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Development and verification of the next generation transgenesis tools

Research Project

Project/Area Number 25290035
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypePartial Multi-year Fund
Section一般
Research Field Laboratory animal science
Research InstitutionTokai University

Principal Investigator

OHTSUKA Masato  東海大学, 医学部, 准教授 (90372945)

Co-Investigator(Kenkyū-buntansha) MOCHIDA Keiji  理化学研究所, バイオリソースセンター, 専任技師 (60312287)
SHITARA Hiroshi  東京都医学総合研究所, 基盤技術研究センター, 主任基盤技術研究職員 (90321885)
Project Period (FY) 2013-04-01 – 2016-03-31
Project Status Completed (Fiscal Year 2015)
Budget Amount *help
¥17,680,000 (Direct Cost: ¥13,600,000、Indirect Cost: ¥4,080,000)
Fiscal Year 2015: ¥5,720,000 (Direct Cost: ¥4,400,000、Indirect Cost: ¥1,320,000)
Fiscal Year 2014: ¥5,720,000 (Direct Cost: ¥4,400,000、Indirect Cost: ¥1,320,000)
Fiscal Year 2013: ¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000)
Keywordsトランスジェニックマウス / PITT / Cre-loxP / PhiC31 / Rosa26 / トランスジェニック / PITT法 / 組織特異的プロモーター / ターゲットトランスジェネシス / C57BL/6N
Outline of Final Research Achievements

We recently developed technology termed “pronuclear injection (PI)-based Targeted Transgenesis (PITT)” that allows targeted insertion of single-copy transgene into the predetermined locus through PI. PITT method enables to generate Tg mice showing reliable transgene expression pattern. Here, we developed an improved-PITT (i-PITT) system by combining Cre-loxP and PhiC31-attP/B systems directly under C57BL/6N inbred strain. The targeted Tg efficiency in the i-PITT was improved up to 67%, which is by-far the best Tg rate reported. Furthermore, the system could generate multiple Tg mice simultaneously: injection of up to three different Tg cassettes in a single injection session into as less as 181 zygotes resulted in production of all three separate Tg DNA containing targeted Tg mice. Standard protocol for PITT was published and the seed mouse strain is available from RIKEN BRC.

Report

(4 results)
  • 2015 Annual Research Report   Final Research Report ( PDF )
  • 2014 Annual Research Report
  • 2013 Annual Research Report
  • Research Products

    (40 results)

All 2016 2015 2014 2013 Other

All Int'l Joint Research (1 results) Journal Article (10 results) (of which Int'l Joint Research: 2 results,  Peer Reviewed: 9 results,  Open Access: 5 results,  Acknowledgement Compliant: 4 results) Presentation (26 results) (of which Int'l Joint Research: 1 results,  Invited: 1 results) Book (2 results) Remarks (1 results)

  • [Int'l Joint Research] ネブラスカ大学医療センター(米国)

    • Related Report
      2015 Annual Research Report
  • [Journal Article] Conditional knockout of Foxc2 gene in kidney: efficient generation of conditional alleles of single-exon gene by double-selection system2016

    • Author(s)
      Motojima, Masaru; Ogiwara, Sanae; Matsusaka, Taiji; Kim, Sang Yong; Sagawa, Nobuho; Abe, Koichiro; Ohtsuka, Masato
    • Journal Title

      Mamm Genome

      Volume: 27 Issue: 1-2 Pages: 62-69

    • DOI

      10.1007/s00335-015-9610-y

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
  • [Journal Article] Establishment of immortalized mesenchymal stem cells derived from the submandibular glands of tdTomato transgenic mice2015

    • Author(s)
      Furukawa S., Kuwajima Y., Chosa N., Satoh K., Ohtsuka M., Miura H., Kimura M., Inoko H., Ishisaki A., Fujimura A. and Miura H.
    • Journal Title

      Exp. Ther. Med.

