Naomi Nakagata

Kumamoto University, Kumamoto, Kumamoto, Japan

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Publications (162)546.11 Total impact

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    ABSTRACT: Human chromosome 21 is known to be associated with the high risk of hematological malignancy but with resistance to breast cancer in the study of Down syndrome. In human cancers, we previously observed the significant alterations of the protein expression encoded by the ganp/MCM3AP gene on human chromosome 21q22.3. Here, we investigated GANP protein alterations in human breast cancer samples (416 cases) at various stages by immunohistochemical analysis. This cohort study clearly demonstrated that the GANP expression is significantly decreased in human breast cancer cases with poor prognosis as an independent risk factor [relapse-free survival; hazard ratio=2.37, 95% confidence interval=1.27 to 4.42, p=0.007 (univariate analysis) and hazard ratio=2.70, 95% confidence interval=1.42 to 5.13, p=0.002 (multivariate analysis)]. To investigate whether the altered GANP expression is associated with mammary tumorigenesis, we created mutant mice that were conditionally deficient in the ganp/MCM3AP gene using wap-cre recombinase transgenic mice. Mammary gland tumors occurred at a very high incidence in female mammary gland-specific GANP-deficient mice after severe impairment of mammary gland development during pregnancy. Moreover, tumor development also occurred in female post parous GANP-heterodeficient mice. GANP has a significant role in the suppression of DNA damage caused by estrogen in human breast cancer cell lines. These results indicated that the GANP protein is associated with breast cancer resistance. This article is protected by copyright. All rights reserved.
    No preview · Article · Jan 2016 · Cancer Science
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    Preview · Article · Nov 2015 · Asian Journal of Pharmaceutical Sciences
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    ABSTRACT: The division of the embryonic cloaca is the most essential event for the formation of digestive and urinary tracts. The defective development of the cloaca results in anorectal malformations (ARMs; 2-5 per 10,000 live births). However, the developmental and pathogenic mechanisms of ARMs are unclear. In the current study, we visualized the epithelia in the developing cloaca and nephric ducts (NDs). Systemic stereoscopic analyses revealed that the ND-cloaca connection sites shifted from the lateral-middle to dorsal-anterior part of the cloaca during cloacal division from E10.5 to E11.5 in mouse embryos. Genetic cell labeling analyses revealed that the cells in the ventral cloacal epithelium in the early stages rarely contributed to the dorsal part. Moreover, we revealed the possible morphogenetic movement of endodermal cells within the anterior part of the urogenital sinus and hindgut. These results provide the basis for understanding both cloacal development and the ARM pathogenesis.
    Full-text · Article · Sep 2015 · Scientific Reports
  • Toru Takeo · Naomi Nakagata
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    ABSTRACT: Actualmente existe una gran cantidad de embriones y esperma de ratones modificados genéticamente (RMG) que se encuentran criopreservados en bancos de recursos genéticos establecidos en diferentes laboratorios del mundo. Las técnicas de criopreservación de embriones y esperma han contribuido a la organización de la producción, preservación y envío eficiente de los ratones almacenados en estos bancos. En nuestro centro, el Center for Animal Resources and Development (CARD) de la Kumamoto University, uno de los bancos genéticos de ratón más grandes de Japón, hemos desarrollado varias técnicas de criopreservación de embriones, de esperma y de ovocitos, así como también de almacenaje en frío de embriones y esperma, y su posterior utilización en fertilización in vitro (FIV). Para ello, hemos tenido que superar varios problemas críticos de la tecnología reproductiva del ratón, tales como la baja fertilidad del esperma criopreservado de la cepa C57BL/6, la variabilidad en la viabilidad y en la capacidad de fertilización de los ovocitos criopreservados, y la duración limitada de los embriones o esperma mantenidos en frío. En la actualidad, las técnicas desarrolladas en nuestro laboratorio están ampliamente reconocidas por la comunidad científica. En este artículo, presentamos una serie de técnicas desarrolladas por nuestro equipo bajo el nombre de Método CARD. Copyright by Animales De Laboratorio
    No preview · Article · Jul 2015
