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Advancement of revolutional cell technology based on the IR/MAR gene amplification method

Research Project

Project/Area Number 23380203
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Applied molecular and cellular biology
Research InstitutionHiroshima University

Principal Investigator

SHIMIZU Noriaki  広島大学, 生物圏科学研究科, 教授 (10216096)

Project Period (FY) 2011-04-01 – 2015-03-31
Project Status Completed (Fiscal Year 2014)
Budget Amount *help
¥19,760,000 (Direct Cost: ¥15,200,000、Indirect Cost: ¥4,560,000)
Fiscal Year 2014: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000)
Fiscal Year 2013: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000)
Fiscal Year 2012: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000)
Fiscal Year 2011: ¥5,330,000 (Direct Cost: ¥4,100,000、Indirect Cost: ¥1,230,000)
Keywords遺伝子増幅 / 細胞工学技術 / 組換え蛋白質生産 / 染色体外遺伝因子 / Double minutes / 細胞癌化 / IR/MAR遺伝子増幅法 / 染色体外因子 / Double Minutes / HSR / 蛋白質生産 / エピソームベクター / 組換え蛋白質発現 / エピソーム / 微小核 / 細胞工学 / 染色体工学 / 遺伝子発現 / Homogeneously Staining Region
Outline of Final Research Achievements

We previously found that a plasmid bearing mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) was amplified spontaneously in animal cells and generate extrachromosomal DMs or chromosomal HSR. This study aimed to develop this method to a novel cell technology. Consequently, 1) we identified minimal required sequence for gene amplification, and clarified the relationship to DNA replication. 2) We clarified the mechanism of gene amplification. 3) We isolated a human genome sequence that enhance the gene expression from tandemly repeated sequence.4) We showed that the pulverization of chromosome de novo generates DMs that had amplified oncogenes. 5) We obtained the understanding about the intracellular maintenance and extracellular elimination of DMs.

Report

(5 results)
  • 2014 Annual Research Report   Final Research Report ( PDF )
  • 2013 Annual Research Report
  • 2012 Annual Research Report
  • 2011 Annual Research Report
  • Research Products

    (34 results)

All 2015 2014 2013 2012 2011 Other

All Journal Article (9 results) (of which Peer Reviewed: 9 results,  Open Access: 1 results,  Acknowledgement Compliant: 1 results) Presentation (24 results) (of which Invited: 2 results) Patent(Industrial Property Rights) (1 results)

  • [Journal Article] How a Replication Origin and Matrix Attachment Region Accelerate Gene Amplification under Replication Stress in Mammalian Cells.2014

    • Author(s)
      Shun-suke Tanaka, Sho-hei Mitsuda, Noriaki Shimizu
    • Journal Title

      PLos ONE

      Volume: 9 Issue: 7 Pages: e103439-e103439

    • DOI

      10.1371/journal.pone.0103439

    • Related Report
      2014 Annual Research Report
    • Peer Reviewed / Open Access / Acknowledgement Compliant
  • [Journal Article] Dissection of the Beta-Globin Replication-Initiation Region Reveals Specific Requirements for Replicator Elements during Gene Amplification.2013

    • Author(s)
      Naoya Okada, Noriaki Shimizu
    • Journal Title

      PLos ONE

      Volume: Volume 8, Issue 10 Issue: 10 Pages: e77350-e77350

    • DOI

      10.1371/journal.pone.0077350

    • Related Report
      2013 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Efficient Recombinant Production in Mammalian Cells Using a Novel IR/MAR Gene Amplification Method2012

    • Author(s)
      Yoshio Araki, Tetsuro Hamafuji, Chiemi Noguchi and Nor iaki Shimizu
    • Journal Title

      PLos ONE

      Volume: olume 7, Issue 7 Issue: 7 Pages: e41787-e41787

    • DOI

      10.1371/journal.pone.0041787

    • URL

      http://www.plosone.org/article/metrics/info%3Adoi%2F10.1371%2Fjournal.pone.0041787

