i.p. with tamoxifen in corn oil (0.36 mg/g body weight) or corn oil alone at 10 weeks of age. Animals were euthanized if found moribund. Autopsies were performed to evaluate the organs.
Delivery of Cre Recombinase by Adv/cre Injection.
A recombinant adenoviral vector that contained the cre gene under control of the herpes simplex virus thymidine kinase promoter (Adv/cre) was a gift from Dr. Leslie Krushel (Scripps Research Institute, La Jolla, CA; 41
). Virus stocks were prepared as described (41)
and titered on 293 cells. Nine VHLf/d and 6 VHLf/+ mice were injected i.v. in the tail vein with 8 x 108 plaque-forming units of Adv/cre. Animals were euthanized at 3-month time points and autopsied for phenotype evaluation.
Production of VHL-conditional Knockout Mice.
Heterozygous VHL floxed mice (VHLf/+) were intercrossed to generate homozygous VHL floxed mice (VHLf/f) and crossed with ß-actin cre transgenic mice (Cre/Cre) to produce mice with one VHL deleted allele and a human ß-actin cre transgene (VHLd/+/Cre). To generate VHL conditional knockout mice, we crossed VHLf/f mice with VHLd/+/Cre mice and selected for VHLf/d/Cre offspring by Southern blot analysis of HindIII-digested tail DNA.
Survival Rate Analysis.
We recorded survival data for all four of the genotypes and introduced the data into the SAS ver.8.0 statistical program for analysis.
Phenotype Evaluation and Histopathology.
Phenotype evaluation was performed on animals from 4–12 months of age. Mice were euthanized when moribund, and age-matched controls were sacrificed at the same time. Body and organ weights were recorded during a complete necropsy. Tissues were fixed in 10% formalin, embedded in paraffin, and 5-µm sections were prepared. The sections were deparaffinized and rehydrated through an ethanol dilution series into water. Sections were stained with H&E.
Immunohistochemistry.
Selected tissues were frozen in OCT with liquid nitrogen and sectioned at a thickness of 10 µm. The sections were fixed in methanol:acetone (1:1) for 10 min at -20°C. Monoclonal anti-HIF-1 (residues 432–528; 1:200; NeoMarkers) was used as the primary antibody for immunohistochemistry. Detection was performed with the UltraVision Mouse Tissue Detection System (NeoMarkers).
Western Analysis.
Mice were killed by CO2 asphyxiation. Liver and testes were collected, immediately snap frozen in liquid nitrogen, and stored at -80°C. Whole-cell extracts were prepared by homogenization and lysis in 20 mM HEPES, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 1 mM Na3VO4, leupeptin 10 µg/ml, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 0.5% deoxycholate, and 0.1% SDS. The homogenate was centrifuged at 2000 rpm for 2 min at 4°C, and the supernatant was stored at -80°C. Total protein was determined with BCA protein assay reagent (Pierce). VHL protein (250 µg/sample per reaction) was immunoprecipitated with monoclonal antihuman VHL Ig32 (1 µg antibody per reaction; PharMingen), separated by 14% SDS-PAGE, and transferred to a nitrocellulose membrane (Millipore). The membranes were blocked with 5% skim milk in 0.1 M PBS buffer (pH 7.4). After incubation with antimouse VHL M-20 antibody (1:400; Santa Cruz Biotechnology), the membrane was washed with PBS for 3 x 10 min, and then incubated with horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech). Signals were detected using the enhanced chemiluminescence system (ECL; Amersham Pharmacia Biotech). To monitor for equal protein loading, ß-actin levels in cell lysates were evaluated by Western blot analysis.
Infertility Testing.
Eight 10–15-week-old VHLf/d/Cre males were bred with 16 age-matched C57BL/6 females over a 4-month time period to evaluate infertility.
Sperm Counts.
The right epididymis was placed into PBS (0.5 ml), the sperm was released with a 27 1/2-gauge needle, and incubated at 37°C in a 5% CO2 atmosphere for 10 min. The sperm were loaded into chamber slides and counted under a dark field microscope with a computer-assisted sperm analysis program (Hamilton-Thorne Research)
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RESULTS |
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. The VHL targeting vector was then introduced into mouse ES cells by electroporation, and homologous recombination was achieved at a frequency of 5%. The neo gene used for positive selection of correctly targeted ES cells was removed by Cre-mediated recombination, and then mice were generated from these targeted ES cells with germ-line transmission of the VHL conditional allele.