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    Toru Takeo · Naomi Nakagata

    Full-text · Article · Jun 2015
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    ABSTRACT: Niemann-Pick type C disease (NPC), an autosomal recessive lysosomal storage disorder, is an inherited disease characterized by the accumulation of intracellular unesterified cholesterol. A solubilizing agent of lipophilic compounds, 2-hydroxypropyl-β-cyclodextrin (HPBCD), is an attractive drug candidate against NPC disease. However, establishment of the optimum dosage of HPBCD remains to be determined. In this study, we evaluated the effective dosage of HPBCD in NPC model (Npc1(-/-)) mice, and determined serum HPBCD concentrations. Subcutaneous injection of 1000-4000 mg/kg HPBCD improved the lifespan of Npc1(-/-) mice. In addition, liver injury and cholesterol sequestration were significantly prevented by 4000 mg/kg HPBCD in Npc1(-/-) mice. Serum HPBCD concentrations, when treated at the effective dosages (1000-4000 mg/kg), were approximately 1200-2500 µg/mL at 0.5 h after subcutaneous injection, and blood HPBCD concentrations were immediately eliminated in Npc1(-/-) mice. Furthermore, we examined serum HPBCD concentrations when treated at 40000 mg (approximately 2500 mg/kg) in a patient with NPC. We observed that the effective concentration in the in vivo study using Npc1(-/-) mice was similar to that in the patient. In the patient, systemic clearance and the volume of distribution of HPBCD were in accordance with the glomerular filtration rate and extracellular fluid volume, respectively. These results could provide useful information for developing the optimal dosage regimen for HPBCD therapy when administered intravenously to NPC patients.
    Full-text · Article · Jun 2015 · Biological & Pharmaceutical Bulletin
  • Toru Takeo · Naomi Nakagata
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    ABSTRACT: Superovulation is a reproductive technique generally used to produce genetically engineered mice. Superovulation in mice involves the administration of equine chorionic gonadotropin (eCG) to promote follicle growth and then that of human chorionic gonadotropin (hCG) to induce ovulation. Previously, some published studies reported that inhibin antiserum (IAS) increased the number of ovulated oocytes in ddY and wild-derived strains of mice. However, the effect of IAS on the C57BL/6 strain, which is the most widely used inbred strain for the production of genetically engineered mice, has not been investigated. In addition, the combined effect of IAS and eCG (IASe) on the number of ovulated oocytes in superovulation treatment has not been examined. In this study, we examined the effect of IAS and eCG on the number of ovulated oocytes in immature female mice of the C57BL/6 strain in superovulation treatment. Furthermore, we evaluated the quality of obtained oocytes produced by superovulation using IASe by in vitro fertilization (IVF) with sperm from C57BL/6 or genetically engineered mice. The developmental ability of fresh or cryopreserved embryos was examined by embryo transfer. The administration of IAS or eCG had a similar effect on the number of ovulated oocytes in C57BL/6 female mice. The number of ovulated oocytes increased to about 3-fold by the administration of IASe than by the administration of IAS or eCG alone. Oocytes derived from superovulation using IASe normally developed into 2-cell embryos by IVF using sperm from C57BL/6 mice. Fresh or cryopreserved 2-cell embryos produced by IVF between oocytes of C57BL/6 mice and sperm from genetically engineered mice normally developed into live pups following embryo transfer. In summary, a novel technique of superovulation using IASe is extremely useful for producing a great number of oocytes and offspring from genetically engineered mice. Copyright at PLoS One
    No preview · Article · May 2015 · PLoS ONE
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    ABSTRACT: Background Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing permits the rapid production of genetically engineered mice. To make the most of this innovative technology, a streamlined procedure is needed for the robust construction of CRISPR/Cas9 vectors, the efficient preparation of mouse oocytes, and refined genotyping strategies. Although we previously demonstrated the applicability of oocyte cryopreservation technologies and various genotyping methods in the production of transcription activator-like effector nuclease-mediated genome editing in mice, it has not yet been clarified whether these techniques can be applied to the CRISPR/Cas9-mediated