Updated on 2024/06/04

写真a

 
KANEKO TAKEHITO
 
Organization
Graduate School of Veterinary Science Department of Veterinary Science Professor
School of Veterinary Science Department of Veterinary Science
Title
Professor
Affiliation
Institute of Veterinary Science

Position

  • Graduate School of Veterinary Science Department of Veterinary Science 

    Professor  2024.04 - Now

  • School of Veterinary Science Department of Veterinary Science 

    Professor  2024.04 - Now

Degree

  • 博士(医学) ( Kumamoto University )

Research Areas

  • Environmental Science/Agriculture Science / Conservation of biological resources

  • Life Science / Laboratory animal science

Research Interests

  • 野生動物保全

  • 配偶子保存

  • 受精技術

  • 動物繁殖

Professional Memberships

  • 関西実験動物研究会

  • 野生動物保全繁殖研究会

  • 東北動物実験研究会

  • JAPANESE SOCIETY OF ZOO AND WILDLIFE MEDICINE

  • SOCIETY FOR REPRODUCTION AND DEVELOPMENT

  • 日本畜産学会

  • JAPANESE ASSOCIATION FOR EXPERIMENTAL ANIMAL TECHNOLOGISTS

  • JAPANESE ASSOCIATION FOR LABORATORY ANIMAL SCIENCE

  • 九州実験動物研究会

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Committee Memberships (off-campus)

  • 評議員   日本実験動物学会  

    2022.06 - 2024.05 

  • 評議員   日本実験動物学会  

    2014.06 - 2020.05 

Awards

  • 日本実験動物学会若手優秀賞

    金子武人 他

    2020   日本実験動物学会  

  • 日本繁殖生物学会技術賞

    金子 武人

    2017  

  • 日本獣医学会獣医繁殖学分科会長賞

    金子 武人

    2015  

  • 日本繁殖生物学会大会優秀発表賞

    金子 武人

    2014  

  • 日本実験動物学会奨励賞

    金子 武人

    2014  

  • 日本実験動物協同組合賞

    金子 武人

    2013  

  • 平成25年度科学技術分野の文部科学大臣表彰若手科学者賞

    金子 武人

    2013  

  • Young Investigator Award

    金子 武人

    2008   Asian Federation of Laboratory Animal Science Associations  

  • The Best Paper featuring cryopreservation

    金子 武人

    2004   Society for Reproduction & Fertility  

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Job Career (off-campus)

  • 東京医科歯科大学   非常勤講師

    2017.04 - Now

Papers

  • Apolipoprotein E-depletion accelerates arterial fat deposition in the spontaneously hypertensive rat.

    Hiroyuki Matsuo, Kohei Kawakami, Hiroki Ohara, Takehito Kaneko, Tomoji Mashimo, Takaya Yamada, Toru Nabika

    Experimental animals   2023.04

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    Publishing type:Research paper (scientific journal)   International / domestic magazine:Domestic journal  

    Hypertension and atherosclerosis are often found in one patient causing serious cardiovascular events. An animal model simultaneously expressing hypertension and atherosclerosis would be useful to study such a complex risk status. We therefore attempted to introduce a null mutation of the apolipoprotein E (ApoE) gene into the spontaneously hypertensive rat (SHR) using CRISPR/Cas9 to establish a genetic model for atherosclerosis with hypertension. We successfully established SHRApoE(-/-) having a 13-bps deletion in the 5'-end of ApoE gene. Deletion of ApoE protein was confirmed by Western blotting. Blood pressure of SHRApoE(-/-) was comparable to that of SHR. Feeding the rats with high fat high cholesterol diet (HFD) caused a significant increase in LDL cholesterol as well as in triglyceride in SHRApoE(-/-). After 8 weeks of HFD loading, superficial fat deposition was observed both in the aorta and the mesenteric arteries of SHRApoE(-/-) instead of mature atheromatous lesions found in humans. In addition, a null mutation of peroxiredoxin 2 (Prdx2) was introduced into SHRApoE(-/-) to examine the effect of increased oxidative stress on the development of atherosclerosis. SHR with the double depletion of ApoE and Prdx2 did not show mature atheroma either. Further, salt loading did not promote development of atheroma although it accelerated the development of fat deposition. These results indicated that when compared with ApoE-knockout mice, SHRApoE(-/-) was more resistant to atherosclerosis even though they have severe hypertension.

    DOI: 10.1538/expanim.23-0012

    PubMed

  • PHF24 is expressed in the inhibitory interneurons in rats.

    Yuki Numakura, Risa Uemura, Miyuu Tanaka, Takeshi Izawa, Jyoji Yamate, Takashi Kuramoto, Takehito Kaneko, Tomoji Mashimo, Takashi Yamamoto, Tadao Serikawa, Mitsuru Kuwamura

    Experimental animals   70 ( 1 )   137 - 143   2021.02

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    Publishing type:Research paper (scientific journal)   International / domestic magazine:Domestic journal  

    Noda epileptic rat (NER) is a mutant model for epilepsy that exhibits spontaneous generalized tonic-clonic seizure. Epileptogenesis of NER remains to be elucidated; but it is detected an insertion of an endogenous retrovirus sequence in intron 2 of the PHD finger protein 24 (Phf24) gene, encoding Gαi-interacting protein (GINIP). Phf24 is a strong candidate gene for epileptogenesis in NER. PHF24 modulates GABAB signaling through interacting with Gαi protein. To clarify the epileptogenesis of NER, we investigated a distribution of PHF24-expressing cells in the central nerve system (CNS). While broad expression of PHF24 was observed in the CNS, characteristic expression was noted in the periglomerular layer of the olfactory bulb and the lamina II of the spinal cord in the control rats. These cells showed co-expression with calbindin or calretinin, inhibitory interneuron markers. In the olfactory bulb, 15.6% and 41.2% of PHF24-positive neurons co-expressed calbindin and calretinin, respectively. Immunoelectron microscopy revealed that PHF24 was located in the presynaptic terminals, synaptic membranes and cytoplasmic matrix of neuronal soma. Our data suggested PHF24 is expressed in the inhibitory interneurons and may play important roles in modulation of the GABAB signaling.

    DOI: 10.1538/expanim.20-0105

    PubMed

  • Pathophysiological significance of Stim1 mutation in sympathetic response to stress and cardiovascular phenotypes in SHRSP/Izm: In vivo evaluation by creation of a novel gene knock-in rat using CRISPR/Cas9 Reviewed

    Batbayar Odongoo, Hiroki Ohara, Davis Ngarashi, Takehito Kaneko, Yayoi Kunihiro, Tomoji Mashimo, Toru Nabika

    Clinical and Experimental Hypertension   23   1 - 8   2020.07

  • Improvement of genome editing by electroporation using embryos artificially removed cumulus cells in the oviducts Reviewed

    Takehito Kaneko, Shungo Tanaka

    Biochemical and Biophysical Research Communications   527   1039 - 1042   2020.07

  • LOX-1 (Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1) Deletion Has Protective Effects on Stroke in the Genetic Background of Stroke-Prone Spontaneously Hypertensive Rat. Reviewed

    Yi-Qiang Liang, Akemi Kakino, Yasunari Matsuzaka, Tomoji Mashimo, Masato Isono, Tomohisa Akamatsu, Hana Shimizu, Michiko Tajima, Takehito Kaneko, Lei Li, Fumihiko Takeuchi, Tatsuya Sawamura, Norihiro Kato

    Stroke   51 ( 6 )   1835 - 1843   2020.06

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    Publishing type:Research paper (scientific journal)   International / domestic magazine:International journal  

    Background and Purpose- oxLDL (oxidized low-density lipoprotein) has been known for its potential to induce endothelial dysfunction and used as a major serological marker of oxidative stress. Recently, LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1), a lectin-like receptor for oxLDL, has attracted attention in studies of neuronal apoptosis and stroke. We aim to investigate the impact of LOX-1-deficiency on spontaneous hypertension-related brain damage in the present study. Methods- We generated a LOX-1 deficient strain on the genetic background of stroke-prone spontaneously hypertensive rat (SHRSP), an animal model of severe hypertension and spontaneous stroke. In this new disease model with stroke-proneness, we monitored the occurrence of brain abnormalities with and without salt loading by multiple procedures including T
    2
    weighted magnetic resonance imaging and also explored circulatory miRNAs as diagnostic biomarkers for cerebral ischemic injury by microarray analysis. Results- Both T
    2
    weighted magnetic resonance imaging abnormalities and physiological parameter changes could be detected at significantly delayed timing in LOX-1 knockout rats compared with wild-type SHRSP, in either case of normal rat chow and salt loading (P<0.005 in all instances; n=11-20 for SHRSP and n=13-23 for LOX-1 knockout rats). There were no significant differences in the form of magnetic resonance imaging findings between the strains. A number of miRNAs expressed in the normal rat plasma, including rno-miR-150-5p and rno-miR-320-3p, showed significant changes after spontaneous brain damage in SHRSP, whereas the corresponding changes were modest or almost unnoticeable in LOX-1 knockout rats. There appeared to be the lessening of correlation of postischemic miRNA alterations between the injured brain tissue and plasma in LOX-1 knockout rats. Conclusions- Our data show that deficiency of LOX-1 has a protective effect on spontaneous brain damage in a newly generated LOX-1-deficient strain of SHRSP. Further, our analysis of miRNAs as biomarkers for ischemic brain damage supports a potential involvement of LOX-1 in blood brain barrier disruption after cerebral ischemia. Visual Overview- An online visual overview is available for this article.

    DOI: 10.1161/STROKEAHA.120.029421

    PubMed

  • Development of feline embryos produced using freeze-dried sperm Reviewed

    Yasunori Tsujimoto, Takehito Kaneko, Takumi Yoshida, Kazuto Kimura, Toshio Inaba, Kikuya Sugiura, Shingo Hatoya

    Theriogenology   147   71 - 76   2020.04( ISSN:0093-691X

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    Publishing type:Research paper (scientific journal)  

    DOI: 10.1016/j.theriogenology.2020.02.021

  • Genome editing of rodents by electroporation of CRISPR/Cas9 into frozen-warmed pronuclear-stage embryos Reviewed

    Takehito Kaneko, Yuki Nakagawa

    Cryobiology   92   231 - 234   2020.02

  • Pathological characteristics of Ccdc85c knockout rats: A rat model of genetic hydrocephalus Reviewed

    Shizuka Konishi, Natsuki Tanaka, Tomoji Mashimo, Takashi Yamamoto, Tetsushi Sakuma, Takehito Kaneko, Miyuu Tanaka, Takeshi Izawa, Jyoji Yamate, Mitsuru Kuwamura

    Experimental Animals   69 ( 1 )   26 - 33   2020.01( ISSN:1341-1357

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    Publishing type:Research paper (scientific journal)  

    Spontaneous hhy mice show hydrocephalus and subcortical heterotopia, and a mutation in the Ccdc85c gene has been identified. To contribute to the comparison of the role of Ccdc85c in different species, we established a Ccdc85c KO rat and investigated its pathological phenotypes. Ccdc85c KO rats were produced by genomic engineering using transcription activator-like effector nuclease (TALEN). The KO rats had an approximately 350-bp deletion in Ccdc85c and lacked CCDC85C protein expression. The KO rats showed non-obstructive hydrocephalus, subcortical heterotopia, and intracranial hemorrhage. The KO rats had many pathological characteristics similar to those in hhy mice. These results indicate that CCDC85C plays an important role in cerebral development in rats, and the function of CCDC85C in the cerebrum are similar in rats and mice.