      Volume: 10 Pages: 1380-1386

    • NAID

      120006334507

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)2015

    • Author(s)
      Ohtsuka M., Miura H., Mochida K., Hirose M., Hasegawa A., Ogura A., Mizutani R., Kimura M., Isotani A., Ikawa M., Sato M. and Gurumurthy C. B.
    • Journal Title

      BMC Genomics

      Volume: 16 Issue: 1 Pages: 274-274

    • DOI

      10.1186/s12864-015-1432-5

    • NAID

      120006957412

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Assessment of artificial miRNA architectures for higher knockdown efficiencies without the undesired effects in mice2015

    • Author(s)
      Miura H., Inoko H., Tanaka M., Nakaoka H., Kimura M., Gurumurthy C. B., Sato M. and Ohtsuka M.
    • Journal Title

      PLoS ONE

      Volume: 10 Issue: 8 Pages: e0135919-e0135919

    • DOI

      10.1371/journal.pone.0135919

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
  • [Journal Article] Insertion of sequences at the original provirus integration site of mouse ROSA26 locus using the CRISPR/Cas9 system2015

    • Author(s)
      Quadros R., Harms D., Ohtsuka M. and Gurumurthy C. B.
    • Journal Title

      FEBS Open Bio

      Volume: 5 Issue: 1 Pages: 191-197

    • DOI

      10.1016/j.fob.2015.03.003

    • Related Report
      2014 Annual Research Report
    • Peer Reviewed / Open Access / Acknowledgement Compliant
  • [Journal Article] トランスジェニックマウスの基礎と応用2014

    • Author(s)
      大塚正人、佐藤正宏
    • Journal Title

      小児外科

      Volume: 46 Pages: 571-574

    • Related Report
      2014 Annual Research Report
  • [Journal Article] Mouse Genome Editing Using the CRISPR/Cas System2014

    • Author(s)
      Harms DW.*, Quadros RM., Seruggia D., Ohtsuka M., Takahashi G., Montoliu L., and Gurumurthy CB
    • Journal Title

      Curr Protoc Hum Genet

      Volume: 83 Issue: 1

    • DOI

      10.1002/0471142905.hg1507s83

    • Related Report
      2014 Annual Research Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Journal Article] PiggyBac-mediated generation of stable transfectants with surface HLA expression from a small number of cells.2013

    • Author(s)
      Miura H., Inoko H., Inoue I., Okada Y., Tanaka M., Sato M. and Ohtsuka M.
    • Journal Title

      Anal. Biochem

      Volume: 437 Issue: 1 Pages: 29-31

    • DOI

      10.1016/j.ab.2013.02.003

    • Related Report
      2013 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Improvement of pronuclear injection-based targeted transgenesis (PITT) by iCre mRNA-mediated site-specific recombination2013

    • Author(s)
      Ohtsuka M, Miura H, Hayashi H, Nakaoka H, Kimura M, Sato M, Gurumurthy CB, Inoko H
    • Journal Title

      Transgenic Research

      Volume: 22 Issue: 4 Pages: 873-875

    • DOI

      10.1007/s11248-013-9703-x

    • Related Report
      2013 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Site-targeted non-viral gene delivery by direct DNA injection into the pancreatic parenchyma and subsequent in vivo electroporation in mice.2013

    • Author(s)
      Sato M, Inada E, Saitoh I, Ohtsuka M, Nakamura S, Sakurai T and Watanabe S
    • Journal Title

      Biotechnology Journal

      Volume: 8 Issue: 11 Pages: 1355-1361

    • DOI

      10.1002/biot.201300169

    • Related Report
      2013 Annual Research Report
    • Peer Reviewed
  • [Presentation] GONAD and Easy (Isi)-CRISPR: novel mouse genome engineering tools2016

    • Author(s)
      Masato Ohtsuka
    • Organizer
      13th Transgenic Technology Meeting
    • Place of Presentation
      Prague, Czech Republic
    • Year and Date
      2016-03-20
    • Related Report
      2015 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] 顕微注入法を介したターゲットトランスジェネシスによるノックダウンマウス作製2015

    • Author(s)
      三浦浩美、木村穣、大塚正人
    • Organizer
      第 29 回モロシヌス研究会
    • Place of Presentation
      神戸 かんぽの宿 有馬 (兵庫県神戸市)
    • Year and Date
      2015-07-03
    • Related Report
      2015 Annual Research Report
  • [Presentation] マウス受精卵への顕微注入を介したターゲットトランスジェネシス法の改良2015

