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Basic Research on Effectiveness of Vibratory Microinjection Method

Research Project

Project/Area Number 16500281
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Laboratory animal science
Research InstitutionTokyo Denki University

Principal Investigator

MIYAWAKI Fujio  Tokyo Denki University, School of Science and Engineering, Professor, 理工学部, 教授 (50174222)

Co-Investigator(Kenkyū-buntansha) KOBAYASHI Kenji  Takusyoku University, Faculty of Engineering, Professor, 工学部, 教授 (40016770)
HASEGAWA Jun  Takusyoku University, Faculty of Engineering, Associate Professor, 工学部, 助教授 (30228449)
糸原 重美  独立行政法人理化学研究所, 行動遺伝学技術開発チーム, チームリーダー (60252524)
Project Period (FY) 2004 – 2006
Project Status Completed (Fiscal Year 2006)
Budget Amount *help
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2006: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2005: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2004: ¥1,100,000 (Direct Cost: ¥1,100,000)
Keywordsvibratory microinjection / longitudinal vibration / gene transfer / pronuclear injection / fertilized egg / blastocyst / ultrasonic vibration / transgenic animal / 振動型マイクロインジェクション法 / 超音波領域振動型 / 安全性評価 / マイクロピペット / 胚盤胞期 / 発生効率 / 死亡率 / 核DNAひきずり出し現象 / 周波数 / 振幅 / 受精卵刺入時変形率 / 核DNA引きずり出し / 内部細胞塊 / venus / 卵細胞培養
Research Abstract

To increase the production rate of transgenic animals, we developed Vibratory Microinjection System (VMS). This system was aimed at facilitating gene transfer by vibrating a micropipette longitudinally during insertion into cells.
1.The VMS providing any frequency less than 18 kHz was evaluated. The vibratory microinjection (VM) at several frequencies was compared with the ordinary (non-vibratory) microinjection (OM). Fertilized eggs obtained from BDF-1 mice were equally allotted to VM and OM groups. The eggs in each group were divided into some sets of 30 eggs. The 30 eggs in each set were injected with Venus genes using a single micropipette.
(1)Compared with OM, VM enabled micropipettes to penetrate zona pellucida with significantly less cellular deformation rates, reducing them by about 20%. (2)After four-day culture of eggs having survived the injection, the VM group showed 2 to 3 times higher rates of development to the blastocyst stage than the OM group did, although it did slightly higher death rates (NS). (3)‘Pulling-out' event, i.e., an event that a micropipette pulled out a part of nuclear DNA before finishing the given number of injections, took place significantly less frequently in VM group than in the OM group. The pulling-out event usually necessitates replacement of the micropipette with a new one.
2.We developed ultrasonic VMS (UVMS) with upper limit of frequency of 100kHz. We conducted safety evaluation of UVMS, and confirmed no cellular damage caused by ultrasonic vibration : extreme change in intracellular pressure, cavitation or fragmentation of DNA.
In conclusion, VMS achieved zygote injection with significantly less cellular deformation and also resulted in significantly better embryonic development, suggesting improvement in production of transgenic animals. VMS can also cut the cost of micropipettes and save the trouble of frequent replacement of micropipettes.

Report

(4 results)
  • 2006 Annual Research Report   Final Research Report Summary
  • 2005 Annual Research Report
  • 2004 Annual Research Report
  • Research Products

    (14 results)

All 2006 2005

All Journal Article (11 results) Patent(Industrial Property Rights) (3 results)

  • [Journal Article] Vibratory Microinjection on Mouse Zygotes Improved Subsequent In vitro Embryonic Development2006

    • Author(s)
      Miyawaki F et al.
    • Journal Title

      The international Journal of Artificial Organs 29(5)

      Pages: 510-510

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Development of Vibratory Microinjection System to increase production rate of transgenic animals2006

    • Author(s)
      Miyawaki F et al.
    • Journal Title

      Reproduction, Fertility and Development 18(1,2)

