| Project/Area Number |
15590259
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General medical chemistry
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| Research Institution | Kawasaki Medical School |
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Principal Investigator |
TAKATA Minoru Kawasaki Medical School, Dept of Immunology and Molecular Genetics, Professor, 医学部, 教授 (30281728)
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| Co-Investigator(Kenkyū-buntansha) |
ISHII Masamichi Kawasaki Med Sch, Dept of Immunol and Mol Genet, Assistant Professor, 医学部, 助教授 (90298844)
MATSUDA Nobuko Kawasaki Med Sch, Dept of Immunol and Mol Genet, Research Associate, 医学部, 助手 (30333222)
HIRANO Seiko Kawasaki Med Sch, Dept of Immunol and Mol Genet, Research Associate, 医学部, 助手 (20368616)
KITAO Hiroyuki Kawasaki Med Sch, Dept of Immunol and Mol Genet, Research Associate, 医学部, 助手 (30368617)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2004: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2003: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | DNA double strand break / DNA repair / estrogen receptor / protein transduction / エストロジェン受容体 / I-SceI / TAT配列 / プロテイントランスダクション / I-Sce I |
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| Research Abstract |
In this study we tried to develop a method to simultaneously introduce a single double strand DNA break (DSB) into defined chromosomal locus. If this is made possible, then we would be able to continuously monitor physical status of the ends by genomic Southern blotting, recruitment of repair and/or checkpoint factors, progression of the repair process itself and so on. In yeast cells, such technology is available and contributes enormously to our understanding of the basic process of the DSB repair.
To this end, we plan to use rare restriction endonuclease I-SceI and cells harbouring the18 base-pair specific recognition sequence in its chromosome. The DSB is introduced in the integrated recombination substrate SCneo by I-SceI, and the repair efficiency can be determined by conversion of the G418-sensitive cells to resistant cells. To introduce I-SceI in high efficiency and in a synchronous manner, the I-SceI coding sequence was fused with two ligand binding domains of estrogen receptor
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or the protein transduction domain derived from HIV TAT sequence and 6XHis sequence for purification tag. In the former case, the fusion protein I-SceI-ER was stably expressed in a cell line. The latter case, the plasmid was introduced into E.coli and the fusion protein TAT-I-SceI was biochemically purifed.
(1)We successfully expressed the I-SceI-ER in the chicken B cell line DT40, and examined localization of the protein before and after treatment with estrogen analog tamoxifen. I-SceI-ER protein was rapidly (within 15min) mobilized from cytoplasm to nucleus with high efficiency. However, relatively few DSB repair events occurred, which is consistent with the possibility that ER may interfere with the endonuclease activity. Nonetheless, this cell fine would be useful for some studies. We are now trying to improve the efficiency by making new fusion proteins.
(2)We purified the recombinant TAT-I-SceI protein but the protein appeared to aggregate spontaneously. We are trying to find optimal condition to purigy and store the protein. Less
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