    • Author(s)
      大塚 正人、三浦 浩美、佐藤 正宏、木村 穣
    • Organizer
      第62回日本実験動物学会総会
    • Place of Presentation
      京都テルサ (京都府京都市)
    • Year and Date
      2015-05-28
    • Related Report
      2015 Annual Research Report
  • [Presentation] 神経障害標的エステラーゼ遺伝子発現マウスの特性と有機リンの影響2015

    • Author(s)
      本杉奈美、山村勇貴、三浦浩美、大塚正人、 大久保朋一、吉野美千代、田中正史、 畑中朋美、坂部貢、木村穣
    • Organizer
      第62回日本実験動物学会総会
    • Place of Presentation
      京都テルサ (京都府京都市)
    • Year and Date
      2015-05-28
    • Related Report
      2015 Annual Research Report
  • [Presentation] 人工 miRNA を用いたマウス遺伝子ノックダウンシステムの検証2015

    • Author(s)
      三浦 浩美、木村 穣、佐藤 正宏、大塚 正人
    • Organizer
      第62回日本実験動物学会総会
    • Place of Presentation
      京都テルサ (京都府京都市)
    • Year and Date
      2015-05-28
    • Related Report
      2015 Annual Research Report
  • [Presentation] Establishment of a co-culture system for simultaneous proliferation of td-Tomato-expressing mouse bone marrow-derived mesenchymal stem cells and undifferentiated blood cells2014

    • Author(s)
      滝沢 尚希、客本 斉子、大久保 直登、帖佐 直幸、衣斐 美歩、藤原 英明、加茂 政晴、大塚 正人、八重柏 隆、石崎 明
    • Organizer
      第37回日本分子生物学会年会
    • Place of Presentation
      パシフィコ横浜
    • Year and Date
      2014-11-25 – 2014-11-27
    • Related Report
      2014 Annual Research Report
  • [Presentation] オートファジー動態の可視化を目的とした新規トランスジェニックマウスの作出2014

    • Author(s)
      秦野 伸二、王 、田中 政之、林 英樹、荻原 早苗、岡田 千沙、伊藤 誠敏、福西 菜穂子、飯田 裕美、中村 彩花、大友 麻子、大塚 正人
    • Organizer
      第37回日本分子生物学会年会
    • Place of Presentation
      パシフィコ横浜
    • Year and Date
      2014-11-25 – 2014-11-27
    • Related Report
      2014 Annual Research Report
  • [Presentation] 薬剤副作用の発症機序解明の為の、HLA遺伝子発現細胞とHLAトランスジェニックマウスの作製2014

    • Author(s)
      三浦 浩美、大塚 正人
    • Organizer
      第37回日本分子生物学会年会
    • Place of Presentation
      パシフィコ横浜
    • Year and Date
      2014-11-25 – 2014-11-27
    • Related Report
      2014 Annual Research Report
  • [Presentation] NTE(神経障害標的エステラーゼ)遺伝子発現マウスと有機リン代謝2014

    • Author(s)
      山村 勇貴、本杉 奈美、三浦 浩美、大塚 正人、大久保 朋一、吉野 美千代、笹川 昇、坂部 貢、木村 穣
    • Organizer
      第37回日本分子生物学会年会
    • Place of Presentation
      パシフィコ横浜
    • Year and Date
      2014-11-25 – 2014-11-27
    • Related Report
      2014 Annual Research Report
  • [Presentation] Rosa26遺伝子座位における組織特異的プロモーター活性の検証2014

    • Author(s)
      田村 沙亜希、大塚 正人、三浦 浩美、松阪 泰二、安岡 有紀子
    • Organizer
      第37回日本分子生物学会年会
    • Place of Presentation
      パシフィコ横浜
    • Year and Date
      2014-11-25 – 2014-11-27
    • Related Report
      2014 Annual Research Report
  • [Presentation] Concurrent production of multiple targeted transgenic mouse lines with C57BL/6N genetic background by improved PITT2014

