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，以确定在单细胞绿色藻类衣原体中使用锌指核酸酶介导的基因靶向的潜力。首次在衣原体中描述了氟法内运输（IFT）的过程，我们对IFT所需的蛋白质的许多了解来自对衣原体鞭毛的组装研究。 IFT中的缺陷已与肾脏发育所需的过程直接相关，以建立侧向性和脊椎动物身体计划，用于视网膜中杆细胞的组装以及哺乳动物发育和细胞功能的许多其他关键过程。在像衣原体这样的简单模型系统中，IFT机械的持续解剖将有助于我们对高等真核生物中的IFT过程的理解。该项目结合了两种新的且非常有前途的技术方法：ITRAQ，一种基于质谱的技术，可确定复杂混合物中单个蛋白质的相对丰度；和使用锌指核酸酶（ZFN）靶向基因来产生感兴趣基因的突变。我们已经确定了一系列新型基因产物 可能参与IFT的衣原体鞭毛，但尚未通过标准生化方法将其鉴定为IFT组件。我们试图通过使用ZFN创建定向基因敲除这些蛋白质的潜在作用。在此过程中，设计用于结合靶基因序列的人工锌指蛋白附着在FOK1核酸酶结构域上，当该融合蛋白在细胞中表达时，基因组中靶序列的裂解会导致基因破坏。我们建议首先，通过破坏和对硝酸盐还原酶基因进行修改，在衣原体中使用这种技术的可行性；其次，确定是否需要三个新鉴定的新鉴定的IFT蛋白来鞭毛组装。 公共卫生相关性：该提案中描述的研究将使单细胞绿藻藻层成为更有用的系统，用于揭示Flagellar运输系统的基本组成部分。 在衣原体中发现IFT的发现使人们对基本生物学过程的理解有了极大的增强，该过程是人类疾病（如多囊肾脏疾病，视网膜退化，Bardet-Biedl综合征以及许多其他遗传性疾病）的基础。 该项目的研究可能会发现有关IFT的更多基本知识，这些知识可应用于人类疾病。