A gene expression control system using a genome exchange technology on a mammalian artificial chromosome vector

• Project/Area Number: 15K12141
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Life / Health / Medical informatics
• Research Institution: Tottori University
• Principal Investigator: Tetsuya Ohbayashi 鳥取大学, 生命機能研究支援センター, 准教授 (80348804)
• Co-Investigator(Kenkyū-buntansha): 古倉 健嗣 鳥取大学, 医学部, 助教 (30344039) ; 中村 和臣 鳥取大学, 生命機能研究支援センター, プロジェクト研究員 (90598137)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000); Fiscal Year 2016: ¥2,600,000 (Direct Cost: ¥2,000,000、Indirect Cost: ¥600,000); Fiscal Year 2015: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
• Keywords: 人工染色体ベクター / 遺伝子工学 / 遺伝子改変技術 / ゲノム改変技術 / ゲノム編集 / インテグレース / システム生物学 / 合成生物学

Outline of Final Research Achievements

Mammalian artificial chromosome (MAC) vectors have several advantages as gene delivery vectors, such as stable and independent maintenance in host cells without integration
Previously, a MAC containing a multi-integrase platform (MI-MAC) was developed to facilitate the transfer of multiple genes into desired cells. To introduce multiple-genes into MI-MAC platform, we developed multiple-gene-loading method by combining multi-integration system-equipped MAC vector and CRISPR-Cas9. Next, we developed multiple expression cassette exchange system via three integrase systems on a MAC vector. We designed three excision cassettes that expressed luciferase, where the luciferase expression could be exchanged to a fluorescent protein by site-specific recombination. The transient expression of an integrase caused the targeted luciferase activity to be lost and fluorescence was activated. Furthermore, we developed in vitro cisplatin nephrotoxicity test using these systems.

Research Products

• [Journal Article] Development of a multiple-gene-loading method by combining multi-integration system-equipped mouse artificial chromosome vector and CRISPR-Cas9 (2018)
  • Author(s): Honma Kazuhisa、Abe Satoshi、Endo Takeshi、Uno Narumi、Oshimura Mitsuo、Ohbayashi Tetsuya、Kazuki Yasuhiro
  • Journal Title: PLOS ONE Volume: 13 Issue: 3 Pages: e0193642-e0193642
  • DOI: 10.1371/journal.pone.0193642
  • Peer Reviewed / Open Access
• [Journal Article] Multiple expression cassette exchange via TP901-1, R4, and Bxb1 integrase systems on a mouse artificial chromosome. (2017)
  • Author(s): Tomimatsu K, Kokura K, Nishida T, Yoshimura Y, Kazuki Y, Narita M, Oshimura M, Ohbayashi T.
  • Journal Title: FEBS Open Bio. Volume: 7 Issue: 3 Pages: 306-317
  • DOI: 10.1002/2211-5463.12169
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Use of a Human Artificial Chromosome for Delivering Trophic Factors in a Rodent Model of Amyotrophic Lateral Sclerosis. (2015)
  • Author(s): Watanabe Y, Kazuki Y, Kazuki K, Ebiki M, Nakanishi M, Nakamura K, Yoshida Yamakawa M, Hosokawa H, Ohbayashi T, Oshimura M, Nakashima K.
  • Journal Title: Mol Ther Nucleic Acids. Volume: 4 Pages: e253-e253
  • DOI: 10.1038/mtna.2015.28
  • Peer Reviewed / Open Access
• [Journal Article] Exploring new gene integration sites for gene knock-in by gene-trapping strategy. (2015)
  • Author(s): Nanchi I, Yoshimura Y, Nakamura K, Masago Y, Ohbayashi T, Okuda T.
  • Journal Title: Transgenic Res Volume: 24(3) Issue: 3 Pages: 549-59
  • DOI: 10.1007/s11248-015-9872-x
  • Peer Reviewed
• [Journal Article] Mouse embryonic stem cells with a multi-integrase mouse artificial chromosome for transchromosomic mouse generation. (2015)
  • Author(s): Yoshimura Y, Nakamura K, Endo T, Kajitani N, Kazuki K, Kazuki Y, Kugoh H, Oshimura M, Ohbayashi T.
  • Journal Title: Transgenic Res. Volume: 24(4) Issue: 4 Pages: 717-727
  • DOI: 10.1007/s11248-015-9884-6
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Phenotype Specific Analyses Reveal Distinct Regulatory Mechanism for Chronically Activated p53. (2015)
  • Author(s): Kirschner K, Samarajiwa SA, Cairns JM, Menon S, Pérez-Mancera PA, Tomimatsu K, Bermejo-Rodriguez C, Ito Y, Chandra T, Narita M, Lyons SK, Lynch AG, Kimura H, Ohbayashi T, Tavaré S, Narita M.
  • Journal Title: PLoS Genet Volume: 11 Issue: 3 Pages: e1005053-e1005053
  • DOI: 10.1371/journal.pgen.1005053
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Exploring the origin and limitations of kidney regeneration. (2015)
  • Author(s): Endo T, Nakamura J, Sato Y, Asada M, Yamada R, Takase M, Takaori K, Oguchi A, Iguchi T, Higashi AY, Ohbayashi T, Nakamura T, Muso E, Kimura T, Yanagita M.
  • Journal Title: J Pathol. Volume: 236(2) Issue: 2 Pages: 251-63
  • DOI: 10.1002/path.4514
  • Peer Reviewed
• [Presentation] ゲノムプログラム&コントロールによる新しい遺伝子改変マウス (2015)
  • Author(s): 大林徹也
  • Organizer: 第5回 合成生物工学シンポジウム
  • Place of Presentation: 神戸大学統合研究拠点コンベンションホール
  • Year and Date: 2015-08-05
  • Invited