generation of knockout mice. In this study, we investigated easy, efficient, and robust methods of creating knockout mice using several CRISPR/Cas9 systems. Results We constructed three types of CRISPR/Cas9 vectors expressing: 1) single guide RNA (gRNA) and Cas9 nuclease, 2) two gRNAs and Cas9 nickase, and 3) two gRNAs and FokI-dCas9, targeting the same genomic locus. These vectors were directly microinjected into the pronucleus of freeze-thawed fertilized oocytes, and surviving oocytes were transferred to pseudopregnant ICR mice. Cas9 nuclease resulted in the highest mutation rates with the lowest birth rates, while Cas9 nickase resulted in the highest birth rates with the lowest mutation rates. FokI-dCas9 presented well-balanced mutation and birth rates. Furthermore, we constructed a single all-in-one FokI-dCas9 vector targeting two different genomic loci, and validated its efficacy by blastocyst analysis, resulting in highly efficient simultaneous targeted mutagenesis. Conclusions Our report offers several choices of researcher-friendly consolidated procedures for making CRISPR/Cas9-mediated knockout mice, with sophisticated construction systems for various types of CRISPR vectors, convenient preparation of in vitro fertilized or mated freeze-thawed oocytes, and an efficient method of mutant screening. Electronic supplementary material The online version of this article (doi:10.1186/s12896-015-0144-x) contains supplementary material, which is available to authorized users.
    Full-text · Article · May 2015 · BMC Biotechnology
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    ABSTRACT: GM1-gangliosidosis is an inherited disorder characterized by the accumulation of GM1-gangliosides in many tissues and organs, particularly in the brain. Currently, there is no treatment available for patients with ganglioside storage diseases. Therefore, we investigated the effects of cyclodextrins (CyDs) on the GM1-ganglioside level in EA1 cells, fibroblasts from patients with GM1-gangliosidosis. The concentrations of cholesterol and phospholipids in supernatants were determined by Cholesterol E-test Wako and Phospholipid C-test Wako, respectively. The effects of CyDs on GM1-ganglioside levels in EA1 cells using fluorescence-labelled cholera toxin B-subunit, which can bind to GM1-gangliosides specifically, were investigated by flow cytometry and confocal laser scanning microscopy. The treatment with methylated CyDs, hydroxypropylated CyDs and branched CyDs decreased GM1-ganglioside levels in EA1 cells at 1 mm for 24 h. Unexpectedly, there was no significant change in the efflux of cholesterol or phospholipids from the cells after treatment with CyDs under the same experimental conditions, indicating that the efflux of membrane components is not associated with down-regulation of GM1-ganglioside levels in EA1 cells upon CyDs treatment. CyDs may have the potential as drugs for GM1-gangliosidosis, although the mechanism should be thereafter clarified. © 2015 Royal Pharmaceutical Society.
    No preview · Article · Apr 2015 · Journal of Pharmacy and Pharmacology
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    ABSTRACT: The developmental processes of the genital tubercle (GT), the anlage of the external genitalia, possess several developmental aspects, including GT outgrowth, urethral tube formation, and epithelial differentiation of the urethra. The GT comprises the mesenchyme derived from the lateral mesoderm, ectodermal epithelium, and endodermal epithelium (embryonic urethral epithelium). The three tissue layers develop the GT coordinately. Around the initial stage of GT outgrowth (E11.5), FGF signaling was detected in the mesenchyme of the GT. FGF signaling was detected in the three tissue layers of the GT around the early stage of urethral formation (E13.5). Subsequently, FGF signaling was predominantly detected in the urethral epithelium (E14.5). Tissue-specific roles of FGF signaling in GT development were revealed by conditional Fgfr gene knockout approaches. Mesenchymal FGF signaling in the early-stage GT is required for its outgrowth. Ectodermal FGF signaling in the GT is required for the differentiation of the ectoderm and urethral epithelium at their junction to form the proper urethral tube. Endodermal FGF signaling in the GT is required for the stratification and cell adhesive characteristics of the urethral epithelium. The current study suggests that spatiotemporally regulated FGF signaling plays tissue-specific roles in multiple processes of external genitalia development. This article is protected by copyright. All rights reserved. © 2015 Wiley Periodicals, Inc.