    • Related Report
      2012 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Fusion of the Dhfr/Mtx and IR/MAR gene amplification methods produces a rapid and efficient method for stable recombinant protein production2012

    • Author(s)
      Chiemi Noguchi, Yoshio Araki, Daisuke Miki, Nor iaki Shimizu
    • Journal Title

      PLos ONE

      Volume: Volume 7, Issue 12 Issue: 12 Pages: e52990-e52990

    • DOI

      10.1371/journal.pone.0052990

    • URL

      http://www.plosone.org/article/metrics/info%3Adoi%2F10.1371%2Fjournal.pone.0052990

    • Related Report
      2012 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Amplification of a plasmid bearing a mammalian replication initiation region in chromosomal and extrachromosomal contexts2011

    • Author(s)
      S.Harada, et al.
    • Journal Title

      Nucleic Acids Research

      Volume: 39 Issue: 3 Pages: 958-969

    • DOI

      10.1093/nar/gkq882

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] High-level expression of human recombinant cyclooxygenase-1 in mammalian cells using a novel gene amplification method2011

    • Author(s)
      H.Yoshimura, et al.
    • Journal Title

      Protein Expression and Purification

      Volume: 80 Issue: 1 Pages: 41-46

    • DOI

      10.1016/j.pep.2011.05.009

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Generation of Micronuclei During Interphase by Coupling between Cytoplasmic Membrane Blebbing and Nuclear Budding2011

    • Author(s)
      K.Utani, et al.
    • Journal Title

      PLosONE

      Volume: 6 Issue: 11 Pages: e27233-e27233

    • DOI

      10.1371/journal.pone.0027233

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] DNA replication occurs in all lamina positive micronuclei, but never in lamina negative micronuclei2011

    • Author(s)
      A.Okamoto, et al.
    • Journal Title

      Mutagenesis

      Volume: (電子版公開) Issue: 3 Pages: 323-327

    • DOI

      10.1093/mutage/ger082

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Molecular mechanisms of the origin of micronuclei from extrachromosomal elements2011

    • Author(s)
      N.Shimizu
    • Journal Title

      Mutagenesis

      Volume: 26 Issue: 1 Pages: 119-123

    • DOI

      10.1093/mutage/geq053

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Presentation] 染色体外遺伝因子と染色体との間で、 _同じ反復配列のエピジェネティック状態はどのように異なるのか?2014

    • Author(s)
      満田 祥平,清水 典明
    • Organizer
      第36回日本分子生物学会
    • Related Report
      2014 Annual Research Report
  • [Presentation] 複製開始配列の逆位反復配列を用いることによる、動物細胞内で安定なエピソームの形成2014

    • Author(s)
      大崎 究, 清水 典明
    • Organizer
      第36回日本分子生物学会
    • Related Report
      2014 Annual Research Report
  • [Presentation] サイレンシングを受けやすい反復配列からの遺伝子発現を高めるヒトゲノム配列の単離と解析2014

    • Author(s)
      福間 美樹,元明 優人,大懸 俊廣,三浦 理,大山 隆,清水 典明
    • Organizer
      第36回日本分子生物学会
    • Related Report
      2014 Annual Research Report
  • [Presentation] ヒト染色体に由来する自律複製する染色体外遺伝因子の、細胞内維持、排出と細胞間伝播2014

    • Author(s)
      坂丸 直人,浪花 修平,Rita Kapoor,宇谷 公一,清水 典明
    • Organizer
      第36回日本分子生物学会
    • Related Report
      2014 Annual Research Report
  • [Presentation] 染色体外での遺伝子増幅とゲノム不安定性に与える低酸素環境の影響2014

    • Author(s)
      山田 拓,小塩 結里恵,清水 典明
    • Organizer
      第36回日本分子生物学会
    • Related Report
      2014 Annual Research Report
  • [Presentation] IR/MAR遺伝子増幅法と、蛋白質生産への応用2013

