| Project/Area Number |
16K14987
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| Research Category |
Grant-in-Aid for Challenging Exploratory Research
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| Allocation Type | Multi-year Fund |
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| Research Field |
Aquatic life science
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| Research Institution | Kumamoto University |
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Principal Investigator |
KITANO TAKESHI 熊本大学, 大学院先端科学研究部(理), 准教授 (40336219)
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| Co-Investigator(Kenkyū-buntansha) |
吉崎 悟朗 東京海洋大学, 学術研究院, 教授 (70281003)
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| Project Period (FY) |
2016-04-01 – 2018-03-31
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| Project Status |
Completed (Fiscal Year 2017)
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| Budget Amount *help |
¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000)
Fiscal Year 2017: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2016: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
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| Keywords | ゲノム編集 / メダカ / ヒラメ / ニジマス / マサバ / CRISPR/Cas9 / ノックアウト |
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| Outline of Final Research Achievements |
Producing complete knockout fish quickly is a great advantage in the breed improvement. This study investigated the generation of F0 knockout medaka using the CRISPR/Cas9 system. To determine whether this editing system induced mutations in the medaka genome at the one-cell stage, recombinant Cas9 protein, tracrRNA and crRNA for dead end (dnd), which is essential for germ cell development, were injected into one-cell stage embryos. Predictably, biallelic mutated sequence patterns in the target sites of dnd were found in the injected embryos. To investigate the phenotypes of the mutated fish, histological observation of germ cells was carried out using fry and adults. The mutations resulted in a complete loss of germ cells, suggesting loss of function of dnd in the injected medaka. Moreover, this system was also functional for the rainbow trout, chub mackerel and Japanese flounder. Thus, this system appears to be extremely effective for the production of F0 knockout fish.
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