    No preview · Article · Mar 2015 · Developmental Dynamics
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    ABSTRACT: Archives of cryopreserved sperm harvested from genetically engineered mice, in mouse resource centers, are a readily accessible genetic resource for the scientific community. We previously reported that exposure of oocytes to reduced glutathione (GSH) greatly improves the fertilization rate of frozen-thawed mouse sperm. Application of GSH to in vitro fertilization techniques is widely accepted as a standard protocol to produce sufficient numbers of mice from cryopreserved sperm. However, the detailed mechanism of the enhancement of fertilization mediated by GSH in vitro is not fully understood. Here we focused on the chemical by determining the effects of its amino acids constituents and cysteine analogs on the fertilization of oocytes by frozen-thawed sperm. Furthermore, we determined the stability of these compounds in aqueous solution. We show here that L-cysteine (L-Cys), D-cysteine (D-Cys), or N-acetyl-L-cysteine (NAC) increased the rate of fertilization when added to the medium but did not adversely affect embryo development in vitro or in vivo. The levels of thiol groups of proteins in the zona pellucida (ZP) and the expansion of the ZP were increased by L-Cys, D-Cys, and NAC. These effects were abrogated by the methylation of the thiol group of L-Cys. NAC was the most stable of these compounds in the fertilization medium at 4°C. These results suggest that the thiol groups of cysteine analogs markedly enhance the fertilization rate of mouse oocytes. Copyright 2015 by The Society for the Study of Reproduction.
    No preview · Article · Feb 2015 · Biology of Reproduction
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    ABSTRACT: The Wolffian duct (WD) is a primordium of the male reproductive tract and kidney collecting duct system. Fibroblast growth factor receptors (FGFRs), members of the receptor tyrosine kinase (RTK) family, are essential for kidney development. Although the functions of FGFR signaling in kidney morphogenesis have been analyzed, their function in WD development has not been comprehensively investigated. Here, we demonstrate that Fgfr2 is the major Fgfr gene expressed throughout the WD epithelia and that it is essential for the maintenance of the WD, specifically in the caudal part of the WD. Hoxb7-Cre mediated inactivation of Fgfr2 in the mouse WD epithelia resulted in the regression of the caudal part of the WD and abnormal male reproductive tract development. Cell proliferation and expression of the downstream target genes of RTK signaling (Etv4 and Etv5) were decreased in the caudal part of the WD epithelia in the mutant embryos. Cranial (rostral) WD formation and ureteric budding were not affected. Ret, Etv4, and Etv5 expression were sustained in the ureteric bud of the mutant embryos. Taken together, these data suggest region-specific requirements for FGFR2 signaling in the developing caudal WD epithelia. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · Feb 2015 · Developmental Biology
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    ABSTRACT: Hydroxypropyl-β-cyclodextrin (HPBCD) is an attractive drug candidate against Niemann–Pick Type C (NPC) disease. However, the safety of HPBCD treatment for NPC patients remains to be elucidated. In this study, we examined the acute toxicity of HPBCD in Npc1-deficient mice. When treated with HPBCD (20,000 mg/kg, subcutaneously), over half of the wild-type (Npc1+/+) or Npc1+/− mice died by 72 h after the injection. In contrast, all of the Npc1−/− mice survived.Marked pathophysiological changes, such as an elevation in serumtransaminase and creatinine levels, hepatocellular necrosis, renal tubular damage, interstitial thickening, and hemorrhages in lungs, were induced by the HPBCD treatment in Npc1+/+ or Npc1+/− mice. However, these pathophysiological changes were significantly alleviated in Npc1−/− mice. In addition, in vitro analysis showed that the Npc1 gene deficiency and treatment with U18666A, an Npc1 inhibitor, remarkably attenuated the cytotoxicity of HPBCD in Chinese hamster ovary cells. These results suggest that the NPC1 genotype exacerbates the cytotoxicity of HPBCD and Npc1−/− mice have substantial resistance to the lethality and the organ injury induced by HPBCD injection compared with Npc1+/+ or Npc1+/− mice.We suggest that the Npc1 genotype should be considered in the safety evaluation of HPBCD using experimental animals and cells.
