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The analysis of the function of long non-coding RNA by reporter gene knock-in via improved CRISPR/Cas9

Research Project

Project/Area Number 17K17773
Research Category

Grant-in-Aid for Young Scientists (B)

Allocation TypeMulti-year Fund
Research Field Laboratory animal science
System genome science
Research InstitutionUniversity of Yamanashi

Principal Investigator

TAIMATSU Kiyohito  山梨大学, 大学院総合研究部, 医学研究員 (10755482)

Research Collaborator KAWAHARA Atsuo  
Project Period (FY) 2017-04-01 – 2019-03-31
Project Status Completed (Fiscal Year 2018)
Budget Amount *help
¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000)
Fiscal Year 2018: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2017: ¥2,990,000 (Direct Cost: ¥2,300,000、Indirect Cost: ¥690,000)
Keywordsゼブラフィッシュ / ゲノム編集 / ノックイン / 長鎖非翻訳RNA / lncRNA / CRISPR/Cas9 / 発生遺伝学 / zebrafish / genome editing / mutant / 動物実験学 / ゲノム生物工学
Outline of Final Research Achievements

To analyze long non-coding RNA (lncRNA) in zebrafish, I chose target genes to generate mutants by checking the genomic location in lncRNA database. I detected target gene’s expression by whole-mount in situ hybridization, and picked up 50 genes which expressed in circulatory system. I performed knock-in of a reporter gene in target loci to generate mutants and visualize target gene’s expression.
Unfortunately, I failed to generate the stable knock-in lines, but I achieved to generate mutant lines. Two lines showed the abnormality in sex determination. Now I am writing the paper to report the phenotype of mutants and the function of target genes. This study will shed light on the function of lncRNA in sex determination. On the other hand, I published the paper about knock-in technology developed via this study.

Academic Significance and Societal Importance of the Research Achievements

RNAのうちタンパク質をコードするmRNAは数%に過ぎず、RNAの機能の全容解明のために大部分を占める非翻訳RNAの理解は大変重要である。これまでの変異体作成技術では非翻訳RNAの変異体の単離が困難だった。非翻訳RNAの変異体作製のために開発したゲノム編集技術は、これまでに変異体の作製が困難であった遺伝子の改変を容易にし、今後の遺伝子研究の推進に役立つものと期待される。
本研究で作製された長鎖非翻訳RNAの変異体のうち2系統は性分化の異常を示した。性決定機構は生殖医療の高まりもあり解明が重要視されている。作製中の論文は長鎖非翻訳RNAの性決定機構における機能に光を当てるものである。

Report

(3 results)
  • 2018 Annual Research Report   Final Research Report ( PDF )
  • 2017 Research-status Report
  • Research Products

    (4 results)

All 2017

All Journal Article (1 results) (of which Peer Reviewed: 1 results) Presentation (3 results) (of which Int'l Joint Research: 3 results,  Invited: 1 results)

  • [Journal Article] Exogenous gene integration mediated by genome editing technologies in zebrafish2017

    • Author(s)
      Hitoshi Morita, Kiyohito Taimatsu, Kanoko Yanagi and Atsuo Kawahara
    • Journal Title

      Bioengineered

      Volume: 4 Issue: 3 Pages: 287-295

    • DOI

      10.1080/21655979.2017.1300727

    • Related Report
      2017 Research-status Report
    • Peer Reviewed
  • [Presentation] CRISPR/Cas9-mediated eGFP reporter integration into zebrafish genome for functional visualization and disruption of target genes (oral)2017

    • Author(s)
      Kiyohito Taimatsu, Satoshi Ota, Shin-ichi Higashijima, Atsuo Kawahara
    • Organizer
      JAPANESE SOCIETY of DEVELOPMENTAL BIOLOGISTS Annual Conference
    • Related Report
      2017 Research-status Report
    • Int'l Joint Research
  • [Presentation] Functional visualization and disruption of target genes by CRISPR/Cas9-mediated eGFP reporter integration into zebrafish genome (poster)2017

    • Author(s)
      Kiyohito Taimatsu
    • Organizer
      Zebrafish Disease Models Society Annual Conference
    • Related Report
      2017 Research-status Report
    • Int'l Joint Research
  • [Presentation] CRISPR/Cas9-mediated eGFP reporter integration into zebrafish genome for functional visualization and disruption of target genes (oral)2017

    • Author(s)
      Kiyohito Taimatsu, Satoshi Ota, Shin-ichi Higashijima, Atsuo Kawahara
    • Organizer
      Zebrafish Disease Models Society Annual Conference
    • Related Report
      2017 Research-status Report
    • Int'l Joint Research / Invited

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Published: 2017-04-28   Modified: 2020-03-30  

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