Development of functional analysis of genes in cells using functional nucleic acids

使用功能核酸对细胞内基因进行功能分析的进展

• 批准号: 12305052
• 负责人: TAIRA Kazunari
• 金额: $ 29.62万
• 依托单位: University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12305052/
• 关键词: 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

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.

现在许多基因组的序列都是可用的，需要快速鉴定功能基因的方法。我们在这里描述了一个简单的系统，用于分离在肿瘤坏死因子-α(TNF-α)介导的凋亡途径中起作用的基因，使用随机底物结合臂的RNA解旋酶相关核酶文库。由于靶位点的可及性极大地限制了细胞内核酶的有效利用，传统的核酶文库的有效性一直很低。为了克服这一障碍，我们将能够与内源性RNA解旋酶相互作用的RNA基序（poly(A)-tail）连接到核酶上，从而使与解旋酶连接的杂交核酶能够更容易地攻击目标位点，而不管其二级或三级结构如何。当引入核酶文库后细胞的表型发生变化时，负责这些变化的基因可以通过对活性核酶克隆进行测序来鉴定。在TNF-α-介导的细胞凋亡中，将核酶文库导入MCF-7细胞后，存活的克隆对TNF-α-诱导的细胞凋亡具有完全或部分抗性。我们用这种方法鉴定了许多促凋亡基因和以前未鉴定的基因的部分序列。我们的基因发现系统应该普遍适用于肿瘤转移等各种系统功能基因的鉴定。

项目成果

Suyama, E., Kawasaki, H., Nakajima, M., Taira, K.: "Identification of genes involved in cell invasion by using a library of randomized hybrid ribozymes."Proc.Natl.Acad.Sci.USA. 100. 5616-5621 (2003)

Suyama, E.、Kawasaki, H.、Nakajima, M.、Taira, K.：“使用随机杂合核酶文库鉴定参与细胞侵袭的基因。”Proc.Natl.Acad.Sci.USA。

Kawasaki H, Onuki R, Suyama E, Taira K: "Identification of genes that function in the TNF-α-mediated pathway to apoptosis by analysis of randomized hybrid-ribozyme libraries"Nature Biotechnology. (in press). (2002)

Kawasaki H、Onuki R、Suyama E、Taira K：“通过分析随机混合核酶文库鉴定在 TNF-α 介导的细胞凋亡途径中起作用的基因”（出版中）。

Kawasaki, H., Onuki, R., Suyama, E., Taira, K.: "Identification of genes that function in the TNF-alpha-mediated apoptotic pathway using randomized hybrid ribozyme libraries."Nature Biotechnology. 20. 376-380 (2002)

Kawasaki, H.、Onuki, R.、Suyama, E.、Taira, K.：“使用随机混合核酶文库鉴定在 TNF-α 介导的细胞凋亡途径中起作用的基因。”《自然生物技术》。

Hiroaki Kawasaki: "A functional gene discovery in the Fas-mediated pathway to apoptosis by analysis of transiently expressed randomized hybrid-ribozyme libraries"Nucleic Acids Res.. 30・16. 3609-3614 (2002)

Hiroaki Kawasaki：“通过分析瞬时表达的随机杂合核酶文库发现 Fas 介导的细胞凋亡途径中的功能基因”Nucleic Acids Res. 30・16 (2002)。

Kawasaki H, Taira K: "A functional gene discovery in the Fas-mediated pathway to apoptosis by analysis of transiently expressed randomized hybrid-ribozyme libraries"EMBO Reports. (in press). (2002)

Kawasaki H、Taira K：“通过分析瞬时表达的随机混合核酶文库发现 Fas 介导的细胞凋亡途径中的功能基因”EMBO 报告。