    Full-text · Article · Dec 2014 · Molecular Genetics and Metabolism Reports
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    ABSTRACT: The ability to interrogate the entire coding sequence of the mouse combined with the tools to manipulate the genome has firmly established the mouse as the model organism of choice for studying the causes of human disease. Consequently, a huge number of novel mouse models are generated each year to support active research programs. However, it is neither ethically justifiable, nor economically viable to maintain mouse colonies on the shelf that are not part of active research programs. This means that novel mouse lines have to be preserved in some way. If this is not done and the line is simply killed off, the genetics will be lost to future generations of scientists. This article describes the current practices used in cryopreservation laboratories to archive and recover mouse embryos frozen using controlled-rate freezing and vitrification techniques. Copyright © 2014 John Wiley & Sons, Inc.
    No preview · Article · Dec 2014
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    ABSTRACT: SNiemann-Pick disease type C (NPC) is a lysosomal storage disease characterized by abnormal accumulation of free cholesterol and glycolipids. Here, we established induced pluripotent stem cell (iPSC) lines from NPC patients. Hepatocyte-like cells (HLCs) and neural progenitors derived from the iPSC lines accumulated cholesterol and displayed impaired autophagy and ATP production. A molecular signature related to lipid metabolism was also impaired in the NPC-iPSC-derived HLCs. These findings indicate that iPSC-derived cells can phenocopy human NPC. We also newly found that 2-hydroxypropyl-γ-cyclodextrin (HPGCD) could reduce the cholesterol accumulation and restore the functional and molecular abnormalities in the NPC patient-derived cells, and do so more effectively than 2-hydroxypropyl-β-cyclodextrin treatment. In addition, NPC model mice showed an improved liver status and prolonged survival with HPGCDs. Thus, iPSC lines derived from patient cells are powerful tools to study cellular models of NPC, and HPGCD is a potential new drug candidate for future treatment of this disease. This article is protected by copyright. All rights reserved.
    No preview · Article · Dec 2014 · Stem Cells
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    ABSTRACT: Hyaluronidase is generally used to remove cumulus cells from mouse oocytes before oocyte cryopreservation, intracytoplasmic sperm injection or DNA injection. In general, use of cumulus-free mouse oocytes decreases in vitro fertilizing ability compared with cumulus-surrounded oocytes. The effect of hyaluronidase exposure on the quality of mouse oocytes is not fully understood. Here, we investigated the effect of hyaluronidase exposure time on the fertilization rate of fresh and vitrified mouse oocytes and their subsequent developmental ability in vitro. We found that the fertilization rate decreased with hyaluronidase treatments. This reduction in the fertilization rate following treatment with hyaluronidase was fully reversed by removal of the zona pellucida. In addition, oocytes treated with hyaluronidase for 5 min or longer had a reduced capacity to develop to the morula and blastocyst stage. The survival, fertilization, and developmental rates of vitrified-warmed oocytes were also reduced by longer exposure to hyaluronidase. In conclusion, these results suggest that prolonged exposure to hyaluronidase decreases the quality of mouse oocytes and shorter hyaluronidase treatment times may help achieve a stable and high fertilization rate in fresh and cryopreserved oocytes.
