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Hideaki Toki,
Maki Inoue,
Hiromi Motegi,
Osamu Minowa,
Hiroaki Kanda,
Noriko Yamamoto,
Ami Ikeda,
Yuko Karashima,
Junko Matsui, Hideki Kaneda,
Ikuo Miura,
Tomohiro Suzuki,
Shigeharu Wakana,
Hiroshi Masuya,
Yoichi Gondo,
Toshihiko Shiroishi,
Tetsu Akiyama,
Ryoji Yao,
Tetsuo Noda
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ABSTRACT: Mutant mouse models are indispensable tools for clarifying the functions of genes and elucidating the underlying pathogenic mechanisms of human diseases. We conducted large-scale mutagenesis employing the chemical mutagen N-ethyl-N-nitrosourea (ENU). One specific aim of our ENU mutagenesis project is to generate novel cancer models. We screened 7,012 animals for dominant traits using a necropsy test and thereby established 17 mutant lines predisposed to cancer. Here, we report on a novel cancer model line that developed osteoma, trichogenic tumor and breast cancer. Using fine mapping and genomic sequencing, we identified a point mutation in the adenomatous polyposis coli (Apc) gene. The Apc1576 mutants bear a nonsense mutation at codon 1,576 in the Apc gene. Although most Apc mutant mice established thus far have multifocal intestinal tumors, mice that are heterozygous for the Apc1576 mutation do not develop intestinal tumors; instead, they develop multifocal breast cancers and trichogenic tumors. Notably, the osteomas that develop in the Apc1576 mutant mice recapitulate the lesion observed in Gardner syndrome, a clinical variant of familial adenomatous polyposis (FAP). Our Apc1576 mutant mice will be valuable not only for understanding the function of the Apc gene in detail but also as models of human Gardner syndrome.
Cancer Science 04/2013; · 3.33 Impact Factor
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ABSTRACT: As part of the RIKEN large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis project, we screened mice with a dominant mutation that exhibited abnormal behavior using an open-field test and a home-cage activity test. We tested 495 male progeny of C57BL/6J males treated with ENU and untreated C3H/HeJ females using the open-field test and isolated behavioral mutant M101736, which exhibited a significant increase in spontaneous locomotor activity. We identified a missense mutation in the Tuba1 gene, which encodes the TUBA1 protein, and designated the mutant gene Tuba1(Rgsc1736). This mutation results in an aspartic acid to glycine substitution in the TUBA1 protein. Detailed analyses revealed that Tuba1(Rgsc1736) heterozygotes exhibited inattention to novel objects and aberrant patterns of home-cage activity. The results of a behavioral pharmacological analysis using methylphenidate and morphological analyses of embryonic and adult brains suggested that Tuba1(Rgsc1736) is a novel animal model for neurodevelopmental disorders.
Behavioural brain research 11/2011; 227(1):167-74. · 3.22 Impact Factor
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Takashi Kohda,
Narumi Ogonuki,
Kimiko Inoue,
Tamio Furuse, Hideki Kaneda,
Tomohiro Suzuki,
Tomoko Kaneko-Ishino,
Teruhiko Wakayama,
Shigeharu Wakana,
Atsuo Ogura,
Fumitoshi Ishino
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ABSTRACT: Faithful transcriptome regulation is important in development and also crucial for applications in reproductive and regenerative medicine. Intracytoplasmic sperm injection (ICSI), one of the human assisted reproductive technologies (ART), has long raised concerns about its influence on development. No clear consensus has been reached, however, in spite of many cohort studies carried out in the last two decades on the children conceived by ICSI and/or in vitro fertilization (IVF). In this study, the pre- and postnatal effects of ICSI were assessed using comprehensive transcriptome and phenotypic analyses in mice under strict conditions. Here we demonstrate that, in contrast to IVF, ICSI induces distinct long-lasting transcriptome change that remains at the neonatal stage. Importantly, no remarkable differences were observed in the ICSI adults in either the gene expression or phenotypic profiles, and there was no indication of transmission to the next generation via natural mating. Our results suggest there are no lifelong or transgenerational effects of ICSI, but the ICSI effects during neonatal period remain to be evaluated.