    • Author(s)
      清水典明
    • Organizer
      日本動物細胞工学会 2013年大会 シンポジウム4「日本発、世界を目指すバイオ医薬生産」
    • Place of Presentation
      福井市
    • Related Report
      2013 Annual Research Report
    • Invited
  • [Presentation] 哺乳動物細胞内で、遺伝子増幅により形成された反復配列からの発現を高めるヒトゲノム配列の、単離と解析2013

    • Author(s)
      元明優人,清水典明
    • Organizer
      第36回日本分子生物学会
    • Place of Presentation
      神戸ポートアイランド
    • Related Report
      2013 Annual Research Report
  • [Presentation] 複製ストレスにより誘導される遺伝子数増減の分子機構と、IR/MAR 配列の影響2013

    • Author(s)
      田中俊介,満田祥平,清水典明
    • Organizer
      第36回日本分子生物学会
    • Place of Presentation
      神戸ポートアイランド
    • Related Report
      2013 Annual Research Report
  • [Presentation] 染色体と染色体外遺伝因子の間で、クロマチンのエピジェネティック修飾状態はどのように異なるか?2013

    • Author(s)
      満田祥平,清水典明
    • Organizer
      第36回日本分子生物学会
    • Place of Presentation
      神戸ポートアイランド
    • Related Report
      2013 Annual Research Report
  • [Presentation] ヒト染色体断片化による、自律複製する安定な染色体外遺伝因子の新生2013

    • Author(s)
      坂丸 直人,浪花 修平,カポール リタ,宇谷 公一,清水 典明
    • Organizer
      第36回日本分子生物学会
    • Place of Presentation
      神戸ポートアイランド
    • Related Report
      2013 Annual Research Report
  • [Presentation] ヒト複製開始配列に含まれる、遺伝子増幅を指示する配列の分子解剖2012

    • Author(s)
      岡田直也、清水典明
    • Organizer
      第35回日本分子生物学会
    • Place of Presentation
      マリンメッセ福岡
    • Year and Date
      2012-12-11
    • Related Report
      2012 Annual Research Report
  • [Presentation] 哺乳動物細胞内で、遺伝子増幅により形成された反復配列からの発現を高めるヒトゲノム配列の、単離と解析2012

    • Author(s)
      元明優人、清水典明
    • Organizer
      第35回日本分子生物学会
    • Place of Presentation
      マリンメッセ福岡
    • Year and Date
      2012-12-11
    • Related Report
      2012 Annual Research Report
  • [Presentation] IR/MAR-Dhfr融合遺伝子増幅法・・・安定な高発現細胞を早く生じる新規方法による、遺伝子増幅の機構2012

    • Author(s)
      田中俊介、野口千笑、荒木義雄、清水典明
    • Organizer
      第35回日本分子生物学会
    • Place of Presentation
      マリンメッセ福岡
    • Year and Date
      2012-12-11
    • Related Report
      2012 Annual Research Report
  • [Presentation] 染色体外遺伝因子の細胞内安定性を支配する遺伝的背景と、染色体断片化による新規染色体外遺伝因子の形成2012

    • Author(s)
      浪花修平、カポール・リタ、宇谷公一、清水典明
    • Organizer
      第35回日本分子生物学会
    • Place of Presentation
      マリンメッセ福岡
    • Year and Date
      2012-12-11
    • Related Report
      2012 Annual Research Report
  • [Presentation] Recombinant protein production in CHO cells using the IR/MAR-amplification based technology2012

    • Author(s)
      Noriaki Shimizu, Yoshio Araki, Chiemi Noguchi and Tetsuro Hamafuji
    • Organizer
      The 25th Annual and International Meeting of the Japanese Association for Animal Cell Technology (JAACT 2012)
    • Place of Presentation
      Nagoya Congress Center, Nagoya, Japan
    • Year and Date
      2012-11-27
    • Related Report
      2012 Annual Research Report
  • [Presentation] ヒト細胞で悪性形質を運搬する染色体外遺伝因子を、核内から細胞質へ、さらに細胞外へと排出する機構…細胞質膜ブレッビングの関与2011