    Full-text · Article · Dec 2014 · Journal of Reproduction and Development
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    ABSTRACT: Masculinization of external genitalia is an essential process in the formation of the male reproductive system. Prominent characteristics of this masculinization are the organ size and the sexual differentiation of the urethra. Although androgen is a pivotal inducer of the masculinization, the regulatory mechanism under the control of androgen is still unknown. Here, we address this longstanding question about how androgen induces masculinization of the embryonic external genitalia through the identification of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B (Mafb) gene. Mafb is expressed prominently in the mesenchyme of male genital tubercle (GT), the anlage of external genitalia. MAFB expression is rarely detected in the mesenchyme of female GTs. However, exposure to exogenous androgen induces its mesenchymal expression in female GTs. Furthermore, MAFB expression is prominently down-regulated in male GTs of androgen receptor (Ar) KO mice, indicating that AR signaling is necessary for its expression. It is revealed that Mafb KO male GTs exhibit defective embryonic urethral formation, giving insight into the common human congenital anomaly hypospadias. However, the size of Mafb KO male GTs is similar with that of wild-type males. Moreover, androgen treatment fails to induce urethral masculinization of the GTs in Mafb KO mice. The current results provide evidence that Mafb is an androgen-inducible, sexually dimorphic regulator of embryonic urethral masculinization.
    Full-text · Article · Oct 2014 · Proceedings of the National Academy of Sciences
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    ABSTRACT: The purpose of this study was to evaluate the therapeutic potential of human induced pluripotent stem (iPS) cell-derived macrophage-like cells for Alzheimer’s disease (AD). In previous studies, we established the technology to generate macrophage-like myeloid lineage cells with proliferating capacity from human iPS cells, and we designated the cells iPS-ML. iPS-ML reduced the level of Aβ added into the culture medium, and the culture supernatant of iPS-ML alleviated the neurotoxicity of Aβ. We generated iPS-ML expressing the Fc-receptor-fused form of a single chain antibody specific to Aβ. In addition, we made iPS-ML expressing Neprilysin-2 (NEP2), which is a protease with Aβ-degrading activity. In vitro, expression of NEP2 but not anti-Aβ scFv enhanced the effect to reduce the level of soluble Aβ oligomer in the culture medium and to alleviate the neurotoxicity of Aβ. To analyze the effect of iPS-ML expressing NEP2 (iPS-ML/NEP2) in vivo, we intracerebrally administered the iPS-ML/NEP2 to 5XFAD mice, which is a mouse model of AD. We observed significant reduction in the level of Aβ in the brain interstitial fluid following administration of iPS-ML/NEP2. These results suggested that iPS-ML/NEP2 may be a potential therapeutic agent in the treatment of AD.
    Full-text · Article · Oct 2014 · Stem Cell Research

  • No preview · Conference Paper · Oct 2014
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    ABSTRACT: Each year, thousands of new mouse models are generated around the world to further biomedical research. Unfortunately, the cost of maintaining mouse colonies makes it uneconomical to keep strains on the shelf that are not part of active research programs. Ideally, these retired strains should be archived. If this is not done and the line is simply killed off, the genetics are lost to future generations of scientists. Traditionally, embryo freezing has been used to cryopreserve mice, but this is expensive, time consuming, requires large numbers of donor females, and usually involves invasive superovulation procedures. Sperm freezing circumvents all of these disadvantages and is rapidly becoming the technique of choice for many repositories. This has been made possible through the use of refined cryoprotective agents and the development of improved in vitro fertilization techniques. This article describes two popular sperm freezing techniques employed by mouse repositories to archive spermatozoa using cryoprotective agents supplemented with either L-glutamine or monothioglycerol. Copyright © 2014 John Wiley & Sons, Inc.
    No preview · Article · Sep 2014

Publication Stats

4k Citations
546.11 Total Impact Points

Institutions

  • 2000-2015
    • Kumamoto University
      • • Department of Reproductive Engineering
      • • Institute of Resource Development and Analysis (IRDA)
      • • Center for Animal Resources and Development
      Kumamoto, Kumamoto, Japan
  • 2002
    • University of Texas Southwestern Medical Center
      • Department of Molecular Genetics
      Dallas, Texas, United States
  • 1997
    • The University of Tokyo
      • Institute of Medical Science
      Tokyo, Tokyo-to, Japan
  • 1995-1996
    • Juntendo University
      • Department of Pathology and Oncology
      Edo, Tokyo, Japan