    DOI: 10.1538/expanim.19-0005

    PubMed

  • Rapid and efficient production of genome-edited animals by electroporation into oocytes injected with frozen or freeze-dried sperm Reviewed

    Yuki Nakagawa, Takehito Kaneko

    Cryobiology   90   71 - 74   2019.10

  • Increased seizure sensitivity, emotional defects and cognitive impairment in PHD finger protein 24 (Phf24)-null rats Reviewed

    Tadao Serikawa, Naofumi Kunisawa, Saki Shimizu, Masaki Kato, Higir Alves Iha, Masato Kinboshi, Hisao Nishikawa, Yu Shirakawa, Birger Voigt, Satoshi Nakanishi, Takashi Kuramoto, Takehito Kaneko, Takashi Yamamoto, Yomoji Mashimo, Masashi Sasa, Yukihiro Ohno

    Behavioural Brain Research   369   111922   2019.04

  • Vitrification of canine ovarian tissues with polyvinylpyrrolidone preserves the survival and developmental capacity of primordial follicles Reviewed

    9 ( 1 )   3970   2019.03

  • Humanized UGT2 and CYP3A transchromosomic rats for improved prediction of human drug metabolism. Reviewed

    Yasuhiro Kazuki, Kaoru Kobayashi, Masumi Hirabayashi, Satoshi Abe, Naoyo Kajitani, Kanako Kazuki, Shoko Takehara, Masato Takiguchi, Daisuke Satoh, Jiro Kuze, Tetsushi Sakuma, Takehito Kaneko, Tomoji Mashimo, Minori Osamura, Mari Hashimoto, Riko Wakatsuki, Rika Hirashima, Ryoichi Fujiwara, Tsuneo Deguchi, Atsushi Kurihara, Yasuko Tsukazaki, Naoto Senda, Takashi Yamamoto, Nico Scheer, Mitsuo Oshimura

    Proceedings of the National Academy of Sciences of the United States of America   116 ( 8 )   3072 - 3081   2019.02

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    Publishing type:Research paper (scientific journal)   International / domestic magazine:International journal  

    Although "genomically" humanized animals are invaluable tools for generating human disease models as well as for biomedical research, their development has been mainly restricted to mice via established transgenic-based and embryonic stem cell-based technologies. Since rats are widely used for studying human disease and for drug efficacy and toxicity testing, humanized rat models would be preferred over mice for several applications. However, the development of sophisticated humanized rat models has been hampered by the difficulty of complex genetic manipulations in rats. Additionally, several genes and gene clusters, which are megabase range in size, were difficult to introduce into rats with conventional technologies. As a proof of concept, we herein report the generation of genomically humanized rats expressing key human drug-metabolizing enzymes in the absence of their orthologous rat counterparts via the combination of chromosome transfer using mouse artificial chromosome (MAC) and genome editing technologies. About 1.5 Mb and 700 kb of the entire UDP glucuronosyltransferase family 2 and cytochrome P450 family 3 subfamily A genomic regions, respectively, were successfully introduced via the MACs into rats. The transchromosomic rats were combined with rats carrying deletions of the endogenous orthologous genes, achieved by genome editing. In the "transchromosomic humanized" rat strains, the gene expression, pharmacokinetics, and metabolism observed in humans were well reproduced. Thus, the combination of chromosome transfer and genome editing technologies can be used to generate fully humanized rats for improved prediction of the pharmacokinetics and drug-drug interactions in humans, and for basic research, drug discovery, and development.

    DOI: 10.1073/pnas.1808255116

    PubMed

  • Effects of the Prdx2 depletion on blood pressure and life span in spontaneously hypertensive rats. Reviewed

    2019.01

  • Tolerance to vitrification of rat embryos at various developmental stages. Reviewed

    Taketsuru H, Kaneko T

    Cryobiology   84   1 - 3   2018.10( ISSN:0011-2240

  • Reproductive technologies for the generation and maintenance of valuable animal strains Reviewed

    Takehito Kaneko

    Journal of Reproduction and Development   64 ( 3 )   209 - 215   2018( ISSN:1348-4400

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    Publishing type:Research paper (scientific journal)  

    Many types of mutant and genetically engineered strains have been produced in various animal species. Their numbers have dramatically increased in recent years, with new strains being rapidly produced using genome editing techniques. In the rat, it has been difficult to produce knockout and knock-in strains because the establishment of stem cells has been insufficient. However, a large number of knockout and knock-in strains can currently be produced using genome editing techniques, including zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system. Microinjection technique has also contributed widely to the production of various kinds of genome edited animal strains. A novel electroporation method, the “Technique for Animal Knockout system by Electroporation (TAKE)” method, is a simple and highly efficient tool that has accelerated the production of new strains. Gamete preservation is extremely useful for maintaining large numbers of these valuable strains as genetic resources in the long term. These reproductive technologies, including microinjection, TAKE method, and gamete preservation, strongly support biomedical research and the bio-resource banking of animal models. In this review, we introduce the latest reproductive technologies used for the production of genetically engineered animals, especially rats, using genome editing techniques and the efficient maintenance of valuable strains as genetic resources. These technologies can also be applied to other laboratory animals, including mice, and domestic and wild animal species.

    DOI: 10.1262/jrd.2018-035

    PubMed

  • In vitro maturation of immature rat oocytes under simple culture conditions and subsequent developmental ability Reviewed

    Hiroaki Taketsuru, Takehito Kaneko

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   62 ( 5 )   521 - 526   2016.10( ISSN:0916-8818

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    Publishing type:Research paper (scientific journal)  

    Rat oocytes can be produced artificially by superovulation. Because some strains show low sensitivity to superovulation treatment, in vitro maturation is an alternative method to produce numerous matured oocytes. Furthermore, establishment of an in vitro maturation system with simple culture conditions is cost effective and leads to easy handling of oocytes. This study examined developmental ability of rat germinal vesicle (GV) oocytes maturing in vitro under simple culture conditions. Significantly different numbers of ovulated oocytes reached the second metaphase of meiosis (MII) among Jcl:Wistar (17.0), F344/Stm (31.0), and BN/SsNS1c (2.2) rats in whom superovulation was induced by pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin. However, similar numbers of GV oocytes were obtained from ovaries of PMSG-injected Wistar (27.7), F344 (34.7), and BN (24.7) rats. These GV oocytes were cultured in vitro in HTF, alpha MEM, and a 1:1 HTF + alpha MEM or TYH + alpha MEM mixture. High proportions of Wistar and F344 oocytes that matured to MII in alpha MEM were parthenogenetically activated by strontium chloride treatment (78% and 74%, respectively). Additionally, 10% of matured oocytes of both strains developed into offspring after intracytoplasmic sperm injection and embryo transfer to foster mothers. Although BN oocytes cultured in aMEM could be parthenogenetically activated and developed into offspring, the success rate was lower than that for Wistar and F344 oocytes. This study demonstrated that numerous GV oocytes were produced in rat ovaries by PMSG injection. This simple in vitro maturation system of immature oocytes could be further developed to maintain valuable rat strains experiencing reproductive difficulties.

  • In vitro maturation of immature rat oocytes under simple culture conditions and subsequent developmental ability Reviewed

    Hiroaki Taketsuru, Takehito Kaneko

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   62 ( 5 )   521 - 526   2016.10( ISSN:0916-8818

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    Publishing type:Research paper (scientific journal)  

    Rat oocytes can be produced artificially by superovulation. Because some strains show low sensitivity to superovulation treatment, in vitro maturation is an alternative method to produce numerous matured oocytes. Furthermore, establishment of an in vitro maturation system with simple culture conditions is cost effective and leads to easy handling of oocytes. This study examined developmental ability of rat germinal vesicle (GV) oocytes maturing in vitro under simple culture conditions. Significantly different numbers of ovulated oocytes reached the second metaphase of meiosis (MII) among Jcl:Wistar (17.0), F344/Stm (31.0), and BN/SsNS1c (2.2) rats in whom superovulation was induced by pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin. However, similar numbers of GV oocytes were obtained from ovaries of PMSG-injected Wistar (27.7), F344 (34.7), and BN (24.7) rats. These GV oocytes were cultured in vitro in HTF, alpha MEM, and a 1:1 HTF + alpha MEM or TYH + alpha MEM mixture. High proportions of Wistar and F344 oocytes that matured to MII in alpha MEM were parthenogenetically activated by strontium chloride treatment (78% and 74%, respectively). Additionally, 10% of matured oocytes of both strains developed into offspring after intracytoplasmic sperm injection and embryo transfer to foster mothers. Although BN oocytes cultured in aMEM could be parthenogenetically activated and developed into offspring, the success rate was lower than that for Wistar and F344 oocytes. This study demonstrated that numerous GV oocytes were produced in rat ovaries by PMSG injection. This simple in vitro maturation system of immature oocytes could be further developed to maintain valuable rat strains experiencing reproductive difficulties.

    DOI: 10.1262/jrd.2016-057

    PubMed

  • Generation of a New Model Rat: Nrf2 Knockout Rats Are Sensitive to Aflatoxin B-1 Toxicity Reviewed

    Keiko Taguchi, Misaki Takaku, Patricia A. Egner, Masanobu Morita, Takehito Kaneko, Tomoji Mashimo, Thomas W. Kensler, Masayuki Yamamoto

    TOXICOLOGICAL SCIENCES   152 ( 1 )   40 - 52   2016.07( ISSN:1096-6080

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    Publishing type:Research paper (scientific journal)  

    The transcription factor Nrf2 (NF-E2-related-factor 2) regulates a battery of antioxidative stress-response genes and detoxication genes, and Nrf2 knockout lines of mice have been contributing critically to the clarification of roles that Nrf2 plays for cell protection. However, there are apparent limitations in use of the mouse models. For instance, rats exhibit more suitable features for toxicological or physiological examinations than mice. In this study, we generated 2 lines of Nrf2 knockout rats by using a genome editing technology; 1 line harbors a 7-bp deletion (Lambda 7) and the other line harbors a 1-bp insertion (+1) in the Nrf2 gene. In the livers of rats homozygously deleting the Nrf2 gene, an activator of Nrf2 signaling, CDDO-Im, could not induce expression of representative Nrf2 target genes. To examine altered toxicological response, we treated the Nrf2 knockout rats with aflatoxin B-1 (AFB(1)), a carcinogenic mycotoxin that elicits gene mutations through binding of its metabolites to DNA and for which the rat has been proposed as a reasonable surrogate for human toxicity. Indeed, in the Nrf2 knockout rat livers the enzymes of the AFB(1) detoxication pathway were significantly downregulated. Single dose administration of AFB(1) increased hepatotoxicity and binding of AFB(1)-N-7-guanine to hepatic DNA in Nrf2 knockout rats compared with wild-type. Nrf2 knockout rats repeatedly treated with AFB(1) were prone to lethality and CDDO-Im was no longer protective. These results demonstrate that Nrf2 knockout rats are quite sensitive to AFB(1) toxicities and this rat genotype emerges as a new model animal in toxicology.

    DOI: 10.1093/toxsci/kfw065

  • Depdc5 knockout rat: A novel model of mTORopathy Reviewed

    Elise Marsan, Saeko Ishida, Adrien Schramm, Sarah Weckhuysen, Giuseppe Muraca, Sarah Lecas, Ning Liang, Caroline Treins, Mario Pende, Delphine Roussel, Michel Le Van Quyen, Tomoji Mashimo, Takehito Kaneko, Takashi Yamamoto, Tetsushi Sakuma, Severine Mahon, Richard Miles, Eric Leguern, Stephane Charpier, Stephanie Baulac

    NEUROBIOLOGY OF DISEASE   89   180 - 189   2016.05( ISSN:0969-9961

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    Publishing type:Research paper (scientific journal)  

    DEP-domain containing 5 (DEPDC5), encoding a repressor of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, has recently emerged as a major gene mutated in familial focal epilepsies and focal cortical dysplasia. Here we established a global knockout rat using TALEN technology to investigate in vivo the impact of Depdc5-deficiency. Homozygous Depdc5(-/-) embryos died from embryonic day 14.5 due to a global growth delay. Constitutive mTORC1 hyperactivation was evidenced in the brains and in cultured fibroblasts of Depdc5(-/-) embryos, as reflected by enhanced phosphorylation of its downstream effectors S6K1 and rpS6. Consistently, prenatal treatment with mTORC1 inhibitor rapamycin rescued the phenotype of Depdc5(-/-) embryos. Heterozygous Depdc5(+/-) rats developed normally and exhibited no spontaneous electroclinical seizures, but had altered cortical neuron excitability and firing patterns. Depdc5(+/-) rats displayed cortical cytomegalic dysmorphic neurons and balloon-like cells strongly expressing phosphorylated rpS6, indicative of mTORC1 upregulation, and not observed after prenatal rapamycin treatment. These neuropathological abnormalities are reminiscent of the hallmark brain pathology of human focal cortical dysplasia. Altogether, Depdc5 knockout rats exhibit multiple features of rodent models of mTORopathies, and thus, stand as a relevant model to study their underlying pathogenic mechanisms. (C) 2016 The Authors. Published by Elsevier Inc.

