| Project/Area Number |
26640050
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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 |
Laboratory animal science
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| Research Institution | University of Tsukuba |
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Principal Investigator |
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| Co-Investigator(Kenkyū-buntansha) |
MIZUNO Seiya 筑波大学, 医学医療系, 助教 (10633141)
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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 |
¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000)
Fiscal Year 2015: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
Fiscal Year 2014: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
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| Keywords | マウス / 複数遺伝子変異 / XYi法 / CRISPR/Cas9 / マウスES細胞 / Y染色体 |
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| Outline of Final Research Achievements |
For producing multiple genes knock-in mouse, we tried to establish novel ES cell line and mouse strain carrying suicide and reporter genes on their Y chromosome.
First, we construct gene targeting vector including drug inducible suicide cassette (SRa-promoter and iCaspase3E gene) and fluorescence reporter cassette (EF1-promoter and EGFP gene). Because Knock-in into Y chromosome happen with very low frequency, we designed two CRISPR-nickase site in each 5’ and 3’homology arm region. These two CRISPR target DNA fragments were inserted in the px335 which carried U6-gRNA cassette and CBh-Cas9 D10A cassette. Then, these 3 vectors (one targeting vector and two CRISPR expression vectors) were electroplated into C57BL/6J mouse ES cells. We obtained one ES clone in which drug inducible suicide and reporter gene were expressed. The KI allele was confirmed by not only PCR but also Southern blotting.
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