    • Author(s)
      岡本敦志、宇谷公一、清水典明
    • Organizer
      第34回日本分子生物学会年会
    • Place of Presentation
      横浜
    • Year and Date
      2011-12-15
    • Related Report
      2011 Annual Research Report
  • [Presentation] ヒト複製開始領域の遺伝子増幅能を指標とした分子解剖2011

    • Author(s)
      岡田直也、荒木義雄、清水典明
    • Organizer
      第34回日本分子生物学会年会
    • Place of Presentation
      横浜
    • Year and Date
      2011-12-15
    • Related Report
      2011 Annual Research Report
  • [Presentation] IR/MAR遺伝子増幅法を核とした動物細胞での抗体等有用蛋白質の高効率発現系2011

    • Author(s)
      荒木義雄、野口千笑、濱藤徹郎、清水典明
    • Organizer
      第34回日本分子生物学会年会
    • Place of Presentation
      横浜
    • Year and Date
      2011-12-13
    • Related Report
      2011 Annual Research Report
  • [Presentation] 染色体外遺伝因子の細胞内安定性を支配する遺伝的背景と、染色体断片化による新規染色体外遺伝因子の形成2011

    • Author(s)
      浪花修平、Kapoor Rita、宇谷公一、清水典明
    • Organizer
      第34回日本分子生物学会年会
    • Place of Presentation
      横浜
    • Year and Date
      2011-12-05
    • Related Report
      2011 Annual Research Report
  • [Presentation] 効率的なタンパク生産技術の可能性2011

    • Author(s)
      清水典明
    • Organizer
      第11回事業創出サロン((合)SARR主催)
    • Place of Presentation
      京都(招待講演)
    • Year and Date
      2011-11-18
    • Related Report
      2011 Annual Research Report
  • [Presentation] Novel, Convenient and Efficient "IR/MAR Gene Amplification Method" for the Production of Recombinant Protein in Animal Cells2011

    • Author(s)
      Noriaki Shimizu
    • Organizer
      10th PEACe (Protein Expression in Animal Cell) conference
    • Place of Presentation
      Cascais, Portugal(oral presentation ; 30 min)(招待講演)
    • Year and Date
      2011-09-25
    • Related Report
      2011 Annual Research Report
  • [Presentation] IR/MAR遺伝子増幅法を核とした、動物細胞での抗体等有用蛋白質の高効率発現系2011

    • Author(s)
      荒木義雄、野口千笑、濱藤徹郎、能勢博、三木大輔、清水典明
    • Organizer
      第11回日本蛋白質科学会年会
    • Place of Presentation
      大阪
    • Year and Date
      2011-06-07
    • Related Report
      2011 Annual Research Report
  • [Presentation] Application of the Novel and Convenient IR/MAR Gene Amplification Technology to the Production of Recombinant Protein Pharmaceuticals2011

    • Author(s)
      Yoshio Araki, Chiemi, Noguchi, Tetsuro Hamafuji, Hiroshi Nose, Daisuke Miki, Noriaki Shimizu
    • Organizer
      22nd ESACT (European Society for Animal Cell Technology) meeting
    • Place of Presentation
      Vienna, Austria
    • Year and Date
      2011-05-16
    • Related Report
      2011 Annual Research Report
  • [Presentation] 動物細胞内で組み換え遺伝子を高度に増幅させるIR/MAR(アイ・アール/マー)法と、蛋白質生産

    • Author(s)
      清水典明
    • Organizer
      第86回日本生化学会大会 Bio-industry Seminar
    • Place of Presentation
      パシフィコ横浜
    • Related Report
      2013 Annual Research Report
    • Invited
  • [Patent(Industrial Property Rights)] 哺乳動物細胞内で増幅された目的遺伝子の発現を高めるポリヌクレオチド2015

    • Inventor(s)
      清水典明
    • Industrial Property Rights Holder
      清水典明
    • Industrial Property Rights Type
      特許
    • Industrial Property Number
      2015-003690
    • Filing Date
      2015-01-09
    • Related Report
      2014 Annual Research Report

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Published: 2011-04-06   Modified: 2021-01-27  

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