Single nucleotide-level genome engineering using transposon cassettes

2013 Fiscal Year Final Research Report

• Project/Area Number: 23710240
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Applied genomics
• Research Institution: Kyoto University
• Principal Investigator: WOLTJEN Knut 京都大学, 白眉センター, 准教授 (50589489)
• Project Period (FY): 2011 – 2012
• Keywords: 遺伝子ターゲティング / ヌクレアーゼ / iPS細胞 / TALEN nuclease / transposon / cardiomyocyte / SNP / ion channel

Research Abstract

In this study, we aimed to combine TALEN (TAL-Effector Nuclease) technology and piggyBac transposon-based selection cassettes to establish novel gene-modification technology in human iPS cells. We succeeded in genetic modification using TALENs, disrupting the HPRT gene and introducing transgene cassettes into the AAVS1 locus in human iPS cells. In addition, we also succeeded in single base substitution within the HPRT gene using TALEN disruption and a single-stranded oligonucleotide repair template. Based on the gene modification technology that we have established in human iPS cells, we propose applications in gene correction of disease-specific iPS cells, or the production of disease models to understand pathogenesis and establish clinical treatments.

Research Products

• [Journal Article] Nuclease-mediated genome editing : At the front-line of functional genomics technology (2014)
  • Author(s): Sakuma, T. and Woltjen, K.
  • Journal Title: Development, growth & differentiation Volume: 56 Pages: 2-13
  • Peer Reviewed
• [Journal Article] Efficient TALEN construction and evaluation methods for human cell and animal applications (2013)
  • Author(s): Sakuma T., Hosoi S., Woltjen K., Suzuki K.I., Kashiwagi K, Wada H., Ochiai H., Miyamoto T., Kawai N., Sasakura Y., Matsuura S., Okada Y., Kawahara A., Hayashi S. and Yamamoto T.
  • Journal Title: Genes to Cells Volume: 18(4) Pages: 315-326
• [Journal Article] Highly efficient site-specific transgenesis in cancer cell lines (2012)
  • Author(s): Michael, I.P., Monetti, C., Chiu, A.C., Zhang, P., Baba, T., Nishino, K., Agha-Mohammadi, S., Woltjen, K., Sung, H.K. and Nagy, A.
  • Journal Title: Molecular Cancer Volume: 11(1) Pages: 89-100
  • Peer Reviewed
• [Presentation] Transposed and targeted gene expression systems for guiding somatic cell reprogramming and differentiation (2013)
  • Author(s): Woltjen, K.
  • Organizer: Conference on Transposition and Genome Engineering
  • Place of Presentation: Budapest, Hungary
  • Year and Date: 2013-09-18
• [Presentation] Transposed and targeted gene expression systems for guiding somatic cell reprogramming and differentiation (2013)
  • Author(s): Woltjen, K.
  • Organizer: Japanese Society for Developmental Biology
  • Place of Presentation: 松江、日本
  • Year and Date: 2013-05-31
• [Presentation] Genome engineering strategies forhuman iPS cells (2012)
  • Author(s): Woltjen, K.
  • Organizer: 分子生物学学会
  • Place of Presentation: 福岡、日本
  • Year and Date: 20121211-14
• [Presentation] Developing TALEN-mediated genome engineering strategies for human iPS cells (2012)
  • Author(s): Woltjen, K.
  • Organizer: 2回のゲノム編集研究会
  • Place of Presentation: 岡崎、日本
  • Year and Date: 20120920-21
  • Invited
• [Book] TALENを用いたヒト iPS細胞におけるゲノム編集; Vol.32 (5) (2013)
  • Author(s): 李紅梅, Knut Woltjen,高橋和利,山中伸弥,堀田秋津
  • Total Pages: 526-531
  • Publisher: 細胞工学
• [Book] Transposon-Based Production of Genetically Unmodified Induced Pluripotent Stem Cells From Human Embryonic Fibroblasts (2011)
  • Author(s): Woltjen, K., Hamalainen, R., Kibschull, M., Mileikovsky, M., Nagy, A.
  • Total Pages: 767,87-103
  • Publisher: Methods in Molecular Biology
• [Remarks]
  • URL: http://www.cira.kyoto-u.ac.jp/woltjen/