    DOI: 10.1016/j.nbd.2016.02.010

  • ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes Reviewed

    Kazuto Yoshimi, Yayoi Kunihiro, Takehito Kaneko, Hitoshi Nagahora, Birger Voigt, Tomoji Mashimo

    NATURE COMMUNICATIONS   7   10431   2016.01( ISSN:2041-1723

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    Publishing type:Research paper (scientific journal)  

    The CRISPR-Cas system is a powerful tool for generating genetically modified animals; however, targeted knock-in (KI) via homologous recombination remains difficult in zygotes. Here we show efficient gene KI in rats by combining CRISPR-Cas with single-stranded oligodeoxynucleotides (ssODNs). First, a 1-kb ssODN co-injected with guide RNA (gRNA) and Cas9 messenger RNA produce GFP-KI at the rat Thy1 locus. Then, two gRNAs with two 80-bp ssODNs direct efficient integration of a 5.5-kb CAG-GFP vector into the Rosa26 locus via ssODN-mediated end joining. This protocol also achieves KI of a 200-kb BAC containing the human SIRPA locus, concomitantly knocking out the rat Sirpa gene. Finally, three gRNAs and two ssODNs replace 58-kb of the rat Cyp2d cluster with a 6.2-kb human CYP2D6 gene. These ssODN-mediated KI protocols can be applied to any target site with any donor vector without the need to construct homology arms, thus simplifying genome engineering in living organisms.

    DOI: 10.1038/ncomms10431

  • Sperm freeze-drying and micro-insemination for biobanking and maintenance of genetic diversity in mammals Reviewed

    Takehito Kaneko

    REPRODUCTION FERTILITY AND DEVELOPMENT   28 ( 8 )   1079 - 1087   2016( ISSN:1031-3613

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    Breeding by natural mating is ideal for maintaining animal populations. However, the lack of breeding space resulting from an increased number of strains and the decline in fertility caused by inbreeding inhibits the reproduction of subsequent generations. Reproductive technologies, such as gamete preservation and artificial fertilisation, have been developed to overcome these problems. These approaches efficiently produce offspring of laboratory, domestic and wild animals, and can also be used to treat human infertility. Gamete preservation using sperm contributes to improvements in reproductive systems and enables the use of smaller breeding spaces. Although cryopreservation with liquid nitrogen has been used to preserve spermatozoa, freeze-drying without liquid nitrogen, a novel method, facilitates long-term storage of spermatozoa. This method has recently been applied to maintain animal strains. Micro-insemination techniques, such as intracytoplasmic sperm injection (ICSI), are exceptional for improving assisted reproduction. ICSI can be used to fertilise oocytes, even with immotile and immature spermatozoa that are unsuitable for AI and IVF. Reproductive technologies provide a substantial advantage for biobanking and maintaining the genetic diversity of laboratory, domestic and wild animals. This review covers the latest method of sperm freeze-drying and micro-insemination, and future possibilities for maintaining animal strains and populations.

    DOI: 10.1071/RD15386

    PubMed

  • Simple gamete preservation and artificial reproduction of mammals using micro-insemination techniques Reviewed

    Takehito Kaneko

    Reproductive Medicine and Biology   14 ( 3 )   99 - 105   2015.12( ISSN:1447-0578

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    Publishing type:Research paper (scientific journal)  

    Assisted reproductive technology (ART) has been applied in various procedures as an effective breeding method in experimental, domestic, and wild animals, and for the treatment of human infertility. Micro-insemination techniques such as intracytoplasmic injection of spermatozoa and spermatids are now routinely used ART tools. With these techniques, even immotile and immature sperm cells can be employed as donors for producing the next generation. Gamete preservation, another ART tool, has contributed to reproductive regulation, worldwide transportation, and disease protection of animal strains, and the preserved gametes have been effectively used for the production of offspring. ART is now an indispensable tool in mammalian reproduction. This review covers the latest ART tools, with a particular emphasis on micro-insemination and gamete preservation, and discusses the future direction of mammalian artificial reproductive technology.

    DOI: 10.1007/s12522-014-0202-4

  • Simple Genome Editing of Rodent Intact Embryos by Electroporation Reviewed

    Takehito Kaneko, Tomoji Mashimo

    PLoS One   10 ( 11 )   e0142755   2015.11( ISSN:1932-6203

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    Publishing type:Research paper (scientific journal)  

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system is a powerful tool for genome editing in animals. Recently, new technology has been developed to genetically modify animals without using highly skilled techniques, such as pronuclear microinjection of endonucleases. Technique for animal knockout system by electroporation (TAKE) method is a simple and effective technology that produces knockout rats by introducing endonuclease mRNAs into intact embryos using electroporation. Using TAKE method and CRISPR/Cas system, the present study successfully produced knockout and knock-in mice and rats. The mice and rats derived from embryos electroporated with Cas9 mRNA, gRNA and single-stranded oligodeoxynucleotide (ssODN) comprised the edited targeted gene as a knockout (67% of mice and 88% of rats) or knock-in (both 33%). The TAKE method could be widely used as a powerful tool to produce genetically modified animals by genome editing.

    DOI: 10.1371/journal.pone.0142755

  • CHD1 acts via the Hmgpi pathway to regulate mouse early embryogenesis Reviewed

    Shinnosuke Suzuki, Yusuke Nozawa, Satoshi Tsukamoto, Takehito Kaneko, Ichiro Manabe, Hiroshi Imai, Naojiro Minami

    DEVELOPMENT   142 ( 13 )   2375 - +   2015.07( ISSN:0950-1991

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    Publishing type:Research paper (scientific journal)  

    The protein CHD1 is a member of the family of ATPase-dependent chromatin remodeling factors. CHD1, which recognizes trimethylated histone H3 lysine 4, has been implicated in transcriptional activation in organisms ranging from yeast to humans. It is required for pre-mRNA maturation, maintenance of mouse embryonic stem cell pluripotency and rapid growth of the mouse epiblast. However, the function(s) of CHD1 in mouse preimplantation embryos has not yet been examined. Here, we show that loss of CHD1 function led to embryonic lethality after implantation. In mouse embryos in which Chd1 was targeted by siRNA microinjection, the expression of the key regulators of cell fate specification Pou5f1 (also known as Oct4), Nanog and Cdx2 was dramatically decreased, starting at mid-preimplantation gene activation (MGA). Moreover, expression of Hmgpi and Klf5, which regulate Pou5f1, Nanog and Cdx2, was also significantly suppressed at zygotic gene activation (ZGA). Suppression of Hmgpi expression in Chd1-knockdown embryos continued until the blastocyst stage, whereas suppression of Klf5 expression was relieved by the morula stage. Next, we rescued HMGPI expression via Hmgpi mRNA microinjection in Chd1-knockdown embryos. Consequently, Pou5f1, Nanog and Cdx2 expression was restored at MGA and live offspring were recovered. These findings indicate that CHD1 plays important roles in mouse early embryogenesis via activation of Hmgpi at ZGA.

    DOI: 10.1242/dev.120493

  • Histone methyltransferase Smyd3 regulates early embryonic lineage commitment in mice Reviewed

    Shinnosuke Suzuki, Yusuke Nozawa, Satoshi Tsukamoto, Takehito Kaneko, Hiroshi Imai, Naojiro Minami

    REPRODUCTION   150 ( 1 )   21 - 30   2015.07( ISSN:1470-1626

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    SET and MYND domain-containing protein 3 (Smyd3) is a histone H3 lysine 4 (H3K4) di- and tri-methyltransferase that forms a transcriptional complex with RNA polymerase II and activates the transcription of oncogenes and cell cycle genes in human cancer cells. However, the study of Smyd3 in mammalian early embryonic development has not yet been addressed. In the present study, we investigated the expression pattern of Smyd3 in mouse preimplantation embryos and the effects of RNA interference (RNAi)-mediated Smyd3 repression on the development of mouse embryos. We showed that Smyd3 mRNA levels increased after the two-cell stage, peaked at the four-cell stage, and gradually decreased thereafter. Moreover, in two-cell to eight-cell embryos, SMYD3 staining was more intense in the nuclei than it was in the cytoplasm. In Smyd3-knockdown embryos, the percentage of inner cell mass (ICM)-derived colony formation and trophectoderm (TE)-derived cell attachment were significantly decreased, which resulted in a reduction in the number of viable offspring. Furthermore, the expression of Oct4 and Cdx2 during mid-preimplantation gene activation was significantly decreased in Smyd3-knockdown embryos. In addition, the transcription levels of ICM and epiblast markers, such as Oct4, Nanog, and Sox2, the transcription levels of primitive endoderm markers, such as Gata6, and the transcription levels of TE markers, such as Cdx2 and Eomes, were significantly decreased in Smyd3-knockdown blastocysts. These findings indicate that SMYD3 plays an important role in early embryonic lineage commitment and peri-implantation development through the activation of lineage-specific genes.

    DOI: 10.1530/REP-15-0019

  • A hyperactive piggyBac transposon system is an easy-to-implement method for introducing foreign genes into mouse preimplantation embryos Reviewed

    Shinnosuke Suzuki, Tomoyuki Tsukiyama, Takehito Kaneko, Hiroshi Imai, Naojiro Minami

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   61 ( 3 )   241 - 244   2015.06( ISSN:0916-8818

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    Transgenic mice are important tools for genetic analysis. A current prominent method for producing transgenic mice involves pronuclear microinjection into 1-cell embryos. However, the total transgenic efficiency obtained using this method is less than 10%. Here, we demonstrate that highly efficient transgenesis in mice can be achieved by cytoplasmic microinjection using a hyperactive piggyBac system. In embryos in which hyPBase mRNA and pPB-CAG-TagRFP DNA were co-injected into the cytoplasm, TagRFP fluorescence was observed after the 2-cell stage; when 30 ng/mu l pPB-CAG-TagRFP DNA and 30 ng/mu l hyPBase mRNA were co-injected, 94.4% of blastocysts were TagRFP positive. Furthermore, a high concentration of hyPBase mRNA resulted in creation of mosaic embryos in which the TagRFP signals partially disappeared. However, suitable concentrations of injected DNA and hyPBase mRNA produced embryos in which almost all blastomeres were TagRFP positive. Thus, the hyperactive piggyBac transposon system is an easy-to-implement and highly effective method that can contribute to production of transgenic mice.

    DOI: 10.1262/jrd.2014-157

  • Engineered nucleases lead to genome editing revolution in rats Reviewed

    Kazuto Yoshimi, Takehito Kaneko, Birger Voigt, Tomoji Mashimo

    Targeted Genome Editing Using Site-Specific Nucleases: ZFNs, TALENs, and the CRISPR/Cas9 System   183 - 195   2015.01

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    Sequence-specific endonucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas), allow simple generation of genetically modified animals. We utilized ZFNs and TALENs to generate several knockout rat models of human diseases. Furthermore, CRISPR/Cas9 used in conjunction with single-stranded oligodeoxyribonucleotides enabled us to generate several types of targeted knockin animal, such as single nucleotide polymorphism substitution, short DNA fragment integration, and large DNA fragment elimination. These powerful technologies have opened new doors for genome manipulation that enable the engineering of animal models of human disease and potential therapeutic applications.

    DOI: 10.1007/978-4-431-55227-7_12

  • Creating knockout and knockin rodents using engineered endonucleases via direct embryo injection Reviewed

    Takehito Kaneko, Tomoji Mashimo

    Methods in Molecular Biology   1239   307 - 315   2015( ISSN:1064-3745

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    Publishing type:Research paper (scientific journal)  

    Genetically engineered rodents have been generated worldwide for biomedical research. Recently, gene targeting techniques have been developed by using engineered endonucleases such as zinc-fi nger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. These endonucleases are useful for simple and rapid production of gene knockout/knockin animals without using embryonic stem (ES) cells. This chapter introduces the latest protocols for producing genetically modifi ed rodents using ZFN, TALEN, and CRISPR/Cas9.

