| Project/Area Number |
16590045
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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 |
Biological pharmacy
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| Research Institution | Kanazawa Medical University (2005)
Kanazawa University (2004) |
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Principal Investigator |
ISHIGAKI Yasuhito Kanazawa Medical University, Medical Research Institute, Lecturer, 総合医学研究所, 講師 (20232275)
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| Co-Investigator(Kenkyū-buntansha) |
MATSUNAGA Tsukasa Kanazawa University, Graduate school of Natural Science and Technology, Professor, 自然科学研究科, 教授 (60192340)
WAKASUGI Mitsuo Kanazawa University, Graduate school of Natural Science and Technology, Assistant Professor, 自然科学研究科, 助手 (80345595)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2005: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2004: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | DNA damage sensor / NMD / SMG1 / DDB1 / p53 / NER / DNA damage / SMGI / DDBI |
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| Research Abstract |
To investigate the biological function of damaged DNA binding 1 (DDB1) and new DNA damage sensor SMG1, we tried knockout or knockdown system of them. At first, to improve the efficiency of shRNA knockdown, we constructed multiple shRNA expression sequences in single plasmid vector, which carries RNA polymerase III promoter. XPA gene was selected for target gene because it is not essential for cell viability and easy to check the functional knockdown by the measurement of repair efficiency or sensitivity. After establishment of stable clones, the efficiency of knockdown was compared among single and triple expression vectors. The single shRNA-expressing vector caused limited knockdown of target protein in stable transfectants, but the multiple expression vectors resulted in significantly increased the frequency of knockdown transfectants. There were significant correlations between knockdown level and EGFP expression in multiple-expressing transfectants, while poor correlations were obs
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erved in singe vector transfectants. Multiple-transfectants showed the reduced repair efficiency of UV-induced DNA damage and the increased sensitivity to UV-irradiation. We concluded that multiple shRNA expression might be useful strategy to establish knockdown cells with shRNA expression vectors. In addition to this, we established the DDB1 knockout heterozygous mouse ES cells by Cre-loxP conditional knockout method. Further work is required to develop the homozygous DDB1 knockout cells. SMG1 knockdown experiments were also carried to know its biological significance in DNA damage response. SMG1 is one of essential factors in nonsense mediated mRNA decay pathway (NMD), but recent reports show the second function of SMG1 as DNA damage sensor. We could not detect the significant role of SMG1 against DNA damaging agents. However we observed the accumulation of other NND components in SMG1 knockdown cells. Ubiquitin-proteasome inhibitors also had similar effect on the amount of NMD factors and we are going to continue the investigation of the mechanism. Less
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