2015 Fiscal Year Research-status Report
Runx-mediated regulation of chemokine CCL5 for lung diseases
| Project/Area Number |
15K08535
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
SEO WOOSEOK 国立研究開発法人理化学研究所, 統合生命医科学研究センター, 国際特別研究員 (40574116)
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| Project Period (FY) |
2015-10-21 – 2018-03-31
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| Keywords | アレルギー / 免疫関連疾患 |
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| Outline of Annual Research Achievements |
The first aim of this project was to generate a knockout mouse by Crispr/Cas9 technology in which a binding region of Runx transcription factor on CCL5 locus is removed. According to the proposal, I have successfully generated the knockout mouse and in the process of the initial examination of the mouse line. Preliminary data by flow cytometry analysis show that CCL5 expression is almost completely eliminated in the absence of the Runx-binding region indicating that this novel DNA region is crucial for CCL5 expression. To gain further insight, I have generated a BAC (Bacterial Artificial Chromosome) transgenic construct in which Runx-binding sequences on this Runx-binding region on CCL5 are mutated. This will be injected into mouse embryo to generated transgenic mouse line as we proposed.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
To propose and prove a novel regulatory pathway of CCL5 expression by Runx transcription factor, my experimental plan was to generate two powerful tools using mouse genetics. The first tool was to construct a mouse line in which Runx-binding to CCL5 locus is abolished. This mouse line was successfully made and its analysis has been generating interesting results. The second tool was to create a BAC transgenic mouse line in which Runx-binding consensus sequences on CCL5 locus are specifically mutated to abolish the influences of Runx on CCL5 expression without removing a chunk of DNA region. This BAC construct is already made and will be submitted to generate the transgenic mouse line. Therefore, the current progress of the project is slightly ahead of its schedule.
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| Strategy for Future Research Activity |
I expect to have this new BAC transgenic mouse line within this fiscal year. While waiting for the successful generation of the mouse line, I will continue to analyze the knockout mouse I generated in the last fiscal year to carefully examine the interesting phenotype. To further prove that this region plays a crucial function in controlling CCL5 expression, I am going to perform ChIP-seq (chromatin immunoprecipitation sequencing) to visualize genomic binding of Runx transcription factor in peripheral CD4+ T help cells and CD8+ T cytotoxic cells. This is quite important since it have been shown that this Runx-binding region on CCL5 has a SNP which is associated with severe liver fibrosis.
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| Causes of Carryover |
27年10月に追加採択されたので、次年度使用額が生ました。
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| Expenditure Plan for Carryover Budget |
27年度に製作すろことだったトランスジェニックマウスを28年度に製作。
研究費使用期間が3年から２年半に少し短くなったが、進捗状況は順調。
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