    DOI: 10.1007/978-1-4939-1862-1_18

    PubMed

  • Sperm Preservation by Freeze-Drying for the Conservation of Wild Animals Reviewed

    Takehito Kaneko, Hideyuki Ito, Hidefusa Sakamoto, Manabu Onuma, Miho Inoue-Murayama

    PLOS ONE   9 ( 11 )   e113381   2014.11( ISSN:1932-6203

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    Sperm preservation is a useful technique for the maintenance of biological resources in experimental and domestic animals, and in wild animals. A new preservation method has been developed that enables sperm to be stored for a long time in a refrigerator at 4 degrees C. Sperm are freeze-dried in a solution containing 10 mM Tris and 1 mM EDTA. Using this method, liquid nitrogen is not required for the storage and transportation of sperm. We demonstrate that chimpanzee, giraffe, jaguar, weasel and the long-haired rat sperm remain viable after freeze-drying. In all species, pronuclei were formed after the injection of freeze-dried sperm into the mouse oocytes. Although preliminary, these results may be useful for the future establishment of "freeze-drying zoo'' to conserve wild animals.

    DOI: 10.1371/journal.pone.0113381

  • Simple knockout by electroporation of engineered endonucleases into intact rat embryos Reviewed

    Takehito Kaneko, Tetsushi Sakuma, Takashi Yamamoto, Tomoji Mashimo

    SCIENTIFIC REPORTS   4   6382   2014.10( ISSN:2045-2322

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    Publishing type:Research paper (scientific journal)  

    Engineered endonucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, provide a powerful approach for genome editing in animals. However, the microinjection of endonucleases into embryos requires a high skill level, is time consuming, and may cause damage to embryos. Here, we demonstrate that the electroporation of endonuclease mRNAs into intact embryos can induce editing at targeted loci and efficiently produce knockout rats. It is noteworthy that the electroporation of ZFNs resulted in an embryonic survival rate (91%) and a genome-editing rate (73%) that were more than 2-fold higher than the corresponding rates from conventional microinjection. Electroporation technology provides a simple and effective method to produce knockout animals.

    DOI: 10.1038/srep06382

  • Allele-specific genome editing and correction of disease-associated phenotypes in rats using the CRISPR-Cas platform Reviewed

    K. Yoshimi, T. Kaneko, B. Voigt, T. Mashimo

    NATURE COMMUNICATIONS   5   4240   2014.06( ISSN:2041-1723

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    The bacterial CRISPR/Cas system has proven to be an efficient gene-targeting tool in various organisms. Here we employ CRISPR/Cas for accurate and efficient genome editing in rats. The synthetic chimeric guide RNAs (gRNAs) discriminate a single-nucleotide polymorphism (SNP) difference in rat embryonic fibroblasts, allowing allele-specific genome editing of the dominant phenotype in (F344 x DA)F1 hybrid embryos. Interestingly, the targeted allele, initially assessed by the allele-specific gRNA, is repaired by an interallelic gene conversion between homologous chromosomes. Using single-stranded oligodeoxynucleotides, we recover three recessive phenotypes: the albino phenotype by SNP exchange; the non-agouti phenotype by integration of a 19-bp DNA fragment; and the hooded phenotype by eliminating a 7,098-bp insertional DNA fragment, evolutionary-derived from an endogenous retrovirus. Successful in vivo application of the CRISPR/Cas system confirms its importance as a genetic engineering tool for creating animal models of human diseases and its potential use in gene therapy.

    DOI: 10.1038/ncomms5240

  • フリーズドライ精子保存法の希少動物への応用 Reviewed

    金子武人

    獣医畜産新報(JVM)   2014

  • ラットにおけるゲノム編集技術革命 ノックアウト・ノックインラットが爆発的に増える! Reviewed

    吉見一人, 金子武人, 真下知士

    実験医学   32 ( 11 )   1715 - 1720   2014

  • Repeating pattern of non-RVD variations in DNA-binding modules enhances TALEN activity Reviewed

    Tetsushi Sakuma, Hiroshi Ochiai, Takehito Kaneko, Tomoji Mashimo, Daisuke Tokumasu, Yuto Sakane, Ken-Ichi Suzuki, Tatsuo Miyamoto, Naoaki Sakamoto, Shinya Matsuura, Takashi Yamamoto

    Scientific Reports   3   2013.11( ISSN:2045-2322

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    Transcription activator-like effector (TALE) nuclease (TALEN) is a site-specific nuclease, which can be freely designed and easily constructed. Numerous methods of constructing TALENs harboring different TALE scaffolds and repeat variants have recently been reported. However, the functionalities of structurally different TALENs have not yet been compared. Here, we report on the functional differences among several types of TALENs targeting the same loci. Using HEK293T cell-based single-strand annealing and Cel-I nuclease assays, we found that TALENs with periodically-patterned repeat variants harboring non-repeat-variable di-residue (non-RVD) variations (Platinum TALENs) showed higher activities than TALENs without non-RVD variations. Furthermore, the efficiencies of gene disruption mediated by Platinum TALENs in frogs and rats were significantly higher than in previous reports. This study therefore demonstrated an efficient system for the construction of these highly active Platinum TALENs (Platinum Gate system), which could establish a new standard in TALEN engineering.

    DOI: 10.1038/srep03379

    PubMed

  • Efficient collection and cryopreservation of embryos in F344 strain inbred rats Reviewed

    Hiroaki Taketsuru, Takehito Kaneko

    CRYOBIOLOGY   67 ( 2 )   230 - 234   2013.10( ISSN:0011-2240

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    In rats, it is now possible to produce genetically engineered strains, not only as transgenic animals but also using gene knockout techniques. Reproductive technologies have been used as indispensable tools to produce and maintain these novel valuable strains. Although studies for collecting and cryopreserving embryos have been reported using outbred rats, efficient methods have not been established in inbred strains. The F344 inbred strain is important in rat breeding and has been used for the production of transgenic/knockout strains and for genome sequencing. Here we studied the optimal conditions for oocyte collection by induction of superovulation, and the development of embryos after cryopreservation in F344 rats. The response to pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) was examined by injection of 150 IU/kg PMSG + 75 IU/kg hCG or 300 IU/kg PMSG + 300 IU/kg hCG. Superovulation was achieved at high efficiency by an injection of 150 IU/kg PMSG + 75 IU/kg hCG. Furthermore, superovulation in this strain showed similar high response as Wistar rats. Of 2-cell embryos cryopreserved by vitrification in a solution containing 10% propylene glycol, 30% ethylene glycol, 20% Percoll and 0.3 M sucrose, more than 90% survived after warming and 32% developed to offspring. However, the freezability of pronuclear stage embryos was extremely low. This study demonstrated that sufficient unfertilized oocytes and embryos can be collected from F344 rats by the induction of superovulation with 150 IU/kg PMSG + 75 IU/kg hCG. Furthermore, cryopreservation of 2-cell embryos using this vitrification protocol can now be applied to maintaining valuable rat strains derived from the F344 inbred strain as genetic resources. (C) 2013 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.cryobiol.2013.07.004

    PubMed

    J-GLOBAL

  • ING3 Is Essential for Asymmetric Cell Division during Mouse Oocyte Maturation Reviewed

    Shinnosuke Suzuki, Yusuke Nozawa, Satoshi Tsukamoto, Takehito Kaneko, Hiroshi Imai, Naojiro Minami

    PLOS ONE   8 ( 9 )   2013.09( ISSN:1932-6203

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    ING3 (inhibitor of growth family, member 3) is a subunit of the nucleosome acetyltransferase of histone 4 (NuA4) complex, which activates gene expression. ING3, which contains a plant homeodomain (PHD) motif that can bind to trimethylated lysine 4 on histone H3 (H3K4me3), is ubiquitously expressed in mammalian tissues and governs transcriptional regulation, cell cycle control, and apoptosis via p53-mediated transcription or the Fas/caspase-8 pathway. Thus, ING3 plays a number of important roles in various somatic cells. However, the role(s) of ING3 in germ cells remains unknown. Here, we show that loss of ING3 function led to the failure of asymmetric cell division and cortical reorganization in the mouse oocyte. Immunostaining showed that in fully grown germinal vesicle (GV) oocytes, ING3 localized predominantly in the GV. After germinal vesicle breakdown (GVBD), ING3 homogeneously localized in the cytoplasm. In oocytes where Ing3 was targeted by siRNA microinjection, we observed symmetric cell division during mouse oocyte maturation. In those oocytes, oocyte polarization was not established due to the failure to form an actin cap or a cortical granule-free domain (CGFD), the lack of which inhibited spindle migration. These features were among the main causes of abnormal symmetric cell division. Interestingly, an analysis of the mRNA expression levels of genes related to asymmetric cell division revealed that only mTOR was downregulated, and, furthermore, that genes downstream of mTOR (e.g., Cdc42, Rac1, and RhoA) were also downregulated in siIng3-injected oocytes. Therefore, ING3 may regulate asymmetric cell division through the mTOR pathway during mouse oocyte maturation.

    DOI: 10.1371/journal.pone.0074749

    PubMed

    CiNii Article

  • Efficient gene targeting by TAL effector nucleases coinjected with exonucleases in zygotes Reviewed

    Tomoji Mashimo, Takehito Kaneko, Tetsushi Sakuma, Junya Kobayashi, Yayoi Kunihiro, Birger Voigt, Takashi Yamamoto, Tadao Serikawa

    SCIENTIFIC REPORTS   3   2013.02( ISSN:2045-2322

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    TAL Effector Nucleases (TALENs) are versatile tools for targeted gene editing in various species. However, their efficiency is still insufficient, especially in mammalian embryos. Here, we showed that combined expression of Exonuclease 1 (Exo1) with engineered site-specific TALENs provided highly efficient disruption of the endogenous gene in rat fibroblast cells. A similar increased efficiency of up to similar to 30% with Exo1 was also observed in fertilized rat eggs, and in the production of knockout rats for the albino (Tyr) gene. These findings demonstrate TALENs with Exo1 is an easy and efficient method of generating gene knockouts using zygotes, which increases the range of gene targeting technologies available to various species.

    DOI: 10.1038/srep01253

    PubMed

    CiNii Article

  • 哺乳動物における生殖技術の過去・現在・未来-マイクロマニピュレーターの出現による生殖技術の発展- Reviewed

    金子武人

    実験動物技術   2013

  • ZFN/TALENを用いた効率的な遺伝子改変ラットの作製法

    金子武人

    LABIO21   2013

  • ラットにおける遺伝子改変技術の新展開

    真下知士, 金子武人

    細胞工学   2013

  • Long-term preservation of freeze-dried mouse spermatozoa Reviewed

    Takehito Kaneko, Tadao Serikawa

    CRYOBIOLOGY   64 ( 3 )   211 - 214   2012.06( ISSN:0011-2240

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    Many genetically engineered mice strains have been generated worldwide and sperm preservation is a valuable method for storing these strains as genetic resources. Freeze-drying is a useful sperm preservation method because it requires neither liquid nitrogen nor dry ice for preservation and transportation. We report here successful long-term preservation at 4 degrees C of mouse spermatozoa freeze-dried using a simple buffer solution (10 mM Iris, 1 mM EDTA, pH 8.0). Offspring with fertility were obtained from oocytes fertilized with freeze-dried spermatozoa from C57BL/6 and B6D2F1 mouse strains stored at 4 degrees C for 3 years. This freeze-drying method is a safe and economical tool for the biobanking of valuable mouse strains. (C) 2012 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.cryobiol.2012.01.010

    PubMed

  • Successful Long-Term Preservation of Rat Sperm by Freeze-Drying Reviewed

    Takehito Kaneko, Tadao Serikawa

    PLOS ONE   7 ( 4 )   2012.04( ISSN:1932-6203

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    Background: Freeze-drying sperm has been developed as a new preservation method where liquid nitrogen is no longer necessary. An advantage of freeze-drying sperm is that it can be stored at 4 degrees C and transported at room temperature. Although the successful freeze-drying of sperm has been reported in a number of animals, the possibility of long-term preservation using this method has not yet been studied.
    Methodology/Principal Findings: Offspring were obtained from oocytes fertilized with rat epididymal sperm freeze-dried using a solution containing 10 mM Tris and 1 mM EDTA adjusted to pH 8.0. Tolerance of testicular sperm to freeze-drying was increased by pre-treatment with diamide. Offspring with normal fertility were obtained from oocytes fertilized with freeze-dried epididymal sperm stored at 4 degrees C for 5 years.
    Conclusions and Significance: Sperm with -SS- cross-linking in the thiol-disulfide of their protamine were highly tolerant to freeze-drying, and the fertility of freeze-dried sperm was maintained for 5 years without deterioration. This is the first report to demonstrate the successful freeze-drying of sperm using a new and simple method for long-term preservation.