Evaluation of ß-Actin Promoter-driven Cre Recombinase Activity Demonstrates Mosaic Expression in ROSA26 Mice.
Cre/lox site-specific recombination mediates deletion of the floxed allele of a target gene in a tissue- or time-dependent manner, thus circumventing embryonic lethality, when homozygous deletion of the gene is lethal (42)
. To produce a mouse model that would closely mimic human VHL disease, we wanted to generate mice in which random VHL deletion events occurred in cells carrying a germ-line VHL deletion. We obtained a transgenic mouse line with Cre recombinase under the control of the human ß actin promoter.4
In general, the human ß-actin promoter is expressed ubiquitously, and it is used to generate transgenic mice expressing high levels of a particular transcript (43)
. However, for this study we chose a line, which expressed cre during early stages of embryogenesis in a stochastic manner, generating an incomplete pattern of Cre expression. When the human ß-actin promoter-driven cre transgenic line was crossed with the ROSA26 reporter mouse (39)
, we found that Cre was expressed and mediated recombination with a mosaic pattern in a variety of organs including liver, heart, kidney, lung, pancreas, brain, retina, spleen, and adrenal gland (Fig. 2)
. Strong expression of human ß-actin promoter-driven Cre recombinase was observed only in the testis and epididymis. The mosaic tissue expression pattern of ß-actin promoter-driven Cre recombinase provided a system for Cre delivery, which was robust in many of the VHL target tissues, but not ubiquitous, thereby circumventing embryonic lethality.
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Comparison of growth curves for VHLf/+ mice, VHLf/f mice, VHLd/+/Cre mice, and VHLf/d/Cre mice indicated that the presence of a VHL floxed allele or deleted allele did not affect growth; these mice grew and developed normally during the rapid growth phase from birth to 8 weeks (Fig. 1C)
.
VHL Protein Levels Were Reduced in Tissues of Mice with VHL Inactivation.
To compare pVHL expression in VHL heterozygous (VHLf/d) and homozygous deleted mice (VHLf/d/Cre) with expression in VHL heterozygous floxed mice (VHLf/+), we evaluated the levels of VHL protein in liver and testis tissues of littermates with different VHL genotypes by Western analysis. Two VHL protein products (long and short forms, Mr 21,000 and Mr 19,000, respectively) were identified in liver and testis tissues. Reduced expression levels of VHL protein were detected in VHLf/d mice compared with VHLf/+ mice (Fig. 3A)
. As a result of mosaic tissue expression of Cre recombinase, we detected an additional reduction in pVHL expression in VHLf/d mice carrying the cre transgene. No differences in pVHL expression between VHL wild-type and floxed mice were observed (data not shown).
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. The median survival time was 26 weeks. Shortly before death (1–2 days), some VHLf/d/Cre mice lost weight, displayed rough fur, and a hunched body position. However, many of these mice died abruptly, with few outward symptoms.
Multiple Hemangiomas, Angiectasis, and Angiogenesis Develop in Livers of 100% of VHL-conditional Knockout Mice.
The most striking phenotypic features of VHLf/d/Cre mice at autopsy were observed in the liver. Surfaces of the livers were usually irregular and contained numerous dark red to black vascular lesions (Fig. 4, A and B)
. Examination of livers of VHLf/d/Cre mice that died revealed hepatic vascular lesions containing multiple, large, thin-walled vessels filled with blood. In VHLf/d/Cre mice that were euthanized at predetermined time points, the appearance of hepatic vascular lesions ranged from multiple, large, blood-filled, vascular vessels to single, small, blood-filled, vascular lesions (Fig. 4, A and B)
. Histological examination of the livers revealed frequent angiectasis, limited new blood vessel formation and the occurrence of multiple hemangiomas, and a single hemangiosarcoma (Fig. 4, C–F)
.
