| Project/Area Number |
15K14366
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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 | Osaka University |
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Principal Investigator |
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| Co-Investigator(Renkei-kenkyūsha) |
IKAWA MASAHITO 大阪大学, 微生物病研究所, 教授 (20304066)
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| Project Period (FY) |
2015-04-01 – 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: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000)
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| Keywords | ゲノム編集 / CRISPR/Cas9 / マウスES細胞 / ノックイン / CRISPR/Casシステム / 遺伝子組換えマウス / ES細胞 / 点変異 |
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| Outline of Final Research Achievements |
The new genome editing CRISPR/Cas9 system can produce genetically modified (GM) animals simply and efficiently. In this project, I attempted more advanced genome editing such as point mutations and knockins using CRISPR/Cas9 in mice.
At first, I tried to introduce knockins using the zygote injection method we established (Sci Rep. 2013, DGD. 2014, Methods Enzymol. 2014). As a result, I found that the knockin-efficiency decreased depending on the size of the knockin cassettes (less than 0.1 kb). Next, when I used the technique in mouse embryonic stem (ES) cells, double-stranded DNA-mediated mutations were more efficient than single-stranded oligonucleotide-mediated mutations. Moreover, ES cell-mediated genome editing was more likely to introduce mutations in both alleles compared to the zygote injection method. The combination of biallelic mutations in ES cells and subsequent chimeric analysis provides an efficient method for rapid phenotypic analysis in GM animals.
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