    DOI: 10.1371/journal.pone.0035043

    PubMed

  • フリーズドライ精子保存法 Reviewed

    金子武人

    日本実験動物技術者協会九州支部会報   2012

  • Essential roles of androgen signaling in Wolffian duct stabilization and epididymal cell differentiation Reviewed

    Murashima Aki, Miyagawa Shinichi, Ogino Yukiko, Nishida-Fukuda Hisayo, Araki Kimi, Matsumoto Takahiro, Kaneko Takehito, Yoshinaga Kazuya, Yamamura Ken-ichi, Kurita Takeshi, Kato Shigeaki, Moon Anne, Yamada Gen

    DEVELOPMENTAL BIOLOGY   356 ( 1 )   166   2011.08( ISSN:0012-1606

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  • Essential Roles of Androgen Signaling in Wolffian Duct Stabilization and Epididymal Cell Differentiation Reviewed

    Aki Murashima, Shinichi Miyagawa, Yukiko Ogino, Hisayo Nishida-Fukuda, Kimi Araki, Takahiro Matsumoto, Takehito Kaneko, Kazuya Yoshinaga, Ken-ichi Yamamura, Takeshi Kurita, Shigeaki Kato, Anne M. Moon, Gen Yamada

    ENDOCRINOLOGY   152 ( 4 )   1640 - 1651   2011.04( ISSN:0013-7227

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    The epididymis is a male accessory organ and functions for sperm maturation and storage under the control of androgen. The development of the epididymis is also androgen dependent. The Wolffian duct (WD), anlagen of the epididymis, is formed in both male and female embryos; however, it is stabilized only in male embryos by testicular androgen. Androgen drives subsequent differentiation of the WD into the epididymis. Although the essential roles of androgen in WD masculinization and epididymal function have been established, little is known about cellular events regulated precisely by androgen signaling during these processes. It is also unclear whether androgen signaling, especially in the epithelia, has further function for epididymal epithelial cell differentiation. In this study we examined the cellular death and proliferation controlled by androgen signaling via the androgen receptor (AR) in WD stabilization. Analyses using AR knockout mice revealed that androgen signaling inhibits epithelial cell death in this process. Analysis of AP2 alpha-Cre;AR(flox/Y) mice, in which AR function is deleted in the WD epithelium, revealed that epithelial AR is not required for the WD stabilization but is required for epithelial cell differentiation in the epididymis. Specifically, loss of epithelial AR significantly reduced expression of p63 that is essential for differentiation of basal cells in the epididymal epithelium. We also interrogated the possibility of regulation of the p63 gene (Trp63) by AR in vitro and found that p63 is a likely direct target of AR regulation. (Endocrinology 152: 1640-1651, 2011)

    DOI: 10.1210/en.2010-1121

    CiNii Article

    J-GLOBAL

  • Improvement in the Development of Oocytes from C57BL/6 Mice after Sperm Injection Reviewed

    Takehito Kaneko, Reiichiro Ohno

    JOURNAL OF THE AMERICAN ASSOCIATION FOR LABORATORY ANIMAL SCIENCE   50 ( 1 )   33 - 36   2011.01( ISSN:1559-6109

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    The C57BL/6 mouse strain is used widely for producing transgenic and knockout strains. Sperm motility is extremely low after a freeze thaw process. Although intracytoplasmic sperm injection (ICSI) can be used to produce embryos from sperm with low or even no motility, its success rate is poor in the C57BL/6 strain. In particular, the survival of C57BL/6 oocytes after ICSI is extremely low compared with that of hybrid strains. We found that the survival percentages of C57BL/6J oocytes (63% and 64%) were lower than those of B6D2F1 oocytes (80% and 80%) when B6D2F1 and C57BL/6J sperm were injected, respectively. For C57BL/6J mice, 87%, 72%, 64%, 56%, and 59% of oocytes survived after ICSI in media containing 61.62, 71.62, 81.62, 91.62, and 101.62 mM NaCl, respectively. In addition, 64%, 81%, and 79% of oocytes survived after ICSI in media with 4.83, 14.83, and 24.83 mM KCl, respectively. Our results suggest that the survival of C57BL/6J oocytes after ICSI is improved by using Na(+)-deficient and K(+) -rich media.

    PubMed

  • Short-Term Storage and Transport at Cold Temperatures of 2-Cell Mouse Embryos Produced by Cryopreserved Sperm Reviewed

    Toru Takeo, Tomoko Kondo, Yukie Haruguchi, Kiyoko Fukumoto, Yoshiko Nakagawa, Yumi Takeshita, Yuko Nakamuta, Shuuji Tsuchiyama, Norihiko Shimizu, Takanori Hasegawa, Motohito Goto, Hitoshi Miyachi, Masayuki Anzai, Rie Fujikawa, Koji Nomaru, Takehito Kaneko, Yoshiaki Itagaki, Naomi Nakagata

    JOURNAL OF THE AMERICAN ASSOCIATION FOR LABORATORY ANIMAL SCIENCE   49 ( 4 )   415 - 419   2010.07( ISSN:1559-6109

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    At refrigerated temperatures, mouse embryos can maintain developmental ability for short periods. Previously, we succeeded in transporting vitrified and warmed 2-cell mouse embryos while maintaining developmental ability at refrigerated temperatures for 50 h. Transport of nonfrozen embryos is an easier and more useful means of exchanging genetically engineered mice between laboratories than is transport of cryopreserved embryos. Here we examined the developmental ability of transported 2-cell embryos that were produced through in vitro fertilization using cryopreserved sperm. Results show that 2-cell embryos produced by cryopreserved sperm can develop into blastocysts after cold storage for 24, 48, and 72 h. Transported 2-cell embryos produced by cryopreserved sperm yielded a favorable number of pups in all of the receiving laboratories after transport lasting 48 to 52 h. In summary, cold storage and transport of 2-cell embryos derived from cryopreserved sperm at refrigerated temperatures provides a novel means of transporting genetically engineered mice as an alternative to the transport of cryopreserved embryos and sperm.

  • Genetic Quality Control of the Rat Strains at the National Bio Resource Project – Rat Reviewed

    T Kuramoto, S Nakanishi, K Yamasaki, K Kumafuji, Y Sakakibara, Y Neoda, A Takizawa, T Kaneko, M Otsuki, R Hashimoto, B Voigt, T Mashimo, T Serikawa

    Interdisciplinary Bio Central   2010

  • Hedgehog,Wnt,FGFの三大増殖因子の相互作用:器官が伸長する(大きくなる)ために どのようなリレーが必要か

    宮川信一, 原田理代, 原口竜摩, 鈴木堅太郎, 中原千彰, 松丸大輔, 金子武人, 中潟直己, 山田 源

    細胞工学   29 ( 2 )   188 - 189   2010

  • Dosage-dependent hedgehog signals integrated with Wnt/beta-catenin signaling regulate external genitalia formation as an appendicular program Reviewed

    Shinichi Miyagawa, Anne Moon, Ryuma Haraguchi, Chie Inoue, Masayo Harada, Chiaki Nakahara, Kentaro Suzuki, Daisuke Matsumaru, Takehito Kaneko, Isao Matsuo, Lei Yang, Makoto M. Taketo, Taisen Iguchi, Sylvia M. Evans, Gen Yamada

    DEVELOPMENT   136 ( 23 )   3969 - 3978   2009.12( ISSN:0950-1991

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    Embryonic appendicular structures, such as the limb buds and the developing external genitalia, are suitable models with which to analyze the reciprocal interactions of growth factors in the regulation of outgrowth. Although several studies have evaluated the individual functions of different growth factors in appendicular growth, the coordinated function and integration of input from multiple signaling cascades is poorly understood. We demonstrate that a novel signaling cascade governs formation of the embryonic external genitalia [genital tubercle (GT)]. We show that the dosage of Shh signal is tightly associated with subsequent levels of Wnt/beta-catenin activity and the extent of external genitalia outgrowth. In Shh-null mouse embryos, both expression of Wnt ligands and Wnt/beta-catenin signaling activity are downregulated. beta-catenin gain-of-function mutation rescues defective GT outgrowth and Fgf8 expression in Shh-null embryos. These data indicate that Wnt/beta-catenin signaling in the distal urethral epithelium acts downstream of Shh signaling during GT outgrowth. The current data also suggest that Wnt/beta-catenin regulates Fgf8 expression via Lef/Tcf binding sites in a 3&apos; conserved enhancer. Fgf8 induces phosphorylation of Erk1/2 and cell proliferation in the GT mesenchyme in vitro, yet Fgf4/8 compound-mutant phenotypes indicate dispensable functions of Fgf4/8 and the possibility of redundancy among multiple Fgfs in GT development. Our results provide new insights into the integration of growth factor signaling in the appendicular developmental programs that regulate external genitalia development.

    DOI: 10.1242/dev.039438

    CiNii Article

    J-GLOBAL

  • Aliskiren Enhances the Protective Effects of Valsartan Against Cardiovascular and Renal Injury in Endothelial Nitric Oxide Synthase-Deficient Mice Reviewed

    Eiichiro Yamamoto, Keiichiro Kataoka, Yi-Fei Dong, Taishi Nakamura, Masaya Fukuda, Yoshiko Tokutomi, Shinji Matsuba, Hisato Nako, Naomi Nakagata, Takehito Kaneko, Hisao Ogawa, Shokei Kim-Mitsuyama

    HYPERTENSION   54 ( 3 )   633 - U360   2009.09( ISSN:0194-911X

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    Publishing type:Research paper (scientific journal)  

    The protective effect of aliskiren, a direct renin inhibitor, against hypertensive cardiovascular and renal injury remains to be defined. This study was undertaken to examine the protective effects of the combination of aliskiren and valsartan, an angiotensin receptor blocker, against cardiovascular and renal injury. Endothelial NO synthase-deficient mice, subjected to cuff injury of femoral artery, were divided into 5 groups and were treated with the following: (1) vehicle; (2) aliskiren (25 mg/kg per day); (3) valsartan (8 mg/kg per day); (4) combined aliskiren (12.5 mg/kg per day) and valsartan (4 mg/kg per day); and (5) hydralazine (10 mg/kg per day) for 4 weeks. Aliskiren and valsartan alone markedly and similarly suppressed cardiac hypertrophy, inflammation and fibrosis, and coronary remodeling; prevented cuff injury-induced arterial intimal thickening; and reduced urinary albumin excretion, glomerular inflammation, and glomerulosclerosis in endothelial NO synthase deficient mice. These beneficial effects of aliskiren and valsartan were associated with the significant attenuation of oxidative stress in these tissues. Hence, aliskiren and valsartan markedly exert the protective effects against cardiovascular and renal injury through the reduction of oxidative stress. Furthermore, compared with monotherapy with aliskiren or valsartan, the combination of a half dose of these drugs more greatly improved the above-mentioned cardiovascular and renal injuries of endothelial NO synthase-deficient mice, which were associated with greater attenuation of tissue oxidative stress by the combination therapy. Thus, the combination of aliskiren and valsartan exerts the synergistic organ-protective effects through synergistic attenuation of oxidative stress. The combination of aliskiren and valsartan seems to be a promising therapeutic strategy for hypertensive organ injury caused by endothelial NO synthase dysfunction. (Hypertension. 2009;54:633-638.)