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. For comparison, we examined age-matched control littermates and observed 4 of 22 (18%) VHLf/d mice with hepatic hemangiomas; these hemangiomas were smaller in size and fewer in number than those observed in VHLf/d/Cre mice. Two of 22 (9%) age-matched VHLf/+/Cre littermates developed hepatic hemangiomas (Table 1)
. The vascular liver lesions that occurred in these control VHLf/d and VHLf/+/Cre mice presumably represent a sporadic loss of heterozygosity event, which would be predicted to inactivate the remaining wild-type VHL allele (one VHL allele is already deleted in the germ-line or by cre expression), predisposing the liver to develop the vascular phenotype as a result of homozygous VHL deletion.
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. We observed that our attempts to breed 8 VHLf/d/Cre male mice with 16 C57BL/6 females over a 4-month period were unsuccessful; only 1 of 8 VHLf/d/Cre male mice was able to impregnate 2 females. This led us to evaluate the male reproductive organs and spermatogenesis in male VHL conditional knockout mice. The testes from 16 VHLf/d/Cre mice were examined at autopsy and by histopathology. We observed reduction in size and weight of the testicle, testes degeneration, and oligospermia in the testes of 16 of 16 VHLf/d/Cre male mice (Fig. 5, A and B
; Table 2
). Sperm counts performed in 9 VHLf/d male mice and 9 VHLf/d/Cre mice showed 2-fold (15.19 ± 6.45 x 106 per testis) and 30-fold (1.13 ± 0.95 x 106/testis; P < 0.0001) reduction in viable sperm, respectively, compared with sperm counts in VHLf/+ control male animals (33.64 ± 4.99 x 106/testis; Table 2
).
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. Enlarged blood vessels occurred in connective tissue surrounding the seminiferous tubules. Cross-sections of the epididymal sac showed large numbers of sperm in control mice but few sperm in the lumen of VHLf/d/Cre mice (data not shown).
Angiectasis and Angiogenesis Are Observed in the Heart, Liver, Pancreas, Lung, and Kidney of the VHLf/d/Cre Mice but not in Other Organs.
In addition to the extensive vascular phenotype in the liver, new blood vessel formation (angiogenesis) in the cardiac muscle was observed in 8 of 10 VHLf/d/Cre mice (Fig. 6A)
. Three of 10 kidneys from VHLf/d/Cre mice displayed abnormally enlarged blood vessels (angiectasis; Fig. 6B
). Increased numbers of blood vessels in the pancreas were observed in 6 of 10 VHLf/d/Cre animals (Fig. 6C)
, and 10 of 10 animals with VHLf/d/Cre genotype showed abnormally enlarged blood vessels in the liver (Fig. 6D)
.
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Up-Regulated HIF1- Expression Is Observed in Hepatic Vascular Lesions in VHLf/d/Cre Mice.
pVHL functions to target HIF1- for ubiquitin-mediated degradation during normoxic conditions. HIF1- is overexpressed in the absence of functional pVHL, up-regulating hypoxia-inducible genes, which support angiogenesis. We evaluated HIF1- expression by immunohistochemistry and found mosaic expression of HIF1- in livers of VHLf/d/Cre mice, especially in endothelial cells lining the vascular lesions, but no HIF1- expression in control VHLf/+ mice (Fig. 3B)
. In serial sections of VHLf/d/Cre livers, we observed some loss of pVHL expression in a mosaic pattern (data not shown).
Alternate Methods of Cre Recombinase Delivery to VHLf/d Mice Resulted in a Similar Vascular Phenotype.
As an alternative to mosaic expression of Cre recombinase driven by the ß-actin promoter, we crossed VHLf/f mice with VHLd/+/CreERTM mice carrying a transgene that produces a tamoxifen-inducible fusion protein between Cre recombinase and a mutant ligand-binding domain of the 17ß-estradiol receptor (40)
. Most of the VHLf/d/CreERTM mice died (21 of 23) within 1 month after tamoxifen induction, with multiple hepatic hemangiomas. Only 3 of 35 VHLf/d mice and 2 of 35 VHLf/+/CreERTM mice died in response to tamoxifen induction (data not shown).