Biochemical and Biophysical Research Communications 05/2011; 410(2):282-8. · 2.48 Impact Factor
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Nobuhiko Tanaka,
Kazunori Waki, Hideki Kaneda,
Tomohiro Suzuki,
Ikuko Yamada,
Tamio Furuse,
Kimio Kobayashi,
Hiromi Motegi,
Hideaki Toki,
Maki Inoue,
Osamu Minowa,
Tetsuo Noda,
Keizo Takao,
Tsuyoshi Miyakawa,
Aki Takahashi,
Tsuyoshi Koide,
Shigeharu Wakana,
Hiroshi Masuya
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ABSTRACT: This article reports the development of SDOP-DB, which can provide definite, detailed and easy comparison of experimental protocols used in mouse phenotypic analyses among institutes or laboratories. Because SDOP-DB is fully compliant with international standards, it can act as a practical foundation for international sharing and integration of mouse phenotypic information. AVAILABILITY: SDOP-DB (http://www.brc.riken.jp/lab/bpmp/SDOP/).
Bioinformatics 03/2010; 26(8):1133-4. · 5.47 Impact Factor
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Tamio Furuse,
Yumiko Wada,
Kotaro Hattori,
Ikuko Yamada,
Tomoko Kushida,
Yoko Shibukawa,
Hiroshi Masuya, Hideki Kaneda,
Ikuo Miura,
Naoki Seno,
Tomoyuki Kanda,
Ryo Hirose,
Shinichiro Toki,
Kousuke Nakanishi,
Kimio Kobayashi,
Hideki Sezutsu,
Yoichi Gondo,
Tetsuo Noda,
Shigeki Yuasa,
Shigeharu Wakana
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ABSTRACT: In the RIKEN large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis project we screened mice with a dominant mutation that exhibited abnormal behavior in the open-field test, passive avoidance test and home-cage activity test. We tested 2045 progeny of C57BL/6J males treated with ENU and untreated DBA/2J females in the open-field test and isolated behavioral mutant M100174, which exhibited a significant increase in spontaneous locomotor activity. We identified a missense mutation in the Grin1 gene, which encodes NMDA receptor subunit 1, and designated the mutant gene Grin1(Rgsc174). This mutation results in an arginine to cysteine substitution in the C0 domain of the protein. Detailed analyses revealed that Grin1(Rgsc174) heterozygote exhibited increased novelty-seeking behavior and slight social isolation in comparison with the wild type. In contrast to other Grin1 mutant mice, this mutant exhibited no evidence of heightened anxiety. These results indicate that this is a unique behavioral Grin1 gene mutant mouse that differs from the known Grin1 mutant mice. The results of immunohistochemical and biochemical analyses suggested that impaired interaction between the glutamatergic pathway and dopaminergic pathway may underlie the behavioral phenotypes of the Grin1(Rgsc174) mutant.
European Journal of Neuroscience 03/2010; 31(7):1281-91. · 3.63 Impact Factor
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Hajime Sato,
Tomohiro Suzuki,
Kyoko Ikeda,
Hiroshi Masuya,
Hideki Sezutsu, Hideki Kaneda,
Kimio Kobayashi,
Ikuo Miura,
Yasuyuki Kurihara,
Shunji Yokokura,
Kohji Nishida,
Makoto Tamai,
Yoichi Gondo,
Tetsuo Noda,
Shigeharu Wakana
[show abstract]
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ABSTRACT: To characterize an N-ethyl-N-nitrosourea-induced dominant mouse mutant, M-1156, that exhibits progressive retinal degeneration and to investigate the pathogenesis of the retinal phenotype in the mutant.
A positional candidate gene approach was used to identify the causative gene in the M-1156 mutant. Funduscopic examination, light microscopy, transmission electron microscopy, and electroretinography were performed to analyze the M-1156 phenotype. Real-time quantitative PCR, immunohistochemistry, and western blotting were also performed.