    DOI: 10.1161/HYPERTENSIONAHA.109.133884

  • Fertilization of C57BL/6 mouse sperm collected from cauda epididymides after preservation or transportation at 4 degrees C using laser-microdissected oocytes Reviewed

    Takehito Kaneko, Kiyoko Fukumoto, Yukie Haruguchi, Tomoko Kondo, Hiromi Machida, Mika Koga, Yoshiko Nakagawa, Shuuji Tsuchiyama, Kiyora Saiki, Shiho Noshiba, Naomi Nakagata

    CRYOBIOLOGY   59 ( 1 )   59 - 62   2009.08( ISSN:0011-2240

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    The C57BL/6 mouse is commonly used to produce transgenic and knockout strains for biomedical research. However, the motility and fertility of its sperm decrease markedly with freezing. Short-term preservation of sperm without freezing can avoid this. Furthermore, such samples can be transported safety without the special skills or equipment needed for the transportation of live animals or frozen products. We evaluated the motility and fertility of sperm collected from cauda epididymides after preservation or transportation at 4 degrees C. Oocytes with the zona pellucida subjected to laser-microdissection were used to assist fertilization in vitro. Although the motility of sperm gradually decreased with storage (P &lt; 0.05), no disruption of the sperm plasma membrane was seen. The proportion of zona-intact oocytes fertilized with sperm preserved for 0, 24, 48 and 72 h were 70, 14, 5 and 1%, respectively. On the other hand, 45, 20 and 14% of laser-microdissected oocytes were fertilized by sperm preserved for 24, 48 and 72 h, respectively (P &lt; 0.05). The fertility of sperm collected from cauda epididymides of two transgenic strains after transportation at 4 degrees C were also significantly increased using laser-microdissected oocytes rather than zona-intact oocytes (57 and 68% vs. 5%, P &lt; 0.05). Efficient production of offspring from sperm preserved or transported at 4 degrees C was achieved using laser-microdissected oocytes. Thus the fertility of sperm preserved or transported at 4 degrees C could be maintained, although motility gradually decreased with storage. Laser-microdissected oocytes will contribute to the efficient production of embryos and offspring using such preserved sperm samples. (C) 2009 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.cryobiol.2009.04.006

  • Importance of primary culture conditions for the development of rat ICSI embryos and long-term preservation of freeze-dried sperm Reviewed

    Takehito Kaneko, Shinya Kimura, Naomi Nakagata

    CRYOBIOLOGY   58 ( 3 )   293 - 297   2009.06( ISSN:0011-2240

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    Rat sperm freeze-dried in a solution containing Tris and ethylenediaminetetraacetic acid (EDTA) (TE buffer) can be preserved at 4 degrees C, and oocytes injected with these sperm developed into offspring though developmental ability was low. We studied the culture conditions to improve the developmental ability of oocytes injected with freeze-dried sperm. After being injected with fresh sperm, the zygotes were cultured in modified Krebs-Ringer bicarbonate (mKRB), modified rat 1-cell embryo culture medium (mR1ECM)/BSA, and mR1ECM with different osmolality, before being cultured in mR1ECM. High proportion of zygotes cultured in mKRB (270 mOsm) before being cultured in mR1ECM developed into blastocysts compared to zygotes cultured only with mR1ECM (50% vs. 28%, P &lt; 0.05). Culturing in mKRB also led to a high proportion of zygotes developing into blastocysts after the injection of freeze-dried sperm than zygotes cultured only with mR1ECM (32% vs. 15%, P &lt; 0.05). Offspring (16%) were obtained when 19 2-cell embryos derived from oocytes that had been injected with freeze-dried sperm preserved at 4 degrees C for I year were transferred. This study demonstrated that the culture conditions soon after the injection of sperm markedly influenced the subsequent development of embryos. Also, rat sperm after freeze-drying in TE buffer were preserved at 4 degrees C for long term without their deterioration. (C) 2009 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.cryobiol.2009.02.004

  • Birth of mice from vitrified/warmed 2-cell embryos transported at a cold temperature Reviewed

    Toru Takeo, Takehito Kaneko, Yukie Haruguchi, Kiyoko Fukumoto, Hiromi Machida, Mika Koga, Yoshiko Nakagawa, Yumi Takeshita, Toyokazu Matsuguma, Shuuji Tsuchiyama, Norihiko Shimizu, Takanori Hasegawa, Motohito Goto, Hitoshi Miyachi, Masayuki Anzai, Ena Nakatsukasa, Koji Nomaru, Naomi Nakagata

    CRYOBIOLOGY   58 ( 2 )   196 - 202   2009.04( ISSN:0011-2240

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    Cryopreservation of 2-cell embryos is an effective technology for storage of genetically engineered mouse strains. Transport of genetically engineered mice between laboratories has frequently been performed using such cryopreserved 2-cell embryos. However, the receiving laboratory requires proficient skills and special instruments to obtain live young from cryopreserved and transported embryos. Therefore, in this study, we tried to address the storage and transport of vitrified/warmed 2-cell embryos at a cold temperature. In cold storage experiments, the development rates of 2-cell embryos stored in M2 medium for 24,48 and 72 h into blastocysts were relatively high (83%, 63% and 43%, respectively). Although, 2-cell embryos stored in PB1 and mWM maintained the developmental potency for 24 h, the rates were markedly decreased to low levels after 48 h (PB1: 0%; mWM: 5%). In transport experiments, many pups were obtained from vitrified/warmed 2-cell embryos transported at a cold temperature in all receiving laboratories (incidence of successful development: 49%; 249/511). In summary, short-term storage and transport of vitrified/warmed 2-cell embryos in M2 medium at a cold temperature can maintain their ability to develop into live young. (C) 2008 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.cryobiol.2008.12.011

    J-GLOBAL

  • Kinetics of Blood Glucose in Mice Carrying Hemizygous Pax6 Reviewed

    Yumiko Nitta, Yasufumi Shigeyoshi, Naomi Nakagata, Takehito Kaneko, Kohsaku Nitta, Toshihide Harada, Fumiko Ishizaki, Jana Townsend

    EXPERIMENTAL ANIMALS   58 ( 2 )   105 - 112   2009.04( ISSN:1341-1357

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    The genotype-phenotype relationship was examined experimentally for the Pax6(Sey-4H) mutant, which carries deletion of its chromosome 2 middle region hemizygously. The genotyping has indicated that this deleted segment is between 102.6 and 109.2 Mb from the centromere. The glucose-6-phosphatase gene followed by the glucagon and carboxyl ester lipase genes were mapped adjacent to the deleted region. Phenotyping indicates that the Pax6(Sey-4H) mutant is more susceptible to diabetes. The glucose tolerance test showed that the mutants were less capable of reducing their level of blood glucose to the standard level than the normal sibs. The insulin-loading test revealed their inability to elevate their blood glucose levels up to normal levels. The time it took for the onset of diabetes induced by streptozotocin was shorter in the mutants than in normal sibs. Both the haploinsufficiency of the genes in the hemizygous segment of chromosome 2 and the quantitative imbalance of the whole genome could contribute the development of this phenotype in the mutant.

    DOI: 10.1538/expanim.58.105

  • In vitro fertilization using cryopreserved laser-microdissected oocytes on the inbred mouse strains Reviewed

    MIYAJI Shiori, ANZAI Masayuki, KODA Yuna, YANAGI Miho, NAKASHIMA Tatsuyuki, KAWABE Toshiaki, KANEKO Takehito, NAKAGATA Naomi

    43 ( 1 )   25 - 29   2008.06( ISSN:0387-978X

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  • Offspring derived from oocytes injected with rat sperm, frozen or freeze-dried without cryoprotection Reviewed

    T. Kaneko, S. Kimura, N. Nakagata

    THERIOGENOLOGY   68 ( 7 )   1017 - 1021   2007.10( ISSN:0093-691X

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    Sperm preservation is a valuable technique for maintaining genetic resources in biomedical research. In the present study, 10 MM Tris-HCl and 1 mM EDTA (TE buffer; a simple solution without cryoprotection), was used to freeze or freeze-dry rat sperm. The results were compared with rat sperm frozen using a solution containing Equex STM and egg yolk. Sperm from Wistar and Sprague-Dawley (SD) rats were evaluated by injecting them individually into oocytes derived from the same strain. Of the oocytes that survived after sperm injection, more than 94% were fertilized in all treatments of both strains. In the Wistar rat, 27, 20, 43, and 30% of 2-cell embryos developed to blastocysts, and 35, 9, 11, and 14% of 2-cell embryos developed to offspring from oocytes injected with fresh, frozen (Equex STM/egg yolk), frozen (TE buffer), and freeze-dried sperm, respectively. Using the analagous sources of sperm in the SD rat, 45, 14, 27, and 7 % of 2-cell embryos developed to blastocysts, and 22,0, 14, and 4% of 2-cell embryos developed to offspring. These results demonstrated that rat sperm could be frozen or freeze-dried using TE buffer. We concluded that this simple preservation method, in which cryoprotection was not required, allowed sperm to be preserved efficiently with maintenance of their fertilizing ability. (C) 2007 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.theriogenology.2007.07.017

  • Application of laser-assisted zona drilling to in vitro fertilization of cryopreserved mouse oocytes with spermatozoa from a subfertile transgenic mouse Reviewed

    Masayuki Anzai, Megumi Nishiwak, Miho Yanagi, Tatsuyuki Nakashima, Takehito Kaneko, Yoshitomo Taguchi, Mikiko Tokoro, Seung-Wook Shin, Tasuku Mitani, Hiromi Kato, Kazuya Matsumoto, Naomi Nakagata, Akira Iritani

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   52 ( 5 )   601 - 606   2006.10( ISSN:0916-8818

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    Development of assisted reproductive technologies is necessary to obtain fertilized oocytes in a subfertile transgenic mouse strain. Here, we showed the application of laser-assisted drilling of the zona pellucida to in vitro fertilization of cryopreserved mouse oocytes with sperm from subfertile transgenic mice (C57BL/6N-Tg(UCP/FAD2)U8 strain). After cryopreservation by vitrification, the recovery and survival rates of the zona-drilled mouse oocytes were 97% (97/100) and 94% (91/97), respectively. In vitro fertilization of the cryopreserved zona-drilled mouse oocytes with sperm from the subfertile transgenic mice was greatly facilitated (60%, 55/91) compared to that of the cryopreserved zona-intact mouse oocytes (11%, 81/768). In vitro fertilized embryos that developed to the 2-cell stage were again cryopreserved by vitrification, and after warming they were transferred into recipient females. Subsequently, six viable offspring were delivered, and all were confirmed to be transgenic mice. These results indicate that laser-assisted zona drilling of oocytes combined with cryopreservation by vitrification may be a useful approach for large-scale production of in vitro fertilized embryos for managing transgenic mouse strains with reproductive disabilities such as subfertile sperm.

    DOI: 10.1262/jrd.18040

    CiNii Article

  • Improvement in the long-term stability of freeze-dried mouse spermatozoa by adding of a chelating agent Reviewed

    Takehito Kaneko, Naomi Nakagata

    CRYOBIOLOGY   53 ( 2 )   279 - 282   2006.10( ISSN:0011-2240

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    This study demonstrates that a small amount of chelating agent in the freeze-drying solution is necessary to prevent the deterioration of spermatozoa during freeze-drying and subsequent preservation at 4 degrees C. We freeze-dried mouse epididymal spermatozoa in the solutions containing Tris-HCl and ethylenediaminetetraacetic acid (EDTA) as a chelating agent. Spermatozoa stored for various times up to 1 year at 4 degrees C were injected intracytoplasmically into individual oocytes, and the normality of chromosomes in fertilized oocytes was analyzed. In addition, embryos derived from freeze-dried spermatozoa were transferred into recipients to determine their developmental ability. Chromosomes were maintained well when spermatozoa were freeze-dried in a solution containing 10mM Tris-HCl and 1 mM EDTA (73%), and 57% of embryos developed to term. Of embryos derived from spermatozoa stored for 1 year, 65% developed into live offspring. On the other hand, when spermatozoa were freeze-dried in a solution containing 10 mM Tris-HCl and 0 or 50 mM EDTA, spermatozoa that maintained karyotypically normal chromosomes were 64% or 22%, and only 16% or 3% of embryos were developed to term, respectively. This finding suggested that mouse spermatozoa can be freeze-dried in a simple solution containing the same composition as that used to preserve extracted DNA.

