Engineering tools for rapid loss of protein function in model organisms

项目总结 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。 现有的大多数基因失活方法，如条件性基因缺失或RNAi，都是针对DNA或mRNA的。 然而，表型不会变得明显，直到来自目标基因的预先存在的蛋白质产物 腐烂了。这个延迟可以是几个小时，甚至几天。然而，在许多情况下，迅速 使基因失活，如在发育中的生物体中进行实验，或在研究基因时 当被移除时会产生一种致命的细胞表型。为了绕过这些限制，有几个 已经建立了直接针对蛋白质基因产物的策略，通常是通过将蛋白质标记为 可被诱导降解标记蛋白质的降解子。但是，这些方法不太适合 对快速发生的发育事件的研究，因为它们要么工作缓慢(几个小时)，要么需要 添加可能难以引入胚胎的药物，或需要特定的长时间光照的药物 有光的细胞。我们最近在线虫中开发了一种基于退化的方法，称为ZF1-Tagging，非常 快速去除标记蛋白以揭示功能丧失的表型。ZF1降解子标记的蛋白质 可以通过表达适配器ZIF-1来诱导降解，该适配器结合ZF1结构域并靶向 标记蛋白质到保守的E3泛素连接酶复合体。在其当前形式中，ZF1-Tagging可用于 通过空间或时间控制来降解蛋白质，但不能两者兼而有之。在这里，我们建议设计重要的 对ZF1标签系统的改进。具体地说，我们将(1)扩展ZF1-Tagging，使其可以使用 利用时间和空间相结合的方法对蛋白质进行降解；(2)采用ZF1标记技术快速杀伤细胞 以及(3)设计ZF1标签以在斑马鱼中发挥作用，其中有效的遗传工具 缺乏特定组织中的失活基因。这些改进将使ZF1-标签成为一个极其重要的 强大而通用的系统，用于在特定时间在特定细胞中快速灭活模型中的基因 并将提供原则证据，证明该方法可适用于任何系统。

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.