| Project/Area Number |
12305052
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
生物・生体工学
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| Research Institution | University of Tokyo |
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Principal Investigator |
TAIRA Kazunari University of Tokyo, School of Engineering, Professor, 大学院・工学系研究科, 教授 (10261778)
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| Co-Investigator(Kenkyū-buntansha) |
KAWASAKI Hiroaki University of Tokyo, School of Engineering, Assistant, 大学院・工学系研究科, 助手 (60332623)
KURATA Hiroyuki University of Kyusyu Institute technology, Faculty of computer Science and System Engineering, Associate Professor, 情報工学部, 助教授 (90251371)
AWAI Shigenori University of Tokyo, School of Engineering, Associate Professor, 大学院・工学系研究科, 助教授 (10168544)
MIYAGISHI Makoto University of Tokyo, School of Engineering, Assistant, 大学院・工学系研究科, 助手 (30323538)
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| Project Period (FY) |
2000 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥46,280,000 (Direct Cost: ¥39,500,000、Indirect Cost: ¥6,780,000)
Fiscal Year 2003: ¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000)
Fiscal Year 2002: ¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000)
Fiscal Year 2001: ¥19,500,000 (Direct Cost: ¥15,000,000、Indirect Cost: ¥4,500,000)
Fiscal Year 2000: ¥16,900,000 (Direct Cost: ¥16,900,000)
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| Keywords | Ribozyme / Gene Discovery / apoptosis / Alzheimer Disease / Metastasis / RNAヘリケース / poly A / アポトーシス / RNAヘリカーゼ / 遺伝子治療 / ジーン・ディスカバリー / ハンマーヘッドリボザイム / 慢性骨髄性白血病 / RNA工学 / 遺伝子破壊 |
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| Research Abstract |
Now that the sequences of many genomes are available, methods are required for the rapid identification of functional genes. We describe here a simple system for the isolation of genes that function in the tumor necrosis factor-α(TNF-α)-mediated pathway of apoptosis, using RNA helicase-associated ribozyme libraries with randomized substrate-binding arms. Because target-site accessibility considerably limits the effective use of intracellular ribozymes, the effectiveness of a conventional ribozyme library has been low. To overcome this obstacle, we attached to ribozymes an RNA motif (poly(A)-tail) able to interact with endogenous RNA helicase(s) so that the resulting helicase-attached, hybrid ribozymes can more easily attack target sites regardless of their secondary or tertiary structures. When the phenotype of cells changes upon introduction of a ribozyme library, genes responsible for these changes may be identified by sequencing the active ribozyme clones. In the case of TNF-α-mediated apoptosis, when a ribozyme library was introduced into MCF-7 cells, surviving clones were completely or partially resistant to TNF-α-induced apoptosis. We identified many pro-apoptotic genes and partial sequences of previously uncharacterized genes using this method. Our gene discovery system should be generally applicable to the identification of functional genes in various systems such as cancer metastasis.
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