    DOI: 10.1016/j.cryobiol.2006.06.004

  • Long-term cryopreservation of mouse sperm Reviewed

    Takehito Kaneko, Ayako Yamamura, Yukie Ide, Mami Ogi, Tomoko Yanagita, Naomi Nakagata

    THERIOGENOLOGY   66 ( 5 )   1098 - 1101   2006.09( ISSN:0093-691X

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    Publishing type:Research paper (scientific journal)  

    The objective was to determine if mouse sperm can maintain their fertilizing ability after being frozen for &gt; 10 y and whether the offspring derived from these sperm had normal fertilizing ability and phenotype. We cryopreserved sperm from six strains of mice (C57BL/6J, DBA/2N, BALB/cA, C3H/HeJ, B6D2F1 and B6C3F1) in a solution containing 18% (w/v) raffinose and 3% (w/v) skim milk, and preserved them in liquid nitrogen for &gt; 10 y. To assess the normality and fertilizing ability of these sperms, they were thawed and used for in vitro fertilization of oocytes of the same strains. Fertilization rates for C57BL/6J, DBA/2N, BALB/cA, C3H/ HeJ, B6D2F1 and B6C3F1 were 66.4, 92.3, 72.8, 32.9, 60.3 and 53.7%, respectively. Furthermore, 38.3, 15.0, 43.3, 26.1, 38.3 and 16.7% of the embryos transferred to pseudopregnant females developed and produced live offspring that had normal phenotype and fertility. (c) 2006 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.theriogenology.2006.02.049

  • The improvement in fertilizing ability of cryopreserved mouse spermatozoa using laser-microdissected oocytes Reviewed

    Takehito Kaneko, Miho Yanagi, Tatsuyuki Nakashima, Naomi Nakagata

    Reproductive Medicine and Biology   5 ( 4 )   249 - 253   2006( ISSN:1447-0578

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    Publishing type:Research paper (scientific journal)  

    Aim: The C57BL/6 mouse strain is now commonly used for producing transgenic/knockout strains. However, the fertilizing ability of these spermatozoa decreases as a result of cryopreservaion. Although the micromanipulation technique has been established to increase their fertilizing ability, it requires a considerable degree of technical skill. In the present report, we investigate the simple microdissection of zona pellucida by laser to increase the fertilizing ability of cryopreserved spermatozoa. Methods: C57BL/6J spermatozoa were cryopreserved using a solution consisting of 18% raffinose/3% skim milk. Oocytes of the same strain were placed in PB1 medium containing 0, 0.25, 0.50 or 0.75 mol sucrose. The zona pellucida of oocytes was microdissected by laser with different pulse lengths selected from 0.45 to 0.65 ms. Microdissected oocytes were then fertilized with cryopreserved spermatozoa, and the subsequent development of embryos was assessed. Results: When oocytes were microdissected in PB1 medium without sucrose, 81.5% of the oocytes were fertilized. The fertilization rates increased significantly as the pulse length was lengthened when compared with oocytes with intact zona pellucida. Furthermore, normal offspring were obtained in all experiments. Conclusion: The fertilizing ability of cryopreserved spermatozoa is improved when oocytes with their zona pellucida microdissected by laser were used. © 2006 The Authors Journal compilation © 2006 Japan Society for Reproductive Medicine.

    DOI: 10.1111/j.1447-0578.2006.00149.x

    CiNii Article

  • Recombinase-mediated mouse transgenesis by intracytoplasmic sperm injection Reviewed

    T Kaneko, S Moisyadi, R Suganuma, B Hohn, R Yanagimachi, P Pelczar

    THERIOGENOLOGY   64 ( 8 )   1704 - 1715   2005.11( ISSN:0093-691X

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    Publishing type:Research paper (scientific journal)  

    The low efficiency of current microinjection-based animal transgenesis techniques is largely the result of poor embryo survival. We have developed a new, bacterial recombinase-based transgenesis method. Intracytoplasmic sperm injection (ICSI) of single stranded DNA (ssDNA) complexed with E. coli recombinase RecA into mouse metaphaseII (MII) arrested oocytes resulted in RecA-dependent transgenesis. This approach offers significant advantages over pronuclear microinjection and previous ICSI-based transgenesis approaches in terms of improved embryo survival, which translates into greater transgenesis efficiency. It also opens the possibility to attempt experiments, which may affect gene targeting by homologous recombination into DNA of mammalian single celled pre-implantation embryos. (c) 2005 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.theriogenology.2005.04.003

  • Relation between storage temperature and fertilizing ability of freeze-dried mouse spermatozoa Reviewed

    T Kaneko, N Nakagata

    COMPARATIVE MEDICINE   55 ( 2 )   140 - 144   2005.04( ISSN:1532-0820

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    The advantage of freeze-dried mouse spermatozoa is that samples can be stored in the refrigerator (+4 degrees C). Moreover, the storage of freeze-dried spermatozoa at ambient temperature would permit spermatozoa to be shipped easily and at low cost around the world. To examine the influence of the storage temperature on freeze-dried spermatozoa, we assessed the fertilizing ability of spermatozoa stored at different temperatures. Cauda epididymal spermatozoa were freeze-dried in buffer consisting of 50 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 50 mM NaCl, and 10 mM Tris-HCI (pH 8.0). Samples of freeze-dried spermatozoa were stored at -70,-20,+4, or +24 degrees C for periods of 1 week and 1, 3, and 5 months. Sperm chromosomes were maintained well at -70, -20, and +4 degrees C for 5 months, and oocytes fertilized with these spermatozoa developed to normal offspring. Moreover, the chromosomal integrity of spermatozoa stored at -20 or +4 degrees C did not decrease even after 17 months. In contrast, the chromosomes of spermatozoa stored at +24 degrees C were maintained well for 1 month but became considerably degraded after 3 months. In addition, to investigate the cause of deterioration of sperm chromosomes during storage at +24 degrees C, spermatozoa were freeze-dried in buffer containing DNase I. The chromosomes of spermatozoa freeze-dried with 1 or 0.2 units/ml of DNase I, 100% or 72%, respectively, exhibited chromosomal abnormalities. Our findings suggest that freeze-dried spermatozoa can be stored long-term with stability at +4 degrees C, and the suppression of nucleases present in the buffer or spermatozoa during storage led to the achievement of long-term storage of freeze-dried spermatozoa.

  • Use of frozen-thawed oocytes for efficient production of normal offspring from cryopreserved mouse spermatozoa showing low fertility Reviewed

    W Sakamoto, T Kaneko, N Nakagata

    COMPARATIVE MEDICINE   55 ( 2 )   136 - 139   2005.04( ISSN:1532-0820

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    Freezing of spermatozoa and unfertilized oocytes is a useful tool for the conservation of mouse genetic resources. However, the proportion of frozen-thawed oocytes fertilized with spermatozoa in vitro is low because spermatozoa, especially those frozen-thawed, can not penetrate into oocytes because of hardening of the zona pellucida following premature release of cortical granules. To produce offspring efficiently from cryopreserved transgenic mouse gametes, we fertilized frozen-thawed gametes by using intracytoplasmic sperm injection (ICSI) and assessed pre- and post-implantation development of embryos. Compared with fresh unfertilized oocytes, frozen-thawed unfertilized oocytes were highly tolerant to damage by injection, as the survival rates after injection of frozen spermatozoa were 51 and 78%, respectively. Frozen-thawed oocytes that survived after sperm injection developed normally to the blastocyst stage and gave rise to offspring. Moreover, offspring with transgenes also were obtained from frozen gametes fertilized by ICSI. These results demonstrate that ICSI is an efficient technique for producing offspring from transgenic spermatozoa showing low fertility and that use of frozen-thawed oocytes leads to conservation of genetic resources because suboptimally preserved gametes are not wasted.

  • Cytoplasmic dysmorphisms in metaphase II chimpanzee oocytes Reviewed

    K Suzuki, N Yoshimoto, K Shimoda, W Sakamoto, Y Ide, T Kaneko, T Nakashima, Hayasaka, I, N Nakagata

    REPRODUCTIVE BIOMEDICINE ONLINE   9 ( 1 )   54 - 58   2004.07( ISSN:1472-6483

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    Prior to fertilization by intracytoplasmic sperm injection (ICSI). cytoplasmic organization was evaluated in metaphase II chimpanzee oocytes obtained from stimulated ovaries. The findings demonstrate a high frequency of anomalies that are remarkably similar to the types of cytoplasmic dysmorphisms reported for human oocytes used in IVF. Similar to the human, the occurrence of these anomalies was oocyte- and animal-specific and associated with reduced competence as indicated by embryo development in vitro to the blastocyst stage.

  • Long-term preservation of mouse spermatozoa after freeze-drying and freezing without cryoprotection Reviewed

    MA Ward, T Kaneko, H Kusakabe, JD Biggers, DG Whittingham, R Yanagimachi

    BIOLOGY OF REPRODUCTION   69 ( 6 )   2100 - 2108   2003.12( ISSN:0006-3363

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    The widespread production of mice with transgenes, disrupted genes and mutant genes, has strained the resources available for maintaining these mouse lines as live populations, and dependable methods for gamete and embryo preservation in these lines are needed. Here we report the results of intracytoplasmic sperm injection (ICSI) with spermatozoa freeze-dried or frozen without a cryoprotectant after storage for periods up to 1.5 years. Freeze-dried samples were stored at VC. Samples frozen without cryoprotection were maintained at -196degreesC. After storage, spermatozoa were injected into the oocytes by ICSI. Zygotic chromosomes and fetal development at Day 15 of gestation were examined after 0, 1, 3, 6, 9, and 12 mo of sperm storage. When fresh spermatozoa were used for ICSI, 96% of resultant zygotes contained normal chromosomes, and 58% of two-cell embryos transferred developed to normal viable fetuses. Similar results were obtained when spermatozoa were frozen without cryoprotection and then used for ICSI (87% and 45%, respectively; P &gt; 0.05) and after 12 mo of sperm storage (mean of six endpoints examined: 87% and 52%, respectively; P &gt; 0.05). Freeze-drying decreased the proportion of zygotes with normal karyoplates (75% vs. 96%; P &lt; 0.001) and the proportion of embryos that developed into fetuses (35% vs. 58%; P &lt; 0.001), but similar to freezing, there was no further deterioration during 12 mo of storage (mean of six endpoints examined: 68% and 34%, respectively; P &gt; 0.05). Live offspring were obtained from both freeze-dried and frozen spermatozoa after storage for 15 yr. The results indicate that 1) the freeze-drying procedure itself causes some abnormalities in spermatozoa but freezing without cryoprotection does not and 2) long-term storage of both frozen and freeze-dried spermatozoa is not deleterious to their genetic integrity. Freezing without cryoprotection is highly successful, simple, and efficient but, like all routine sperm storage methods, requires liquid nitrogen. Liquid nitrogen is also required for freeze-drying, but sperm can then he stored at 4degreesC and shipped at ambient temperatures. Both preservation methods are successful, but rapid freezing without cryoprotection is the preferred method for preservation of spermatozoa from mouse strains carrying unique genes and mutations.

    DOI: 10.1095/biolreprod.103.020529

  • Tolerance of the mouse sperm nuclei to freeze-drying depends on their disulfide status Reviewed

    T Kaneko, DG Whittingham, JW Overstreet, R Yanagimachi

    BIOLOGY OF REPRODUCTION   69 ( 6 )   1859 - 1862   2003.12( ISSN:0006-3363

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    Mouse spermatozoa from the caudae epididymides could be freeze-dried without losing their ability to support normal development. Immature spermatozoa from the testes, in contrast, were damaged by freeze-drying. However, immature spermatozoa became resistant to freeze-drying after their treatment with diamide, which oxidizes free -SH groups. Conversely, epididymal spermatozoa were damaged by freeze-drying if first treated with dithiothreitol (DTT), which reduces -SS- bonds. The potential for freeze-drying damage seems likely to relate to the -SS- status of sperm proteins, in particular its protamines.

    DOI: 10.1095/biolreprod.103.019729

  • Effect of pH value of freeze-drying solution on the chromosome integrity and developmental ability of mouse spermatozoa Reviewed

    T Kaneko, DG Whittingham, R Yanagimachi

    BIOLOGY OF REPRODUCTION   68 ( 1 )   136 - 139   2003.01( ISSN:0006-3363

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    Publishing type:Research paper (scientific journal)  

    The nuclei of freeze-dried mouse spermatozoa are able to retain their chromosome integrity and developmental potential. To optimize the conditions of freeze-drying, we examined whether pH values of the freeze-drying solution affect the chromosome integrity and developmental potential of sperm nuclei. The sperm freeze-drying solution we used contained a, high concentration (50 mM) of calcium-chelating EGTA. Sperm chromosomes were examined at the metaphase of the first mitosis after injection of freeze-dried spermatozoa into matured oocytes. The developmental potential of sperm nuclei was assessed by examining the development of fetuses in midgestation. The results showed that both sperm chromosomes and sperm developmental potential are maintained better when the freeze-drying solution was slightly alkaline (pH 8.0) rather than near neutral or acidic (pH 7.4-6.0). The data indicated that the chromosome integrity and developmental ability of mouse spermatozoa are affected by the pH value of freeze-drying solution.