Linkage analysis enabled the mutant gene to be mapped to a region of chromosome 19 containing Rom1, which encodes rod outer segment membrane protein 1. Sequence analysis demonstrated that the mutation consisted of a single base T-->C substitution at position 1,195 in Rom1 (M96760, National Center for Biotechnology Information [NCBI]) and that the mutant allele was expressed. A putative missense mutation designated Rom1(Rgsc1156) that was identified in the M-1156 mutant mouse causes a Trp to Arg substitution at position 182 in the translated protein. Rom1(Rgsc1156) heterozygotes were found to have a mottled retina and narrowed arteries in the fundus oculi. Photomicrographs of the retina revealed significant differences among the genotypes in the thickness of the outer nuclear layer and in the length of the outer segments of the photoreceptors. The alterations were more marked in the homozygotes than in the heterozygotes. Electron micrographs showed that the diameters of the discs varied in the heterozygotes and that the discs were more compactly stacked than in the wild type. There were significant differences among the genotypes in the amplitude of the a-wave in single-flash electroretinograms, but there were no significant differences among the photopic electroretinograms. Real-time quantitative PCR showed that there were no significant differences among the genotypes in Rom1 or peripherin/rds (Prph2) mRNA levels relative to the rhodopsin (Rho) mRNA level. Rom1 and Prph2 immunoreactivity were decreased in the retinas of the Rom1(Rgsc1156) mutants. Semiquantitative western blot analysis of retinas from 3-week-old Rom1(Rgsc1156) mutants demonstrated significant decreases in Rom1, Prph2, and Rho protein levels in all of the genotypes.
The Trp182Arg substitution in Rom1(Rgsc1156) mutants causes retinal degeneration. The results suggested that Trp182Arg mutant Rom1 causes a decrease in the levels of wild-type Prph2 and Rom1, which in turn cause a reduction in the level of Prph2 containing tetramers in the disc rim region and ultimately result in unstable, disorganized outer segments and photoreceptor degeneration.
Molecular vision 01/2010; 16:378-91. · 2.20 Impact Factor
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Yumiko Wada,
Tamio Furuse,
Ikuko Yamada,
Hiroshi Masuya,
Tomoko Kushida,
Yoko Shibukawa,
Yuji Nakai,
Kimio Kobayashi, Hideki Kaneda,
Yoichi Gondo,
Tetsuo Noda,
Toshihiko Shiroishi,
Shigeharu Wakana
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ABSTRACT: To establish the cutoff values for screening ENU-induced behavioral mutations, normal variations in mouse behavioral data were examined in home-cage activity (HA), open-field (OF), and passive-avoidance (PA) tests. We defined the normal range as one that included more than 95% of the normal control values. The cutoffs were defined to identify outliers yielding values that deviated from the normal by less than 5% for C57BL/6J, DBA/2J, DBF(1), and N(2) (DXDB) progenies. Cutoff values for G1-phenodeviant (DBF(1)) identification were defined based on values over +/- 3.0 SD from the mean of DBF(1) for all parameters assessed in the HA and OF tests. For the PA test, the cutoff values were defined based on whether the mice met the learning criterion during the 2nd (at a shock intensity of 0.3 mA) or the 3rd (at a shock intensity of 0.15 mA) retention test. For several parameters, the lower outliers were undetectable as the calculated cutoffs were negative values. Based on the cutoff criteria, we identified 275 behavioral phenodeviants among 2,646 G1 progeny. Of these, 64 were crossed with wild-type DBA/2J individuals, and the phenotype transmission was examined in the G2 progeny using the cutoffs defined for N(2) mice. In the G2 mice, we identified 15 novel dominant mutants exhibiting behavioral abnormalities, including hyperactivity in the HA or OF tests, hypoactivity in the OF test, and PA deficits. Genetic and detailed behavioral analysis of these ENU-induced mutants will provide novel insights into the molecular mechanisms underlying behavior.
Experimental Animals 01/2010; 59(4):495-510. · 0.92 Impact Factor
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Shigeharu Wakana,
Tomohiro Suzuki,
Tamio Furuse,
Kimio Kobayashi,
Ikuo Miura, Hideki Kaneda,
Ikuko Yamada,
Hiromi Motegi,
Hideaki Toki,
Maki Inoue,
Osamu Minowa,
Tetsuo Noda,
Kazunori Waki,
Nobuhiko Tanaka,
Hiroshi Masuya,
Yuichi Obata
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ABSTRACT: A systematic and comprehensive phenotyping platform has been developed by the RIKEN ENU-mutagenesis project between 1999 and 2007. As a result of phenotype screening on this platform, we have discovered about 400 mutants as animal models for human diseases. All information regarding these mouse mutants is now available to the public through our home page (http://www.brc.riken.jp/lab/gsc/mouse/indexJ.html). In 2008, we reconstructed the existing phenotyping platform and built a new platform. The new system has a hierarchical structure, consisting of a fundamental pipeline that utilizes the existing platform and an additional pipeline, which is optimized for more in-depth phenotyping assays. Using this system, we have started to perform more comprehensive phenotyping of mouse mutants. We have opened this system to Japanese scientists as the Japanese Mouse Clinic. It is anticipated that existing mouse mutants will be reevaluated as disease models by identifying novel phenotypes on the new platform. We will share detailed information about the standard operating procedures (SOPs) of our phenotyping analyses with other related large-scale projects, such as the European Mouse Disease Clinic (EUMODIC) and the German Mouse Clinic (GMC). Moreover, we will contribute to international efforts to standardize mouse phenotype data by sharing annotation of mutant phenotypes, which are made by internationally standardized methods, with other related projects.
