Directed gene targeting to study intraflagellar transport

描述(申请人提供)：这项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。鞭毛内转运(IFT)的过程最早是在衣藻中被描述的，我们对IFT所需蛋白质的大部分知识都来自于对衣藻组装的研究。IFT的缺陷直接与肾脏发育所需的过程、建立偏侧性和脊椎动物的身体计划、视杆细胞在视网膜中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有关。在像衣藻这样的简单模型系统中继续剖析IFT的机制将有助于我们了解高等真核生物中的IFT过程。该项目结合了两种非常有前景的新技术方法：iTRAQ，一种基于质谱学的技术，用于确定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)进行基因靶向，以产生目标基因的突变。我们发现了一系列新的基因产物 鞭毛衣藻可能与IFT有关，但标准生化方法尚未将它们鉴定为IFT成分。我们试图通过使用ZFN创建定向基因敲除来检查这些蛋白质的潜在作用。在这一过程中，旨在与目标基因中的序列结合的人造锌指蛋白被连接到Fok1核酸酶结构域，当该融合蛋白在细胞中表达时，基因组中目标序列的切割会导致基因中断。我们建议首先，通过破坏和修改硝酸还原酶基因来证明在衣藻中使用这项技术的可行性；其次，确定是否有三个新发现的潜在IFT蛋白是鞭毛组装所必需的。 公共卫生相关性：该提案中描述的研究将使单细胞绿藻衣藻成为一个更有用的系统，以揭示鞭毛内运输系统的基本组成部分。衣藻中IFT的发现极大地提高了人们对多囊肾病、视网膜变性、Bardet-Biedl综合征和许多其他遗传性疾病的基本生物学过程的理解。该项目的研究可能会揭示更多关于IFT的基本知识，这些知识可以应用于人类疾病。

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.