2013 Fiscal Year Annual Research Report
トランスポゾンを利用した一塩基レベル遺伝子ターゲティング
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23710240
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| Research Institution | Kyoto University |
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Principal Investigator |
ウォルツェン クヌート 京都大学, 白眉センター, 准教授 (50589489)
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| Keywords | Gene targeting / TALEN nuclease / CRISPR/Cas9 nuclease / transposon / iPS Cell / cardiomyocyte / SNP |
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| Research Abstract |
This WakateB grant has helped us to establish efficient nuclease technology for genome editing of human iPS cells. In the original proposal, we set out to use selection cassettes based on the piggyBac transposon to enrich gene targeting, and allow later removal by transposase expression. However, we chose to focus on the development new gene targeting technologies based on TALEN and CRISPR/Cas9 nucleases. Now, we are able to target genes directly with oligonucleotide templates at high efficiency, obviating the need for transposon selection cassettes.
Using TALEN and CRISPR/Cas9 nucleases, we successfully targeted both the AAVS1 and HPRT genes. For AAVS1, we introduced a GFP reporter that is constitutively expressed in iPS cells and Tuj1 positive neurons. We are checking the differentiation capacity of these cells along the cardiomyocyte lineage. Constitutive GFP expression will allow cell tracking in transplantation studies.
HPRT gene disruption and correction using oligonucleotides has allowed us to establish the best conditions for targeting non-selectable genes, such as KCNE1.
Along with an expanded list of candidate genes and alleles, SNPs in the KCNE1 gene are being modified to determine the contribution of each SNP to KCNE1 function. Finally, we published our research on TALEN mediated genome engineering in human iPS cells in a research article (Sakuma et al., Genes to Cells, 2013), and invited review (Sakuma and Woltjen, Dev. Growth and Diff., 2014).
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[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.
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Journal Title
Genes to Cells.
Volume: 18(4)
Pages: 315-326
DOI
Peer Reviewed
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