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:
- 金额:$ 21.19万
- 依托单位:
- 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:
- 关键词:Adaptor Signaling ProteinAddressAnimal ModelApoptosisBindingBiochemicalBiological ModelsCaenorhabditis elegansCaenorhabditis elegans ProteinsCaspaseCellsCessation of lifeChimeric ProteinsCommunitiesComplexCre-LoxPDNADevelopmentDevelopmental GeneEmbryoEngineeringEventGene DeletionGene ProteinsGene SilencingGene TargetingGenesGeneticGenetic RecombinationGenetic ResearchGenetic studyGoalsHeat-Shock ResponseHourHuman DevelopmentHybridsInvertebratesLearningLifeLightLightingMaintenanceMasksMessenger RNAMethodsMolecular ProfilingOrganismOutcomePharmaceutical PreparationsPharmacotherapyPhenotypePhototherapyProteinsRNA InterferenceRoleSiteSystemTechniquesTestingTimeTissuesTranslatingUncertaintyWorkZebrafishZebrafish ProteinsZinc Fingersbasecell killingdevelopmental geneticselongin Cgene functiongene productgenetic analysishuman diseaseinsightkillingsknockout geneloss of functionpreventprogramspromoterprotein degradationprotein functionresearch studytoolubiquitin-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。
项目成果
期刊论文数量(0)
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Holger Knaut其他文献
Holger Knaut的其他文献
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{{ truncateString('Holger Knaut', 18)}}的其他基金
Engineering Tools for Rapid Loss of Protein Function with Spatio-Temporal Control in Zebrafish
通过时空控制斑马鱼蛋白质功能快速丧失的工程工具
- 批准号:
10571350 - 财政年份:2023
- 资助金额:
$ 21.19万 - 项目类别:
Molecular and Cellular Control of Collective Cell Migration.
集体细胞迁移的分子和细胞控制。
- 批准号:
10357669 - 财政年份:2018
- 资助金额:
$ 21.19万 - 项目类别:
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
- 批准号:
9356570 - 财政年份:2016
- 资助金额:
$ 21.19万 - 项目类别:
Molecular Regulation of Trigeminal Sensory Ganglia Development
三叉神经感觉神经节发育的分子调控
- 批准号:
8669500 - 财政年份:2013
- 资助金额:
$ 21.19万 - 项目类别:
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