| Project/Area Number |
26670153
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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 |
Pathological medical chemistry
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| Research Institution | Shinshu University |
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Principal Investigator |
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| Co-Investigator(Kenkyū-buntansha) |
SHINDO Takayuki 信州大学, 学術研究院医学系, 教授 (90345215)
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| Project Period (FY) |
2014-04-01 – 2016-03-31
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| Project Status |
Completed (Fiscal Year 2015)
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| Budget Amount *help |
¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000)
Fiscal Year 2015: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2014: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
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| Keywords | 疾患モデル動物 / 遺伝子改変マウス / CRISPR / Cas9 / ゲノム編集 / 発生工学 / アドレノメデュリン / RAMP / 受容体修飾因子 / 血管作動性ペプチド / CRISPR/Cas9 / 血管作動性ペプチト |
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| Outline of Final Research Achievements |
The aim of this study was to develop new strategies to generate multiple-gene edited mice by using CRISPR/Cas9 system. First, we established a single blastocyst-based assay for rapidly determining whether CRISPR/Cas9-mediated genome editing worked in mouse preimplantation embryos. Then, we established transgenic (Tg) mice with systemic Cas9 overexpression (sCAT) to use non-inheritable maternal Cas9 (maCas9) protein derived from the zygotes. The maCas9 protein in zygotes derived from mating or in vitro fertilization of Tg/+ oocytes and +/+ sperm could successfully edit the target genome, and modified nine target genes simultaneously after injection with nine different gRNAs alone. Furthermore, we demonstrated the creation of “Cas9 transgene-free” gene-edited mice using non-Tg (+/+) zygotes carrying maCas9. These results suggest that sCAT zygotes have the potential to create multiple gene-modified mice both with and without the Cas9 transgene, enabling repeatable genome engineering.
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