Directed gene targeting to study intraflagellar transport

描述（由申请人提供）：本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。鞭毛衣单胞菌中首次描述了鞭毛衣单胞菌的鞭毛内运输过程，我们对鞭毛衣单胞菌鞭毛内运输所需蛋白质的了解大多来自对鞭毛衣单胞菌组装的研究。IFT缺陷直接与肾脏发育所需的过程、侧边性和脊椎动物身体计划的建立、视网膜中杆状细胞的组装以及哺乳动物发育和细胞功能的许多其他关键过程相关。在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；以及利用锌指核酸酶（ZFNs）进行基因靶向，在感兴趣的基因中产生突变。我们发现了一系列新的基因产物

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.