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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：
9163926
负责人：
Holger Knaut
金额：
$ 21.19万
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 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9163926
关键词：
Adaptor Signaling Protein Address Animal Model Apoptosis Binding Biochemical Biological Models Caenorhabditis elegans Caenorhabditis elegans Proteins 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 gene function gene product genetic analysis human disease insight killings knockout gene loss of function prevent programs promoter protein degradation protein function research study 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.

项目概要模式生物的遗传分析依赖于在特定时间使特定细胞中的基因失活的工具。大多数现有的基因失活方法，例如条件性基因删除或RNAi，目标DNA或mRNA。然而，直到来自目标基因的预先存在的蛋白质产物出现后，表型才会变得明显。腐烂了。这种滞后可能会持续数小时甚至数天。然而，在许多情况下，必须迅速使基因失活，例如在发育中的生物体中进行实验或研究基因时去除后会产生细胞致死表型。为了规避这些限制，一些已经创建了直接靶向蛋白质基因产物的策略，通常通过用可以诱导降解标记蛋白的降解决定子。然而，这些方法不太适合研究快速发生的发育事件，因为它们要么缓慢发生（几个小时），要么需要添加可能难以引入胚胎的药物，或需要长时间照射特定的药物细胞与光。我们最近在秀丽隐杆线虫中开发了一种基于降解决定子的方法，称为 ZF1 标记，非常适合快速去除标记的蛋白质以揭示功能丧失的表型。用 ZF1 降解决定子标记的蛋白质可以通过表达接头 ZIF-1 来诱导降解，该接头结合 ZF1 结构域并靶向将蛋白质标记到保守的 E3 泛素连接酶复合物上。在目前的形式中，ZF1 标记可用于通过空间或时间控制来降解蛋白质，但不能同时控制两者。在这里，我们建议设计重要的对 ZF1 标签系统的改进。具体来说，我们将 (1) 扩展 ZF1 标记，以便可以使用它通过空间和时间的联合控制来降解蛋白质； (2) 采用ZF1标签快速杀死细胞遗传上； (3) 设计 ZF1 标签使其在斑马鱼中发挥作用，这是一种有效的遗传工具缺乏使特定组织中的基因失活的方法。这些改进将使 ZF1 标记成为极其强大且多功能的系统，可在模型中的特定时间快速灭活特定细胞中的基因生物体，并将提供该方法可以适用于任何系统的原理验证。