Directed gene targeting to study intraflagellar transport

定向基因打靶研究鞭毛内运输

• 批准号: 7772559
• 负责人: PAUL A. LEFEBVRE
• 金额: $ 22.65万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-06 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7772559
• 关键词: Bardet-Biedl Syndrome; Binding; Biochemical; Biological Models; Biological Process; Carrier Proteins; Cell physiology; Cells; Chimeric Proteins; Chlamydomonas; Complex Mixtures; Defect; Development; Dissection; Eukaryota; Flagella; Gene Targeting; Genes; Genome; Green Algae; Handedness; Inborn Genetic Diseases; Individual; Knowledge; Mass Spectrum Analysis; Mediating; Modification; Monomeric GTP-Binding Proteins; Mutation; Nitrate Reductases; Play; Polycystic Kidney Diseases; Procedures; Process; Proteins; Relative (related person); Research; Retina; Retinal Degeneration; Role; Series; System; Techniques; Technology; Transport Process; Zinc Fingers; base; design; gene replacement; homologous recombination; human disease; interest; knockout gene; nephrogenesis; nitrate reductase; novel; nuclease; public health relevance; retinal rods

DESCRIPTION (provided by applicant): This proposal describes a project to ascertain the potential for using zinc finger nuclease-mediated gene targeting in the unicellular green alga Chlamydomonas. The process of intraflagellar transport (IFT) was first described in Chlamydomonas, and much of our knowledge of the proteins required for IFT comes from studies of the assembly of Chlamydomonas flagella. Defects in IFT have been directly associated with processes required for kidney development, for establishment of laterality and the vertebrate body plan, for the assembly of rod cells in the retina, and many other key processes in mammalian development and cell function. The continued dissection of the machinery of IFT in a simple model system like Chlamydomonas will help inform our understanding of IFT processes in higher eukaryotes. This project combines two new and very promising technical approaches: iTRAQ, a mass spectrometry-based technique to identify the relative abundance of individual proteins in complex mixtures; and gene targeting using zinc finger nucleases (ZFNs) to generate mutations in genes of interest. We have identified a series of novel gene products in Chlamydomonas flagella that are likely to be involved in IFT, but they have not been identified as IFT components by standard biochemical approaches. We seek to examine a potential role for these proteins by creating directed gene knockouts using ZFNs. In this procedure, artificial zinc finger proteins designed to bind to a sequence in a target gene are attached to a Fok1 nuclease domain, and when this fusion protein is expressed in cells, cleavage of the target sequence in the genome leads to gene disruption. We propose to first, demonstrate the feasibility of using this technique in Chlamydomonas by disrupting and making modifications in the nitrate reductase gene; and second, to determine whether three newly identified potential IFT proteins are required for flagellar assembly. PUBLIC HEALTH RELEVANCE: The research described in this proposal will make the unicellular green algae Chlamydomonas an even more useful system for uncovering fundamental components of the intraflagellar transport system. The discovery of IFT in Chlamydomonas has led to a greatly enhanced understanding of basic biological process that underlies human diseases such as polycystic kidney disease, retinal degeneration, Bardet-Biedl syndrome, and many other inherited disorders. The research in this project will likely uncover more basic knowledge about IFT that can be applied to human diseases.

描述（由申请人提供）：该提案描述了一个项目，以确定在单细胞绿藻衣藻中使用锌指核酸酶介导的基因靶向的潜力。鞭毛内运输 (IFT) 过程首先在衣藻中被描述，我们对 IFT 所需蛋白质的了解大部分来自对衣藻鞭毛组装的研究。 IFT 缺陷与肾脏发育、侧向性和脊椎动物身体规划的建立、视网膜中杆状细胞的组装以及哺乳动物发育和细胞功能中的许多其他关键过程所需的过程直接相关。在像衣藻这样的简单模型系统中继续剖析 IFT 机制将有助于我们了解高等真核生物中的 IFT 过程。该项目结合了两种新的、非常有前途的技术方法：iTRAQ，一种基于质谱的技术，用于识别复杂混合物中单个蛋白质的相对丰度；以及使用锌指核酸酶 (ZFN) 进行基因打靶以在感兴趣的基因中产生突变。我们已经鉴定出一系列新的基因产物 衣藻鞭毛可能参与 IFT，但标准生化方法尚未将其鉴定为 IFT 成分。我们试图通过使用 ZFN 进行定向基因敲除来研究这些蛋白质的潜在作用。在此过程中，设计用于与靶基因中的序列结合的人工锌指蛋白被附着到 Fok1 核酸酶结构域上，当这种融合蛋白在细胞中表达时，基因组中靶序列的切割会导致基因破坏。我们建议首先通过破坏和修饰硝酸还原酶基因来证明在衣藻中使用该技术的可行性；其次，确定鞭毛组装是否需要三种新发现的潜在 IFT 蛋白。 公共健康相关性：本提案中描述的研究将使单细胞绿藻衣藻成为揭示鞭毛内运输系统基本组成部分的更有用的系统。 衣藻中 IFT 的发现极大地加深了对人类疾病（如多囊肾病、视网膜变性、Bardet-Biedl 综合征和许多其他遗传性疾病）基础生物学过程的了解。 该项目的研究可能会揭示更多有关 IFT 的基础知识，这些知识可应用于人类疾病。