Experimental Animals 10/2009; 58(5):443-50. · 0.92 Impact Factor
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ABSTRACT: A large number of genetically modified mouse strains have been produced in recent years. Sperm cryopreservation is the most effective means of preserving these valuable strains, most of which have a C57BL/6 genetic background. However, the fertilization efficiency of sperm from several cryopreserved strains, including C57BL/6, is quite low. While new and improved methods of cryopreservation have been developed, the majority of sperm stocks have already been cryopreserved using traditional methods, such as storage in 18% raffinose and 3% skim milk (R18S3). Therefore, new thawing methods for these frozen stocks are needed. We have developed a new thawing method that involves selective collection of motile sperm and a preincubation medium that enhances capacitation. Motile sperm are selected simply by collecting a sample from the center of a dish, and capacitation is induced by the addition of methyl-beta-cyclodextrin, D-penicillamine, sodium citrate, and hypotaurine to modified Tyrode's solution. The fertilization rate of sperm prepared using this method was increased significantly compared to that of sperm thawed using the traditional method (63.9 vs 16.5%, P<0.01). These results demonstrate that this new in vitro fertilization method is an effective means of reviving C57BL/6 sperm cryopreserved in R18S3.
Experimental Animals 07/2009; 58(4):395-401. · 0.92 Impact Factor
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ABSTRACT: The DBA/2J mouse strain is a standard laboratory strain that is widely used for biomedical research. This strain, however, suffers from poor reproductive performance. In addition, the conditions for reliable embryo transfer (ET) of this strain have not been elucidated. The intention of this study was to determine the optimal number of embryos for transfer that allow the effective production of DBA/2J offspring. In the experiment, 7 to 15 embryos per oviduct were transferred into pseudopregnant ICR females. A relatively high success rate for pup production was observed when a large number of DBA/2J embryos (30 embryos per female) were transferred. This result shows that the ET efficiency of the DBA/2J strain can be improved by increasing the number of transferred embryos.
Experimental Animals 11/2007; 56(5):385-8. · 0.92 Impact Factor
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Steven J Clapcote,
Tatiana V Lipina,
J Kirsty Millar,
Shaun Mackie,
Sheila Christie,
Fumiaki Ogawa,
Jason P Lerch,
Keith Trimble,
Masashi Uchiyama,
Yoshiyuki Sakuraba, Hideki Kaneda,
Toshihiko Shiroishi,
Miles D Houslay,
R Mark Henkelman,
John G Sled,
Yoichi Gondo,
David J Porteous,
John C Roder
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ABSTRACT: To support the role of DISC1 in human psychiatric disorders, we identified and analyzed two independently derived ENU-induced mutations in Exon 2 of mouse Disc1. Mice with mutation Q31L showed depressive-like behavior with deficits in the forced swim test and other measures that were reversed by the antidepressant bupropion, but not by rolipram, a phosphodiesterase-4 (PDE4) inhibitor. In contrast, L100P mutant mice exhibited schizophrenic-like behavior, with profound deficits in prepulse inhibition and latent inhibition that were reversed by antipsychotic treatment. Both mutant DISC1 proteins exhibited reduced binding to the known DISC1 binding partner PDE4B. Q31L mutants had lower PDE4B activity, consistent with their resistance to rolipram, suggesting decreased PDE4 activity as a contributory factor in depression. This study demonstrates that Disc1 missense mutations in mice give rise to phenotypes related to depression and schizophrenia, thus supporting the role of DISC1 in major mental illness.