    DOI: 10.1095/biolreprod.102.008706

  • Experimental evaluation of cross-contamination between cryotubes containing mouse 2-cell embryos and murine pathogens in liquid nitrogen tanks Reviewed

    S Kyuwa, T Nishikawa, T Kaneko, T Nakashima, K Kawano, N Nakamura, K Noguchi, T Urano, T Itoh, N Nayagata

    EXPERIMENTAL ANIMALS   52 ( 1 )   67 - 70   2003.01( ISSN:1341-1357

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    It has been suspected that embryos stored in liquid nitrogen tanks may become contaminated with murine pathogens, if some pathogens had been introduced to the tanks accidentally. To examine this, we stored tubes containing embryos with tubes containing mouse hepatitis virus (MHV) or Pasteurella pneumotropica in liquid nitrogen tanks and examined whether progeny mice derived from the embryos were contaminated with the pathogens or not After storing for 6 months or 1 year the frozen embryos were thawed and implanted into the oviducts of pseudopregnant female mice, and the mice were bred in vinyl isolators. We could not detect serum antibodies to MHV and isolate Pasteurella pneumotropica in the progeny mice, suggesting that cross-contamination between tubes in a liquid nitrogen tank scarcely occurs.

    DOI: 10.1538/expanim.52.67

    CiNii Article

  • Detection of Messenger Ribonucleic Acid Coding for Glyceraldehyde-3-Phosphate Dehydrogenase in Single Bovine Oocytes and Early Embryos.

    Kaneko Takehito, Saeki Kazuhiro, Matsumoto Kazuya, Nakagami Kayoko, Hosoi Yoshihiko, Kato Hiromi, Iritani Akira

    Journal of Mammalian Ova Research   16 ( 3 )   118 - 123   1999( ISSN:1341-7738

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    The reverse transcription-polymerase chain reaction (RT-PCR) method is widely used for studying mRNA expression in cells and tissues. In this study, we examined whether the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene could be used as an endogenous control for gene expression in single bovine oocytes and early embryos. First, sequencing of partial bovine G3PDH cDNA from ovarian tissue was performed. The sequence analysis of bovine G3PDH cDNA after subcloning indicated a high homology with human, mouse and rat G3PDH cDNA. Next, we examined whether G3PDH could be detected in single bovine oocytes and early embryos by using the RT-PCR method. Signals for G3PDH mRNA were detected in single immature and mature oocytes and single embryos at the one-cell to blastocyst stages. Thus, G3PDH is suitable as an endogenous control for examining mRNA expression even with single bovine oocytes or early embryos by using the RT-PCR method.<br>

    DOI: 10.1274/jmor.16.118

    CiNii Article

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Books and Other Publications

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MISC

  • Reproductive technologies for the generation and maintenance of valuable animal strains

    64 ( 3 )   209 - 215   2018.06

  • Reproductive technologies for the generation and maintenance of valuable animal strains

    64 ( 3 )   209 - 215   2018.01

  • Functional analysis of histone demethylase Kdm5b in mouse preimplantation development. Reviewed

    Kurasaka M, Kaneko T, Suzuki S, Tsukamoto S, Imai H, Minami N

    4th World Congress of Reproductive Biology   2017.09

  • The role of SMYD3 in methylation of H3K4 of Hmgpi promoter regions in mouse preimplantation embryos. Reviewed

    Honda S, Suzuki S, Tsukamoto S, Kaneko T, Imai H, Minami N

    4th World Congress of Reproductive Biology   2017.09

  • DEVELOPMENT OF A NEW MODEL FOR COMBINED ATHEROSCLEROSIS AND HYPERTENSION: APOE-KNOCKOUT SPONTANEOUSLY HYPERTENSIVE RAT

    Hiroyuki Matsuo, Takehito Kaneko, Koichi Fujikawa, Hiroki Ohara, Tomoji Mashimo, Toru Nabika

    ATHEROSCLEROSIS   263   E86 - E86   2017.08( ISSN:0021-9150 ( eISSN:1879-1484

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  • Sperm freeze-drying and micro-insemination for biobanking and maintenance of genetic diversity in mammals

    Takehito Kaneko

    Reproduction Fertility and Development   2016.02

  • CRISPR/CasシステムによるGFPノックインラットの作製

    MASHIMO TOMOJI, YOSHIMI KAZUTO, KANEKO TAKEHITO

    日本実験動物学会総会講演要旨集   62nd   76   2015.04

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  • Precisely engineered rodent models of Parkinson's with CRISPR technology

    Karamjit Singh Dolt, Seiya Mizuno, Takehito Kaneko, Satoru Takahashi, Tomoji Mashimo, Tilo Kunath

    TRANSGENIC RESEARCH   23 ( 5 )   903 - 903   2014.10( ISSN:0962-8819 ( eISSN:1573-9368

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  • CHD1 is the zygotic determinant of Oct4 and Cdx2 expression via the Hmgpi pathway during preimplantation development. Reviewed

    Suzuki, S, Nozawa Y, Tsukamoto S, Kaneko T, Imai H, Minami N

    2014 World Congress of Reproductive Biology   2014.09

  • CRISPR/Casシステムを用いたノックインラットの作製

    YOSHIMI KAZUTO, KANEKO TAKEHITO, MASHIMO TOMOJI

    日本実験動物学会総会講演要旨集   61st   242   2014.05

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  • 希少動物におけるフリーズドライ精子保存法の確立及び配偶子バンクの設立.

    金子武人, 伊藤英之, 坂本英房, 大沼学, 村山美穂

    第20回日本野生動物医学会大会、2014年9月16-19日、つくば   2014

  • The role of Ing3 during oocyte maturation in the mouse

    SUZUKI Shinnosuke, NOZAWA Yusuke, KANEKO Takehito, IMAI Hiroshi, MINAMI Naojiro

    30 ( 2 )   S57   2013.04( ISSN:1341-7738

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  • 実験用ラットにおけるゲノム編集技術:ZFN,TALENそしてCRISPRへ

    MASHIMO TOMOJI, YOSHIMI KAZUHITO, KANEKO TAKEHITO

    日本分子生物学会年会プログラム・要旨集(Web)   36th   1AW2-7 (WEB ONLY)   2013

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  • Ing3 has an important role during oocyte maturation in the mouse Reviewed

    Suzuki S, Nozawa Y, Tsukamoto S, Kaneko T, Imai H, Minami N

    The 46th Annual Meeting of the Society for the Study of Reproduction   2013

  • マウス初期胚におけるヒストンメチル化酵素Smyd3の役割について

    野澤佑介, 鈴木伸之介, 塚本智史, 金子武人, 今井 裕, 南 直治郎

    第105回日本繁殖生物学会、講演要旨集、j186   2012.09

  • Effects of chromatin remodeling factor Chd1 on in vitro differentiation of mouse preimplantation embryos Reviewed

    Suzuki S, Nozawa Y, Tsukamoto S, Kaneko T, Imai H, Minami N

    10th Annual Meetinf of International Socirety for Stem Cell Reseach   2012

  • マウス初期胚における黒町ん再構成タンパク質Chd1に関する研究

    鈴木伸之介, 野澤佑介, 塚本智史, 金子武人, 今井 裕, 南直治郎

    第105回日本繁殖生物学会、講演要旨集、j91   2011.09

  • Cryopreservation of sperm in genetically engineered mice

    TAKEO Toru, FUKUMOTO Kiyoko, KONDO Tomoko, HARUGUCHI Yukie, TAKESHITA Yumi, NAKAMUTA Yuko, UMEZAWA Miyuki, TSUCHIYAMA Shuuji, KANEKO Takehito, NAKAGATA Naomi

    27 ( 2 )   S47   2010.04( ISSN:1341-7738

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  • Birth of mice from vitrified/warmed 2-cell embryos transported at a cold temperature

    NAKAGATA Naomi, TAKEO Toru, KANEKO Takehito, HARUGUCHI Yukie, FUKUMOTO Kiyoko, MACHIDA Hiromi, KOGA Mika, NAKAGAWA Yoshiko, TAKESHITA Yumi, MATSUGUMA Toyokazu, TSUCHIYAMA Shuuji, SHIMIZU Norihiko, HASEGAWA Takanori, GOTO Nobuhito, MIYACHI Hitoshi, ANZAI Masayuki, NAKATSUKASA Ena, NOMARU Kouji

    26 ( 2 )   2009.04( ISSN:1341-7738

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  • Fertility of mouse sperm preserved at low temperature

    KANEKO Takehito, SAIKI Kiyora, NOSHIBA Shiho, NAKAGATA Naomi

    26 ( 2 )   S42   2009.04( ISSN:1341-7738

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  • Tissue compartment-specific androgen receptor functions in the male reproductive tract development

    村嶋亜紀, 宮川信一, 西田(福田)尚代, 荻野由紀子, 荒木喜美, 松本高広, 金子武人, 吉永一也, 山村研一, 加藤茂明, MOON Anne. M, 山田源

    日本分子生物学会年会講演要旨集   32nd ( Vol.4 )   173   2009

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  • Improvement of culture conditions for the development of oocytes injected with freeze-dried rat sperm

    Takehito Kaneko, Shinya Kimura, Naomi Nakagata

    BIOLOGY OF REPRODUCTION   153 - 153   2008( ISSN:0006-3363

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    Publishing type:Research paper, summary (international conference)  

  • Offspring derived from frozen rat spermatozoa using laser-microdissected oocytes

    KANEKO Takehito, IMAGAWA Takanari, NAKAGATA Naomi

    24 ( 2 )   S64   2007.04( ISSN:1341-7738

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  • Mouse embryo/sperm bank at the center for animal resources and development (CARD), kumamoto university.

    Takehito Kaneko, Yukie Haruguchi, Tomoko Yana-Gita, Hiromi Machida, Kiyoko Fukumoto, Yoshiko Nakagawa, Mika Koga, Shuuji Tsuchiyama, Naoko Nakamura, Satomi Yoshizumi, Mari Koga, Toru Urano, Naomi Nakagata

    BIOLOGY OF REPRODUCTION   83 - 83   2007( ISSN:0006-3363

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  • Improvement of preservation solution for freeze-drying of mouse spermatozoa

    KANEKO Takehiro, NAKAGATA Naomi

    23 ( 2 )   S29   2006.04( ISSN:1341-7738

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  • Necessity of chelating agent in freeze-drying of mouse spermatozoa.

    Takehito Kaneko, Naomi Nakagata

    BIOLOGY OF REPRODUCTION   180 - 180   2006( ISSN:0006-3363

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  • Use of frozen oocytes for production of offspring from frozen spermatozoa showing low fertility

    KANEKO Takehito, SAKAMOTO Wataru, NAKAGATA Naomi

    22 ( 2 )   S36   2005.04( ISSN:1341-7738

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  • In vitro fertilization and cryopreservation of embryos in genetically engineered mice

    KANEKO Takehito, YAMAMURA Ayako, OGAWA Mami, IDE Yukie, YANAGITA Tomoko, TAKEICHI Miwako, NAKASHIMA Tatsuyuki, NAKAGATA Naomi

    21 ( 2 )   S37   2004.04( ISSN:1341-7738

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  • Developmental potential of freeze-dried mouse spermatozoa stored at low or ambient temperature.

    T Kaneko, N Nakagata

    BIOLOGY OF REPRODUCTION   237 - 237   2004( ISSN:0006-3363

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  • Studies on the storage of mouse embryo at 4℃

    NAKASHIMA Tetsuyuki, KANEKO Takehito, YAMAMURA Ayako, OGAWA Mami, KOUYAMA Takashi, KAWANO Kayo, NAKAGATA Naomi

    18 ( 2 )   S16   2001.04( ISSN:1341-7738

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  • Injection of rabbit sperm head into oocyte and subsequent embryonic development in vitro.

    KUSAKA NAOKO, HOSOI YOSHIHIKO, KANEKO TAKEHITO, KATO HIROMI, MATSUMOTO KAZUYA, SAEKI KAZUHIRO, IRITANI AKIRA

    日本胚移植学雑誌   21 ( 2 )   85 - 91   1999.05( ISSN:1341-2965

  • Onset of transcription and translation of embryonic gene in early bovine embryos following introduction of exogenous gene into pronuclei

    SAEKI Kazuhiro, MATSUMOTO Kazuya, KANEKO Takehito, HARADA Mayumi, NOICHI Teruhisa, HOSOI Yoshihiko, KATO Hiromi, IRITANI Akira

    21   604 - 604   1998.12

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