We also delivered Cre recombinase to VHLf/d mice and VHLf/+ control mice by tail vein injection of Adv/cre, a replication-defective adenovirus carrying Cre recombinase (41) . At 3-, 6-, and 9-month time points, mice were euthanized and examined by autopsy. By 9 months, multiple hemangiomas had appeared in the livers of 5 of 9 Adv/cre-injected VHLf/d mice but in only 2 of 6 uninjected VHLf/d mice and 0 of 6 VHLf/+ Adv/cre-injected mice had developed a vascular phenotype (data not shown).
Modifier Genes Present in Different Mouse Strains May Enhance the Phenotype of the VHL-conditional Knockout Mice.
Reports in the literature suggest that mouse models for human disease may develop variable phenotypes, which are strain-dependent. It has been proposed that these differences in phenotype are because of the presence of polymorphic variants in certain modifier genes in some strains, which may contribute to the development of the disease phenotype. In this report we describe a VHL conditional knockout mouse model produced in a C57BL/6 background, which developed liver hemangiomas in 100% (22 of 22) of animals by 1 year. Only 18% (4 of 22) of VHLf/d control littermates developed liver hemangiomas during that period. We evaluated the effect of different strains (modifier genes) on phenotype development by introducing the VHL deleted allele into BALB/c and A/J mouse strains, and observed first generation offspring for phenotypic changes. After 18 months, 88% (7 of 8) of VHLd/+BALB/c mice developed liver hemangiomas, and 67% (4 of 6) of VHLd/+ A/J mice developed hepatic vascular lesions, representing a substantial increase in the number of vascular lesions when compared with C57BL/6 VHLf/d mice.
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DISCUSSION |
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is disrupted by conditional inactivation of the VHL gene in our mouse model. Single layers of endothelial cells, which line the multiple, dilated blood vessels, were seen as sites of HIF1- up-regulation resulting in increased angiogenic signaling and a multiorgan vascular phenotype. In mice, unlike humans, the vascular phenotype was most extensive in the liver, resulting in the development of hemangiomas and hemangiosarcomas in 100% of VHL conditional knockout mice.
Here we present important new findings not reported previously in VHL knockout mouse models, which suggest a novel role for pVHL in mouse spermatogenesis. Oligospermia, reduction in testicular weight, and infertility were observed in male VHLf/d/Cre mice. Histological evaluation of the testes suggests that loss of pVHL function may interfere with maturation of spermatogonia cells to mature sperm. VHL expression was shown to be high in developing seminiferous tubules of the mouse testis at embryonic days 10–13, but low in the ovary (44)
. An oxygen-dependent isoform of HIF (HIF1-1.2) is expressed in testis (45
, 46)
, permitting one to speculate that impaired regulation of HIF may result in defects in spermatogenesis.
Evidence is accumulating for a role of VHL in induction of cell differentiation and growth arrest through integration of cell-cell and cell-extracellular matrix signals. In the absence of pVHL, renal cells do not differentiate, and the cell cycle proceeds (47) . In addition, pVHL was shown to play a role in neuronal differentiation (48 , 49) . Inactivation of pVHL may also inhibit differentiation in the testis, resulting in defects in spermatogenesis. Inability to exit the cell cycle, a "gatekeeper" function of pVHL, may induce apoptotic cell death.
The VHL protein was shown recently to bind to and stabilize microtubules (30)
. The testicular degeneration seen in the male VHLf/d/Cre mice may be a consequence of loss of microtubule stability within the testis, which is important in mitosis as well as meiosis. MutS homologue 4 mouse knockouts, defective in postreplicative DNA mismatch repair and meiotic recombination, display testicular degeneration and are infertile (50)
. Msh4, which is expressed predominantly in the testis, associates with VBP1 (51)
in vivo and in vitro. Disruption of the VBP1 homologue in yeast (GIM2/PAC10) has been shown to interfere with -tubulin biogenesis; the lethal phenotype can be rescued by mammalian VBP1 (52)
, identified as a VHL binding partner in a yeast-two hybrid system (53)
. Additional studies will be required to ascertain the mechanism by which absence of pVHL leads to defective spermatogenesis.