Neuron 06/2007; 54(3):387-402. · 14.74 Impact Factor
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Hiroshi Masuya,
Hideki Sezutsu,
Yoshiyuki Sakuraba,
Tomoko Sagai,
Masaki Hosoya, Hideki Kaneda,
Ikuo Miura,
Kimio Kobayashi,
Kenta Sumiyama,
Aya Shimizu,
Junko Nagano,
Haruka Yokoyama,
Satoko Kaneko,
Noriko Sakurai,
Yuka Okagaki,
Tetsuo Noda,
Shigeharu Wakana,
Yoichi Gondo,
Toshihiko Shiroishi
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ABSTRACT: Mammal-fish-conserved-sequence 1 (MFCS1) is a highly conserved sequence that acts as a limb-specific cis-acting regulator of Sonic hedgehog (Shh) expression, residing 1 Mb away from the Shh coding sequence in mouse. Using gene-driven screening of an ENU-mutagenized mouse archive, we obtained mice with three new point mutations in MFCS1: M101116, M101117, and M101192. Phenotype analysis revealed that M101116 mice exhibit preaxial polydactyly and ectopic Shh expression at the anterior margin of the limb buds like a previously identified mutant, M100081. In contrast, M101117 and M101192 show no marked abnormalities in limb morphology. Furthermore, transgenic analysis revealed that the M101116 and M100081 sequences drive ectopic reporter gene expression at the anterior margin of the limb bud, in addition to the normal posterior expression. Such ectopic expression was not observed in the embryos carrying a reporter transgene driven by M101117. These results suggest that M101116 and M100081 affect the negative regulatory activity of MFCS1, which suppresses anterior Shh expression in developing limb buds. Thus, this study shows that gene-driven screening for ENU-induced mutations is an effective approach for exploring the function of conserved, noncoding sequences and potential cis-regulatory elements.
Genomics 03/2007; 89(2):207-14. · 3.02 Impact Factor
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Hiroshi Masuya,
Maki Inoue,
Yumiko Wada,
Aya Shimizu,
Junko Nagano,
Akiko Kawai,
Ayako Inoue,
Tomoko Kagami,
Taeko Hirayama,
Ayako Yamaga, Hideki Kaneda,
Kimio Kobayashi,
Osamu Minowa,
Ikuo Miura,
Yoichi Gondo,
Tetsuo Noda,
Shigeharu Wakana,
Toshihiko Shiroishi
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ABSTRACT: SHIRPA is a three-stage protocol for the comprehensive assessment of primarily mouse behavior. The first stage consists of high-throughput phenotyping of 33 behavioral observations and 7 metabolic or disease observations. We modified this part of the protocol by integrating new morphologic observations into the initial phenotype assay of behavior and dysmorphology. Behavioral observations assessed by this protocol, now referred to as the "modified-SHIRPA," are compatible with the original "SHIRPA" protocol. Using modified-SHIRPA, we screened dominant phenotypes of more than 10,000 G(1) progeny generated by crossing DBA/2J females with ENU-treated C57BL/6J males. To date, we have obtained 136 hereditary-confirmed mutants that exhibit behavioral and morphologic defects. Some independent mutant lines exhibited similar phenotypes, suggesting that they may represent alleles of the same gene or mutations in the same genetic pathway. They could hold great potential for the unraveling of the molecular mechanisms of certain phenotypes.
Mammalian Genome 12/2005; 16(11):829-37. · 2.89 Impact Factor
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Yoshiyuki Sakuraba,
Hideki Sezutsu,
K Ryo Takahasi,
Keiko Tsuchihashi,
Rie Ichikawa,
Naomi Fujimoto,
Satoko Kaneko,
Yuji Nakai,
Masashi Uchiyama,
Noriko Goda, [......], Hideki Kaneda,
Hiroshi Masuya,
Osamu Minowa,
Hideki Noguchi,
Atsushi Toyoda,
Yoshiyuki Sakaki,
Shigeharu Wakana,
Tetsuo Noda,
Toshihiko Shiroishi,
Yoichi Gondo
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ABSTRACT: The large-scale mouse mutagenesis with ENU has provided forward-genetic resources for functional genomics. The frozen sperm archive of ENU-mutagenized generation-1 (G1) mice could also provide a "mutant mouse library" that allows us to conduct reverse genetics in any particular target genes. We have archived frozen sperm as well as genomic DNA from 9224 G1 mice. By genome-wide screening of 63 target loci covering a sum of 197 Mbp of the mouse genome, a total of 148 ENU-induced mutations have been directly identified. The sites of mutations were primarily identified by temperature gradient capillary electrophoresis method followed by direct sequencing. The molecular characterization revealed that all the identified mutations were point mutations and mostly independent events except a few cases of redundant mutations. The base-substitution spectra in this study were different from those of the phenotype-based mutagenesis. The ENU-based gene-driven mutagenesis in the mouse now becomes feasible and practical.
