Engineering Tools for Rapid Loss of Protein Function with Spatio-Temporal Control in Zebrafish

项目摘要 在斑马鱼中，遗传工具箱仅限于全局和组织特异性基因失活。然而，它往往是 期望在特定组织和特定时间表达基因。此外，表型不会成为 直到来自靶基因的预先存在的蛋白质产物已经衰变。这种滞后可能是几个小时， 甚至几天这是一个问题，当研究快速的生物过程或基因所需的细胞 生存近年来，已经制定了一些战略来克服这些限制。大部分 这些策略直接靶向蛋白质基因产物，通常通过用降解决定子标记蛋白质， 被诱导降解标记的蛋白质。我们最近将deGradFP系统从苍蝇调整到斑马鱼， 叫做zGrad。zGrad靶向GFP标记的蛋白质进行降解，并揭示功能丧失的表型。 zGrad由抗GFP纳米抗体与F-box结构域的融合物组成。zGrad结合GFP标记的蛋白 通过其抗GFP纳米抗体部分，并将标记的蛋白质靶向E3泛素连接酶复合物， 通过其F-box结构域降解。在其目前的形式中，zGrad可用于降解蛋白质， 空间或时间控制，但不能两者兼而有之。在这里，我们建议对zGrad进行重大改进 系统具体来说，我们将（1）扩展zGrad，以便它可以用于降解蛋白质， 和时间控制，和（2）工程zGrad，使其通过光调节，以诱导降解， 特定的细胞和亚细胞区室。这些改进将使zGrad成为 一种非常强大和通用的系统，可以在特定时间在特定细胞中快速灭活基因， 斑马鱼，并将提供原理证明，deGradFP/zGrad方法可以适用于功能 在任何系统中。

PROJECT SUMMARY In zebrafish, the genetic toolkit is limited to global and tissue-specific gene inactivation. However, often it is desirable to inactivate genes in specific tissues and at specific times. Moreover, 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. This is a problem when studying rapid biological processes or genes that are required for cell survival. In recent years, several strategies have been developed to overcome these limitations. Most of the strategies target the protein gene product directly, typically by tagging the protein with a degron that can be induced to degrade the tagged protein. We recently adapted the deGradFP system from flies to zebrafish and called it zGrad. zGrad targets GFP-tagged proteins for degradation and reveals loss-of-function phenotypes. zGrad consists of a fusion of an anti-GFP nanobody to a F-box domain. zGrad binds GFP-tagged proteins through its anti-GFP nanobody part and targets the tagged protein to the E3 ubiquitin ligase complex for degradation through its F-box domain. In its current form, zGrad can be used to degrade proteins with either spatial or temporal control, but not both. Here we propose to engineer significant improvements to the zGrad system. Specifically, we will (1) expand zGrad so that it can be used to degrade proteins with combined spatial and temporal control, and (2) engineer zGrad so that it is regulated through light to induce degradation in specific cells and subcellular compartments at desired times. These improvements will make zGrad an extremely powerful and versatile system for inactivating genes rapidly in specific cells at specific times in zebrafish, and would provide proof-of-principle that the deGradFP/zGrad method could be adapted to function in any system.