The VHL conditional knockout mouse model described in this report is the second attempt to create an animal model for the VHL disease using conditionally targeted VHL alleles. Haase et al. (34) used albumin promoter driven-Cre expression previously to target VHL inactivation in the liver, which produced enlarged blood vessels and premature death. In our mouse model, ß-actin promoter-driven mosaic expression of Cre recombinase, resulting in homozygous inactivation of VHL in certain cells within target organs, more closely mimics the human disease condition in which loss of the wild-type VHL allele occurs in cells carrying a germ-line VHL mutation. Stochastic cre expression produced not only an extensive vascular phenotype in the liver, but also angiectasis and angiogenesis in the pancreas, kidney, spleen, and heart, and defective spermatogenesis, reflecting a novel role for pVHL.
Different modifier gene alleles present in the different mouse genetic backgrounds may enhance or alter phenotype development in mouse models of human cancer. Strain differences were shown to contribute to the disease phenotype in a mouse model of hereditary hemorrhagic telangiectasis (54) and in a mouse model for tuberous sclerosis (55 , 56) . The differences in phenotypic consequence of the heterozygous VHL deleted allele, which we observed among three different mouse strains in our study, suggest that strain-specific modifier genes may provide protection against the formation of hepatic hemangiomas in VHLf/d C57BL/6 mice, or may enhance the development of the vascular phenotype in VHLd/+ BALB/c or A/J mice. Identification of these modifier genes may provide insight into the events leading to the development of the vascular phenotype and suggest additional angiogenic effectors to knockout in our VHL mouse model.
Multiple hepatic hemangiomas produced by conditional inactivation of the VHL gene resulted in premature death, and the lethality of this phenotype prevented long-term observation of mice for possible changes in the kidneys or CNS. Delivery of Cre recombinase either by i.v. injection with adenovirus carrying Cre recombinase, or tamoxifen induction of CreERTM recombinase resulted in the formation of hepatic hemangiomas and increased mortality. Organ-specific (i.e. kidney or CNS) delivery of Cre recombinase will be required to circumvent the lethality of the hepatic vascular phenotype produced by inactivation of the VHL gene in mice.
In summary, we have generated a VHL conditional knockout mouse model with an extensive vascular phenotype in multiple organs, and infertility and reduced sperm count in males, which will prove useful for testing antiangiogenic drug treatments, as well as understanding the novel role for VHL in spermatogenesis. Renal cell carcinomas, pheochromocytomas, and CNS hemangioblastomas, which are observed in patients affected with VHL, were not observed in the VHL conditional knockout mouse model described in this report or by Haase et al. (34) . This observation raises the question of whether other events, perhaps inactivation of other modifier genes, may be essential to the development of these tumor phenotypes. Breeding experiments introducing null alleles of modifier genes (i.e. tumor suppressor genes) into the VHL conditional knockout mouse background are under way to test this hypothesis. Cre expression and targeted deletion of a VHL floxed allele in renal cells await the development of a kidney-specific Cre transgenic animal.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This publication has been funded in whole or in part with federal funds from the National Cancer Institute, NIH, under Contract # N01-C0–12400. 
2 To whom requests for reprints should be addressed, at Laboratory of Immunobiology, National Cancer Institute-Frederick, Building 560, Room 12-69, Frederick, MD 21702. Phone: (301) 846-5856; Fax: (301) 846-6145; E-mail: schmidtl{at}mail.ncifcrf.gov
3 The abbreviations used are: VHL, von Hippel-Lindau; f, VHL floxed allele; d, VHL deleted allele; +, VHL wild-type allele; Cre, human ß actin Cre recombinase transgene; CreERTM, tamoxifen-inducible Cre recombinase-mutant estrogen receptor fusion protein transgene; CNS, central nervous system; p, protein; HIF, hypoxia-inducible factor; ES, embryonic stem; floxed, flanked by loxP sites; VBP1, von Hippel-Lindau binding protein-1.
4 M. E. Palko and L. Tessarollo, unpublished observations.
Received 3/27/03. Revised 6/ 6/03. Accepted 6/ 9/03.