      Pages: 296-296

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Vibratory Microinjection on Mouse Zygotes Improved Subsequent In Vitro Embryonic Development2006

    • Author(s)
      Miyawaki F, Hasegawa J, Kikuchi T, Okada R, Saito 4, Suzuki H, Ippongi T, Kobayashi K, Itohara S
    • Journal Title

      The international Journal of Artificial Organs 29(5)

      Pages: 510-510

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Development of Vibratory Microinjection System to increase production rate of transgenic animals2006

    • Author(s)
      Miyawaki F, Hasegawa J, Kikuchi T, Tsuchiya M, Saito Y, Suzuki H, Ippongi T, Kobayashi K, Itohara S
    • Journal Title

      Reproduction, Fertility and Development 18(1,2)

      Pages: 296-296

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Development of Vibratory Microinjection System to increas production rate of transgenic animals2006

    • Author(s)
      Miyawaki F et al.
    • Journal Title

      Reproduction, Fertility and Development 18

      Pages: 296-296

    • Related Report
      2005 Annual Research Report
  • [Journal Article] マウス受精卵への振動型マイクロインジェクションの適用とその後の胚への影響2005

    • Author(s)
      菊池智行 他
    • Journal Title

      第21回ライフサポート学会大会 講演予稿集

      Pages: 104-104

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Effects of Vibratory Microinjection on Mouse Zygotes and Subsequent In vitro Development.2005

    • Author(s)
      Miyawaki F et al.
    • Journal Title

      6th EMBL Mouse Molecular Genetics Meeting

      Pages: 95-95

    • NAID

      130001056295

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Effects of Vibratory Microinjection on Mouse Zygotes and Subsequent In vitro Development2005

    • Author(s)
      Kikuchi T et al.
    • Journal Title

      The Society of Life Support Technology Suppl 104

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Effects of Vibratory Microinjection on Mouse Zygotes and Subsequent In vitro Development2005

    • Author(s)
      Miyawaki F et al.
    • Journal Title

      6th EMBL Mouse Molecular Genetics Meeting 95

    • NAID

      130001056295

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Effects of Vibratory Microinjection on Mouse Zygotes and Subsequent In vitro Development2005

    • Author(s)
      Miyawaki F et al.
    • Journal Title

      6th EMBL Mouse Molecular Genetics Meeting

      Pages: 95-95

    • NAID

      130001056295

    • Related Report
      2005 Annual Research Report
  • [Journal Article] マウス受精卵への振動型マイクロインジェクションの適用とその後の胚への影響2005

    • Author(s)
      菊池智行, 宮脇富士夫 他
    • Journal Title

      第21回ライフサポート学会大会講演予稿集

      Pages: 104-104

    • Related Report
      2005 Annual Research Report
  • [Patent(Industrial Property Rights)] Vibration Type Micro Injection Device2006

    • Inventor(s)
      Fujio Miyawaki, Kenji Kobayashi, Jun Hasegawa
    • Industrial Property Rights Holder
      Fujio Miyawaki, Kenji Kobayashi, Jun Hasegawa
    • Filing Date
      2006-06-16
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Patent(Industrial Property Rights)] 振動型マイクロインジェクション装置2005

    • Inventor(s)
      宮脇富士夫, 小林健二, 長谷川淳
    • Industrial Property Rights Holder
      宮脇富士夫, 小林健二, 長谷川淳
    • Industrial Property Number
      2005-191613
    • Filing Date
      2005-06-30
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Patent(Industrial Property Rights)] 振動型マイクロインジェクション装置2005

    • Inventor(s)
      宮脇 富士夫, 小林 健二, 長谷川 淳
    • Industrial Property Rights Holder
      宮脇 富士夫, 小林 健二, 長谷川 淳
    • Industrial Property Number
      2005-191613
    • Filing Date
      2005-06-30
    • Related Report
      2005 Annual Research Report

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Published: 2004-04-01   Modified: 2016-04-21  

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