RNA-based tools for developmental and regenerative biology

项目摘要 基于RNA的技术使多细胞中基因功能的扰动和观察成为可能 生物体，导致了生物医学研究的重大发现。然而，获得机械的洞察力 进入发育和再生过程需要用空间和空间来操纵基因功能 时间精度，这仍然是脊椎动物模型中的一项主要挑战。的总目标是 这项探索性的技术开发方案是为了开发出一种具有创新性和广泛适用性的 分子工具包，它将使基因表达能够在空间和时间上精确控制。 具体地说，我们将引入一类新的基于RNA的通用遗传条件开关来调节 在发育中的斑马鱼中进行翻译。在我们的第一个目标中，我们将开发可控制的RNA开关 通过非侵入性的无毒小分子给药。这些化学诱导的开关 实现对目标基因的动态、可滴定和有条件的控制。我们将建立一种快速的哺乳动物- 基于原型平台，为斑马鱼模型的开发确定理想的切换候选对象的优先顺序。在……里面 我们的第二个目标是，我们将建立一个概念上的新的光遗传开关，它可以使用 蓝光。这些新工具将在斑马鱼身上开发，并以现有的光遗传学为基准 包括光激活转录工具在内的工具。该项目的成功实施将为 简化流程以开发新的RNA交换机，以响应各种正交的非侵入性 投入。考虑到物种独立的机制，开发的RNA开关工具包很可能是 广泛适用于动物模型。通过特别关注斑马鱼开关的发育， 我们将贡献一种新的方法来重新编程和审问发展和再生 生物学。

Project Summary RNA-based technologies have enabled perturbation and observation of gene function in multicellular organisms, resulting in major discoveries in biomedical research. However, gaining mechanistic insights into developmental and regenerative processes requires the manipulation of gene functions with spatial and temporal precision, a task that remains a major challenge in vertebrate animal models. The overall goal of this exploratory technology development proposal is to develop an innovative and widely applicable molecular toolset, which will enable control of gene expression with spatial and temporal precision. Specifically, we will introduce a new class of versatile RNA-based genetic conditional switches to regulate translation within the developing zebrafish. In our first aim, we will develop RNA switches that are controlled through non-invasive administration of non-toxic small molecules. These chemically-induced switches enable dynamic, titratable, and conditional control of a target gene. We will establish a rapid mammalian- based prototyping platform to prioritize ideal switch candidates for development in the zebrafish model. In our second aim, we will establish a conceptually new optogenetic switch that can be turned on and off using blue light. These novel tools will be developed in zebrafish and benchmarked against existing optogenetic tools including light-activated transcriptional tools. The successful execution of this project will provide a streamlined pipeline to develop new RNA switches that respond to a diversity of orthogonal non-invasive inputs. Given the species-independent machinery, the toolkit of RNA switches developed are likely to be broadly applicable to animal models. By specifically focusing the development of the switches in zebrafish, we will we will contribute a new approach to reprogram and interrogate developmental and regenerative biology.