Biochemical and Biophysical Research Communications 11/2005; 336(2):609-16. · 2.48 Impact Factor
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Hiroshi Masuya,
Kunihiko Shimizu,
Hideki Sezutsu,
Yoshiyuki Sakuraba,
Junko Nagano,
Aya Shimizu,
Naomi Fujimoto,
Akiko Kawai,
Ikuo Miura, Hideki Kaneda,
Kimio Kobayashi,
Junko Ishijima,
Takahide Maeda,
Yoichi Gondo,
Tetsuo Noda,
Shigeharu Wakana,
Toshihiko Shiroishi
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ABSTRACT: Amelogenesis imperfecta (AI) is a group of commonly inherited defects of dental enamel formation, which exhibits marked genetic and clinical heterogeneity. The genetic basis of this heterogeneity is still poorly understood. Enamelin, the affected gene product in one form of AI (AIH2), is an extracellular matrix protein that is one of the components of enamel. We isolated three ENU-induced dominant mouse mutations, M100395, M100514 and M100521, which caused AI-like phenotypes in the incisors and molars of the affected individuals. Linkage analyses mapped each of the three mutations to a region of chromosome 5 that contained the genes encoding enamelin (Enam) and ameloblastin (Ambn). Sequence analysis revealed that each mutation was a single-base substitution in Enam. M100395 (Enam(Rgsc395)) and M100514 (Enam(Rgsc514)) were putative missense mutations that caused S to I and E to G substitutions at positions 55 and 57 of the translated protein, respectively. Enam(Rgsc395) and Enam(Rgsc514) heterozygotes showed severe breakage of the enamel surface, a phenotype that resembled local hypoplastic AI. The M100521 mutation (Enam(Rgsc521)) was a T to A substitution at the splicing donor site in intron 4. This mutation resulted in a frameshift that gave rise to a premature stop codon. The transcript of the Enam(Rgsc521) mutant allele was degraded, indicating that Enam(Rgsc521) is a loss-of-function mutation. Enam(Rgsc521) heterozygotes showed a hypomaturation-type AI phenotype in the incisors, possibly due to haploinsufficiency of Enam. Enam(Rgsc521) homozygotes showed complete loss of enamel on the incisors and the molars. Thus, we report here that the Enam gene is essential for amelogenesis, and that mice with different point mutations at Enam may provide good animal models to study the different clinical subtypes of AI.
Human Molecular Genetics 04/2005; 14(5):575-83. · 7.64 Impact Factor
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Maki Inoue,
Yoshiyuki Sakuraba,
Hiromi Motegi,
Naoto Kubota,
Hideaki Toki,
Junko Matsui,
Yukiyasu Toyoda,
Ichitomo Miwa,
Yasuo Terauchi,
Takashi Kadowaki, [......],
Ayako Inoue, Hideki Kaneda,
Junko Ishijima,
Hiroshi Masuya,
Tomohiro Suzuki,
Shigeharu Wakana,
Yoichi Gondo,
Osamu Minowa,
Toshihiko Shiroishi,
Tetsuo Noda
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ABSTRACT: Mutant mouse models are indispensable tools for clarifying the functions of genes and for elucidating the underlying pathogenic mechanisms of human diseases. Currently, several large-scale mutagenesis projects that employ the chemical mutagen N-ethyl-N-nitrosourea (ENU) are underway worldwide. One specific aim of our ENU mutagenesis project is to generate diabetic mouse models. We screened 9375 animals for dominant traits using a clinical biochemical test and thereby identified 11 mutations in the glucokinase (Gk) gene that were associated with hyperglycemia. GK is a key regulator of insulin secretion in the pancreatic beta-cell. Approximately 190 heterozygous mutations in the human GK gene have been reported to cause maturity onset diabetes of the young, type 2 (MODY2). In addition, five mutations have been reported to cause permanent neonatal diabetes mellitus (PNDM) when present on both alleles. The mutations in our 11 hyperglycemic mutants are located at different positions in Gk. Four have also been found in human MODY2 patients, and another mutant bears its mutation at the same location that is mutated in a PNDM patient. Thus, ENU mutagenesis is effective for developing mouse models for various human genetic diseases, including diabetes mellitus. Some of our Gk mutant lines displayed impaired glucose-responsive insulin secretion and the mutations had different effects on Gk mRNA levels and/or the stability of the GK protein. This collection of Gk mutants will be valuable for understanding GK gene function, for dissecting the function of the enzyme and as models of human MODY2 and PNDM.