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. Genes Dev., 12: 149-162, 1998. is encoded by two different mRNA isoforms: expression from a tissue-specific and a housekeeping-type promoter. Blood, 91: 3471-3480, 1998. during the late stages of mouse spermiogenesis. Mol. Endocrinol., 16: 234-243, 2002.- and 
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D. J. Liepinsh, S. I. Grivennikov, K. D. Klarmann, M. A. Lagarkova, M. S. Drutskaya, S. J. Lockett, L. Tessarollo, M. McAuliffe, J. R. Keller, D. V. Kuprash, et al. Novel Lymphotoxin Alpha (LT{alpha}) Knockout Mice with Unperturbed Tumor Necrosis Factor Expression: Reassessing LT{alpha} Biological Functions. Mol. Cell. Biol., June 1, 2006; 26(11): 4214 - 4225. [Abstract] [Full Text] [PDF]
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N. Zanesi, R. Mancini, C. Sevignani, A. Vecchione, M. Kaou, M. Valtieri, G. A. Calin, Y. Pekarsky, J. R. Gnarra, C. M. Croce, et al. Lung Cancer Susceptibility in Fhit-Deficient Mice Is Increased by Vhl Haploinsufficiency Cancer Res., August 1, 2005; 65(15): 6576 - 6582. [Abstract] [Full Text] [PDF]
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E. B. Rankin, D. F. Higgins, J. A. Walisser, R. S. Johnson, C. A. Bradfield, and V. H. Haase Inactivation of the Arylhydrocarbon Receptor Nuclear Translocator (Arnt) Suppresses von Hippel-Lindau Disease-Associated Vascular Tumors in Mice Mol. Cell. Biol., April 15, 2005; 25(8): 3163 - 3172. [Abstract] [Full Text] [PDF]
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 B. I. Rini and E. J. Small Biology and Clinical Development of Vascular Endothelial Growth Factor-Targeted Therapy in Renal Cell Carcinoma J. Clin. Oncol., February 10, 2005; 23(5): 1028 - 1043. [Abstract] [Full Text] [PDF]
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 W.G. KAELIN JR. The von Hippel-Lindau Tumor Suppressor Protein: Roles in Cancer and Oxygen Sensing Cold Spring Harb Symp Quant Biol, January 1, 2005; 70(0): 159 - 166. [Abstract] [PDF]
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 C. Graveel, Y. Su, J. Koeman, L.-M. Wang, L. Tessarollo, M. Fiscella, C. Birchmeier, P. Swiatek, R. Bronson, and G. Vande Woude Activating Met mutations produce unique tumor profiles in mice with selective duplication of the mutant allele PNAS, December 7, 2004; 101(49): 17198 - 17203. [Abstract] [Full Text] [PDF]
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W. K. Rathmell, M. M. Hickey, N. A. Bezman, C. A. Chmielecki, N. C. Carraway, and M. C. Simon In vitro and In vivo Models Analyzing von Hippel-Lindau Disease-Specific Mutations Cancer Res., December 1, 2004; 64(23): 8595 - 8603. [Abstract] [Full Text] [PDF]
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 S. Richard, R. Lidereau, S. Giraud, and on behalf of the French inherited kidney tumours c The growing family of hereditary renal cell carcinoma Nephrol. Dial. Transplant., December 1, 2004; 19(12): 2954 - 2958. [Full Text] [PDF]
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 V. Coppola, C. A. Barrick, E. A. Southon, A. Celeste, K. Wang, B. Chen, E.-B. Haddad, J. Yin, A. Nussenzweig, A. Subramaniam, et al. Ablation of TrkA function in the immune system causes B cell abnormalities Development, October 15, 2004; 131(20): 5185 - 5195. [Abstract] [Full Text] [PDF]
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W. G. Kaelin Jr. The Von Hippel-Lindau Tumor Suppressor Gene and Kidney Cancer Clin. Cancer Res., September 15, 2004; 10(18): 6290S - 6295S. [Abstract] [Full Text] [PDF]
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 R. Depping, S. Hagele, K. F. Wagner, R. J. Wiesner, G. Camenisch, R. H. Wenger, and D. M. Katschinski A Dominant-Negative Isoform of Hypoxia-Inducible Factor-1{alpha} Specifically Expressed in Human Testis Biol Reprod, July 1, 2004; 71(1): 331 - 339. [Abstract] [Full Text] [PDF]
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 J. M. Ward and D. E. Devor-Henneman Mouse Models of Human Familial Cancer Syndromes Toxicol Pathol, January 1, 2004; 32(1_suppl): 90 - 98. [Abstract] [PDF]
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