Engineering tools for rapid loss of protein function in model organisms

模型生物中蛋白质功能快速丧失的工程工具

基本信息

批准号：
9356570
负责人：
Holger Knaut
金额：
$ 25.43万
依托单位：
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-23 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9356570
关键词：
Acute Adaptor Signaling Protein Address Alpha Cell Animal Model Apoptosis BCL-2 Protein Binding Biochemical Biological Models Caenorhabditis elegans Caspase Cells Cessation of life Chimeric Proteins Communities Complex Cre-LoxP DNA Development Developmental Gene Embryo Engineering Event Gene Deletion Gene Proteins Gene Silencing Gene Targeting Genes Genetic Genetic Recombination Genetic Research Genetic study Goals Heat-Shock Response Hour Human Development Hybrids Invertebrates Learning Life Light Lighting Maintenance Masks Messenger RNA Methods Molecular Profiling Organism Outcome Pharmaceutical Preparations Pharmacotherapy Phenotype Phototherapy Proteins RNA Interference Role Site System Techniques Testing Time Tissues Translating Uncertainty Work Zebrafish Zebrafish Proteins Zinc Fingers base cell killing developmental genetics elongin C experimental study gene function gene product genetic analysis human disease insight killings knockout gene loss of function prevent promoter protein degradation protein function tool ubiquitin-protein ligase

项目摘要

PROJECT SUMMARY Genetic analysis in model organisms relies on tools to inactivate genes in particular cells at specific times. Most existing methods for gene inactivation, such as conditional gene deletion or RNAi, target DNA or mRNA. However, phenotypes do not become evident until pre-existing protein product from the targeted gene has decayed. This lag can be many hours or even days. However, in many cases, it is essential to rapidly inactivate genes, such as when performing experiments in developing organisms, or when studying a gene that produces a cell lethal phenotype when removed. In an attempt to circumvent these limitations, several strategies have been created that target protein gene product directly, typically by tagging the protein with a degron that can be induced to degrade the tagged protein. However, these methods are poorly suited for the study of rapidly occurring developmental events, as they either work slowly (several hours), necessitate the addition of drugs that may be difficult to introduce into embryos, or require the prolonged illumination of specific cells with light. We recently developed a degron-based method in C. elegans, called ZF1-tagging, that very rapidly removes tagged proteins to reveal loss-of-function phenotypes. Proteins tagged with the ZF1 degron can be induced to degrade by expressing the adaptor ZIF-1, which binds the ZF1 domain and targets the tagged protein to a conserved E3 ubiquitin ligase complex. In its current form, ZF1-tagging can be used to degrade proteins with either spatial or temporal control, but not both. Here we propose to engineer significant improvements to the ZF1-tagging system. Specifically, we will (1) expand ZF1-tagging so that it can be used to degrade proteins with combined spatial and temporal control; (2) adapt ZF1-tagging to rapidly kill cells genetically; and (3) engineer ZF1-tagging to function in zebrafish, where an effective genetic tool for inactivating genes in specific tissues is lacking. These improvements will make ZF1-tagging an extremely powerful and versatile system for inactivating genes rapidly in specific cells at specific times in model organisms, and would provide proof-of-principle that the method could be adapted to function in any system.

项目摘要模型生物中的遗传分析依赖于在特定时间在特定细胞中灭活基因的工具。大多数现有的基因失活方法，例如条件基因缺失或RNAi，靶DNA或mRNA。然而，直到靶向基因的蛋白质产物的蛋白质产物具有腐烂。这个滞后可能是数小时甚至几天。但是，在许多情况下，要迅速灭活基因，例如在开发生物或研究基因时进行实验时去除时会产生细胞致命的表型。为了规避这些限制，有几个已经创建了直接靶向蛋白基因产物的策略，通常是通过用A标记蛋白质可以诱导降解标记蛋白的Degron。但是，这些方法不适合研究快速发生的发展事件，因为它们要么缓慢工作（几个小时），因此需要添加可能难以引入胚胎的药物，或需要长时间照明特定的药物带有光的细胞。我们最近在秀丽隐杆线虫中开发了一种基于Degron的方法，称为ZF1标记，非常迅速去除标记的蛋白质以揭示功能丧失表型。用ZF1 Degron标记的蛋白质可以通过表达适配器ZIF-1来诱导降解，该适配器ZIF-1结合ZF1域并靶向将蛋白质标记为保守的E3泛素连接酶复合物。以目前的形式，ZF1标记可用于降解具有空间或时间控制的蛋白质，但并非两者兼而有之。在这里，我们建议设计重要的 ZF1标记系统的改进。具体而言，我们将（1）扩展ZF1标记，以便可以使用它降解具有空间和时间对照组合的蛋白质；（2）适应ZF1标记以快速杀死细胞遗传上；（3）工程师ZF1标记在斑马鱼中的功能，其中有效的基因工具缺乏特定组织中的灭活基因。这些改进将使ZF1标记非常在模型中，特定时间在特定细胞中迅速灭活基因的功能强大且通用的系统生物体，并将提供原理证明该方法可以适应任何系统的功能。