Human Molecular Genetics 07/2004; 13(11):1147-57. · 7.64 Impact Factor
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Hiroshi Masuya,
Yuji Nakai,
Hiromi Motegi,
Norio Niinaya,
Yuichiro Kida,
Yoshiharu Kaneko,
Haruhiko Aritake,
Nobuaki Suzuki,
Jun Ishii,
Koji Koorikawa, [......],
Hideaki Toki,
Yumiko Wada, Hideki Kaneda,
Junko Ishijima,
K Ryo Takahashi,
Osamu Minowa,
Tetsuo Noda,
Shigeharu Wakana,
Yoichi Gondo,
Toshihiko Shiroishi
[show abstract]
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ABSTRACT: A mouse ENU-mutagenesis program at RIKEN GSC has been initiated to conduct a large-scale, genome-wide, early- and late-onset phenotypic screen of mutant mice. We screened about a hundred mice every week with a comprehensive set of phenotype assays including behavioral tests based on a modified SHIRPA protocol, blood tests (both clinical biochemical testing and hemogram), and measurement of locomotor activity in their home cages. To manage the entire program, we developed a client/server architecture database system and named it MUSDB (Mutagenesis Universal Support DataBase). It manages mouse husbandry, mating protocols, procedures for ENU injection and phenotypic screens, phenotype inheritance tests, preservation of sperm and organs, and other materials generated during the program. We have implemented MUSDB in quite a large-scale system that includes 150 client computers. It has, helped reduce typographical errors and provided simple and efficient operation via its front-end user interface. It significantly contributed to the communication within and between workgroups in the program and in the accumulation of various phenotypic and inheritance data.
Mammalian Genome 06/2004; 15(5):404-11. · 2.89 Impact Factor
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ABSTRACT: P19 embryonic carcinoma (EC) cells are one of the simplest systems for analyzing the neuronal differentiation. To identify the membrane-associated molecules on the neuronal cells involved in the early neuronal differentiation in mice, we generated two monoclonal antibodies, SKY-1 and SKY-2, by immunizing rats with a membrane fraction of the neuronally committed P19 EC cells as an antigen. SKY-1 and SKY-2 recognized the carbohydrate moiety of a 90 kDa protein (RANDAM-1) and the polypeptide core of a 40 kDa protein (RANDAM-2), respectively. In the P19 EC cells, the expression of RANDAM-1 was colocalized to a part of Nestin-positive cells, whereas that of RANDAM-2 was observed in most Nestin-positive cells as well as beta-III-tubulin positive neurons. In the embryonic and adult brain of mice, RANDAM-1 was expressed at embryonic day 8.5 (E8.5), and the localization of antigen was restricted on the neuroepithelium and choroid plexus. The RANDAM-2 expression commenced at E6.0, and the antigen was distributed not only on the neuroepithelium of embryonic brain but on the neurons of adult brain. Collectively, it was concluded that RANDAM-1 is a stage specific antigen to express on the neural stem cells, and RANDAM-2 is constitutively expressed on both the neural stem cells and differentiated neuronal cells in mouse central nervous system (CNS).
Journal of Neuroscience Research 04/2002; 67(5):595-606. · 2.74 Impact Factor
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Steven J. Clapcote,
Tatiana V. Lipina,
J. Kirsty Millar,
Shaun Mackie,
Sheila Christie,
Fumiaki Ogawa,
Jason P. Lerch,
Keith Trimble,
Masashi Uchiyama,
Yoshiyuki Sakuraba, Hideki Kaneda,
Toshihiko Shiroishi
