Engineering tools for rapid loss of protein function in model organisms

项目摘要 模式生物中的遗传分析依赖于在特定时间在特定细胞中检测基因的工具。 大多数现有的基因失活方法，如条件性基因缺失或RNAi，靶DNA或mRNA。 然而，表型不会变得明显，直到来自靶基因的预先存在的蛋白质产物被去除。 腐烂了这种延迟可能是几个小时甚至几天。然而，在许多情况下，必须迅速 研究基因，例如在发育中的生物体中进行实验时，或在研究基因时 移除后会产生致命的细胞表型为了规避这些限制，一些 已经产生了直接靶向蛋白质基因产物的策略，通常通过用 可以被诱导降解标记蛋白质的降解决定子。然而，这些方法不太适合于 研究快速发生的发育事件，因为它们要么工作缓慢（几个小时），需要 添加可能难以引入胚胎的药物，或需要长时间的特定光照， 光的细胞。我们最近开发了一个基于degron的方法在C。elegans，称为ZF 1-tagging， 快速去除标记的蛋白质以揭示功能丧失的表型。用ZF 1降解决定子标记的蛋白质 可以通过表达衔接子ZIF-1来诱导降解，该衔接子结合ZF 1结构域并靶向 标记的蛋白质与保守的E3泛素连接酶复合物。在其当前形式中，ZF 1标记可用于 降解蛋白质的空间或时间的控制，但不是两个。在这里，我们建议工程师显着 ZF 1标记系统的改进。具体来说，我们将（1）扩展ZF 1标记，以便它可以用于 结合空间和时间控制降解蛋白质;（2）使ZF 1标记适应快速杀死细胞 遗传学上;和（3）工程化ZF 1-标记以在斑马鱼中发挥作用，其中有效的遗传工具用于 缺乏特定组织中的失活基因。这些改进将使ZF 1标记成为一种非常有效的方法。 一种强大的通用系统，用于在模型中的特定时间在特定细胞中快速灭活基因 生物，并将提供该方法可以适用于任何系统的原理证明。

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.