Directed gene targeting to study intraflagellar transport

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衣原体中使用锌指核酸酶介导的基因靶向的潜力。鞭毛内转运（IFT）的过程首先在衣原体中被描述，我们对IFT所需蛋白质的大部分知识来自衣原体鞭毛组装的研究。IFT的缺陷与肾脏发育、侧化和脊椎动物身体平面的建立、视网膜中视杆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。在一个简单的模型系统，如衣原体IFT机制的继续解剖将有助于我们了解IFT过程中的高等真核生物。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）的基因靶向，以产生感兴趣的基因突变。我们已经鉴定了一系列新的基因产物， 衣原体鞭毛可能参与IFT，但它们尚未被标准的生物化学方法确定为IFT组分。我们试图通过使用ZFN创建定向基因敲除来研究这些蛋白质的潜在作用。在该方法中，将被设计为与靶基因中的序列结合的人工锌指蛋白连接到Fok 1核酸酶结构域，并且当该融合蛋白在细胞中表达时，基因组中的靶序列的切割导致基因破坏。我们建议，首先，证明使用这种技术在衣原体的可行性，破坏和修改硝酸还原酶基因，第二，以确定是否需要三个新发现的潜在IFT蛋白鞭毛组装。 公共卫生相关性：在这项建议中所描述的研究将使单细胞绿色藻类衣原体一个更有用的系统，揭示基本组成部分的鞭毛内运输系统。 IFT在衣原体中的发现极大地增强了对人类疾病（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）的基础生物过程的理解。 该项目的研究可能会发现更多关于IFT的基础知识，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.