    • Author(s)
      Masato Ohtsuka, Hiromi Miura, Minoru Kimura, Ayako Isotani, Masahito Ikawa, Masahiro Sato, Channabasavaiah Gurumurthy
    • Organizer
      12th Transgenic Technology Meeting (TT2014)
    • Place of Presentation
      Edinburgh, Scotland, United Kingdom
    • Year and Date
      2014-10-06 – 2014-10-08
    • Related Report
      2014 Annual Research Report
  • [Presentation] RepLiCre: Reporter-linked-Cre transgenic mice for efficient use of conditional knockout mouse resources2014

    • Author(s)
      Channabasavaiah Gurumurthy, Masato Ohtsuka, Rolen Quadros, Donald Harms, Andras Nagy, Kent Lloyd
    • Organizer
      12th Transgenic Technology Meeting (TT2014)
    • Place of Presentation
      Edinburgh, Scotland, United Kingdom
    • Year and Date
      2014-10-06 – 2014-10-08
    • Related Report
      2014 Annual Research Report
  • [Presentation] Targeted insertion of short sequences in the mouse ROSA 26 locus using CRISPR/Cas9 system2014

    • Author(s)
      Channabasavaiah Gurumurthy, Rolen Quadros, Donald Harms, Masato Ohtsuka
    • Organizer
      12th Transgenic Technology Meeting (TT2014)
    • Place of Presentation
      Edinburgh, Scotland, United Kingdom
    • Year and Date
      2014-10-06 – 2014-10-08
    • Related Report
      2014 Annual Research Report
  • [Presentation] Presence/absence of a marker gene in artificial microrna expression constructs affects knockdown efficiencies in a targeted transgenic system2014

    • Author(s)
      Hiromi Miura, Minoru Kimura, Masahiro Sato, Masato Ohtsuka
    • Organizer
      12th Transgenic Technology Meeting (TT2014)
    • Place of Presentation
      Edinburgh, Scotland, United Kingdom
    • Year and Date
      2014-10-06 – 2014-10-08
    • Related Report
      2014 Annual Research Report
  • [Presentation] tdTomato遺伝子導入マウス唾液腺由来間葉系幹細胞株の樹立2014

    • Author(s)
      古川真司、桑島幸紀、畠山慧、帖佐直幸、佐藤和朗、大塚正人、石崎明、藤村朗、三浦廣行
    • Organizer
      第56回歯科基礎医学会学術大会・総会
    • Place of Presentation
      福岡国際会議場
    • Year and Date
      2014-09-25 – 2014-09-27
    • Related Report
      2014 Annual Research Report
  • [Presentation] マウス骨髄由来異種細胞共培養系の確立2014

    • Author(s)
      滝沢尚希、客本斉子、大久保直登、帖佐直幸、衣斐美歩、加茂政晴、大塚正人、八重柏隆、石崎明
    • Organizer
      第56回歯科基礎医学会学術大会・総会
    • Place of Presentation
      福岡国際会議場
    • Year and Date
      2014-09-25 – 2014-09-27
    • Related Report
      2014 Annual Research Report
  • [Presentation] ファイブロサイトに注目した顎関節領域疾患発症機構の解明2014

    • Author(s)
      衣斐美歩、堀江沙和、帖佐直幸、吉田茉莉子、加茂政晴、客本斉子、大塚正人、佐原資謹、藤村朗、石崎明
    • Organizer
      第56回歯科基礎医学会学術大会・総会
    • Place of Presentation
      福岡国際会議場
    • Year and Date
      2014-09-25 – 2014-09-27
    • Related Report
      2014 Annual Research Report
  • [Presentation] マウス受精卵への顕微注入法を介したRosa26 遺伝子座位へのターゲットトランスジェネシス2014

    • Author(s)
      大塚正人
    • Organizer
      第28回モロシヌス研究会
    • Place of Presentation
      修善寺総合会館
    • Year and Date
      2014-06-27 – 2014-06-28
    • Related Report
      2014 Annual Research Report
  • [Presentation] Neuropathy target esterase(神経障害標的エステラーゼ)遺伝子導入マウスの特徴2014

