| Project/Area Number |
25650006
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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 |
Molecular biology
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| Research Institution | Kyoto University |
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Principal Investigator |
TAKEDA Shunichi 京都大学, 医学(系)研究科(研究院), 教授 (60188191)
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| Co-Investigator(Renkei-kenkyūsha) |
SASANUMA Hiroyuki 京都大学, 大学院医学研究科, 准教授 (00531691)
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| Project Period (FY) |
2013-04-01 – 2015-03-31
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| Project Status |
Completed (Fiscal Year 2014)
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| Budget Amount *help |
¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
Fiscal Year 2014: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Fiscal Year 2013: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
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| Keywords | ゲノム編集 / CRISPR/Cas9 / DNA組換え / 標的組換え / DT40 / ニワトリBリンパ細胞 / プロテオーム解析 |
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| Outline of Final Research Achievements |
The CRISPR/Cas9 system, an artificial restriction enzyme, dramatically increases the efficiency of genetic manipulation, such as knock-in of designed point mutations, in human cell lines. However, genetic manipulation in primary cells is still a formidable challenge, because the efficiency of homologous recombination (HR) between genomic DNA and transfected DNA is still very low despite the usage of the CRISPR/Cas9 system. This study is to further increase the efficiency of genetic manipulation. To this end, I have explored molecular mechanisms underlying the extremely efficient HR of a chicken B cell line, DT40. HR is a very complex biochemical reaction, which is carried out by more than 50 proteins including many unidentified proteins. Having a support of Grant-in-Aid for Challenging Exploratory Research, we have established methods of biochemically purifying proteins involved in HR. In future, I will purify proteins involved in HR from DT40 (wild-type and HR deficient cell lines).
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