    • Author(s)
      木村 穣、加賀谷 徹、本杉奈美、割田貴之、三浦浩美、大塚正人、大久保朋一、坂部 貢
    • Organizer
      第23回日本臨床環境医学会学術集会
    • Place of Presentation
      京都大学百周年時計台記念館国際交流ホール
    • Year and Date
      2014-06-14 – 2014-06-15
    • Related Report
      2014 Annual Research Report
  • [Presentation] C57BL/6Nを遺伝的背景に持つターゲットトランスジェネシス用種マウスの作製と評価2014

    • Author(s)
      大塚 正人, 三浦 浩美, 磯谷 綾子, 伊川 正人, 佐藤 正宏, 木村 穣
    • Organizer
      第61回日本実験動物学会総会
    • Place of Presentation
      札幌コンベンションセンター
    • Year and Date
      2014-05-15 – 2014-05-17
    • Related Report
      2014 Annual Research Report
  • [Presentation] 受精卵へのiCre mRNA注入によるターゲットトランスジェネシス法(PITT法)の効率改善2013

    • Author(s)
      大塚正人、三浦浩美、佐藤正宏、木村穣
    • Organizer
      第60回日本実験動物学会総会
    • Place of Presentation
      つくば国際会議場
    • Related Report
      2013 Annual Research Report
  • [Presentation] Neuropathy target esterase(神経障害標的エステラーゼ)遺伝子導入マウスの作成2013

    • Author(s)
      加賀谷徹、割田貴之、三浦浩美、大塚正人、大久保朋一、坂部貢、木村穣
    • Organizer
      第22回日本臨床環境医学会学術集会
    • Place of Presentation
      北里大学白金キャンパス
    • Related Report
      2013 Annual Research Report
  • [Presentation] トランスポゾンPiggyBacシステムによる複数遺伝子のブタ細胞への同時導入2013

    • Author(s)
      郡山美優、稲田絵美、齋藤一誠、三浦浩美、大塚正人、中村伸吾、桜井敬之、渡部聡、三好和睦、佐藤正宏
    • Organizer
      第106回日本繁殖生物学会
    • Place of Presentation
      東京農工大学農学部府中キャンパス
    • Related Report
      2013 Annual Research Report
  • [Presentation] Generation of Knockdown Mice Using Pronuclear Injection-Based Targeted Transgenesis: Advantages and Limitations2013

    • Author(s)
      Hiromi Miura, Masato Ohtsuka
    • Organizer
      The 27th International Mammalian Genome Conference
    • Place of Presentation
      Salamanca, Spain
    • Related Report
      2013 Annual Research Report
  • [Presentation] 赤色蛍光強発現 tg マウス唾液腺細胞の蛍光発現に関する研究2013

    • Author(s)
      古川真司、畠山慧、堀智樹、石崎明、大塚正人、藤村朗、金野吉晃、清野幸男、三浦廣行
    • Organizer
      第55回歯科基礎医学会学術大会・総会
    • Place of Presentation
      岡山コンベンションセンター
    • Related Report
      2013 Annual Research Report
  • [Presentation] 赤色蛍光強発現tgマウスの唾液腺細胞の蛍光発現 第2報2013

    • Author(s)
      古川真司、畠山慧、木村仁迪、青松恵美子、石崎明、大塚正人、藤村朗、金野吉晃、清野幸男、三浦廣行
    • Organizer
      第72回日本矯正歯科学会大会
    • Place of Presentation
      キッセイ文化ホール(長野県松本文化会館)
    • Related Report
      2013 Annual Research Report
  • [Book] 遺伝子改変法①部位特異的組換え酵素システム.ES iPS細胞実験スタンダード2014

    • Author(s)
      大塚正人、角田茂
    • Publisher
      羊土社
    • Related Report
      2013 Annual Research Report
  • [Book] Development and recent progress in the pronuclear injection-based targeted transgenesis method by site-specific recombination in mice. in Proceedings of the Genetic Transformation Technologies (plants and animals)2013

    • Author(s)
      Ohtsuka M.
    • Publisher
      MEDIMOND
    • Related Report
      2013 Annual Research Report
  • [Remarks]

    • URL

      http://masato2.wix.com/ohtsuka-lab

    • Related Report
      2015 Annual Research Report

URL: 

Published: 2013-05-21   Modified: 2022-01-27  

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