Switchable Systems for Spatio-Temporal Control of Gene Expression in Zebrafish

描述(申请人提供)：斑马鱼(Danio Rerio)已成为研究人类疾病和发育遗传学的强大模型；然而，目前还没有以可切换的时空方式解剖胚胎基因功能的机制。该项目的总体目标是开发多种机制，通过光照射对斑马鱼的基因和转基因表达进行时空调控。其具体目的是利用小型可光激活(光笼)和光可切换的有机化合物，利用紫外光严格调控核酶和核糖开关功能。这些基于RNA的方法将被用于控制斑马鱼胚胎发育中的基因和转基因表达。我们的策略基于：1)合成可光激活和光可切换化合物的能力，2)在体外选择变构核酶的能力，其活性依赖于特定有机化合物的存在，3)在体外选择选择性结合有机化合物的短RNA序列(RNA适配子)的能力，以及4)结合1-3来创建光响应开关的能力，以激活和失活斑马鱼的基因表达。具体目标是：1.建立RNA功能的光化学调控系统。我们将合成无毒、细胞可穿透、对光敏感的小有机分子，并在体外选择可被这些分子结合紫外线激活的核酶。此外，RNA适配子的进化方式是，它们与这些分子的结合可以通过紫外光进行调节。2.开发光化学介导的基因调控系统。具体地说，我们将利用紫外光照射，以不可逆和可逆的方式展示对斑马鱼胚胎基因敲除的时空控制。3.开发光化学介导的转基因调控系统。具体地说，我们将利用紫外光照射，以不可逆和可逆的方式展示对斑马鱼胚胎中转基因表达的时空控制。所有已开发的成分(例如，光敏性小有机分子、变构核酶、RNA)。适配子、表达质粒等)和协议(例如，用于有机合成、紫外线照射等)将通过一个专门的网页向科学界提供。

DESCRIPTION (provided by applicant): The zebrafish (Danio rerio) has become a powerful model for investigating the genetics of human disease and development; however, there is currently no mechanism for dissecting embryonic gene function in a switchable, spatio-temporal manner. The overall goal of this project is to develop multiple mechanisms for spatio-temporal control of gene and transgene expression in zebrafish via light irradiation. The specific intent is to utilize small photo-activatable (photo-caged) and photo-switchable organic compounds to tightly regulate ribozyme and riboswitch function using UV light. These RNA based methods will be utilized to control gene and transgene expression within the developing zebrafish embryo. Our strategies are based on: 1) the ability to synthesize photo-activatable and photo-switchable compounds, 2) the ability to select allosteric ribozymes in vitro whose activity relies on the presence of specific organic compounds, 3) the ability to select short RNA sequences (RNA aptamers) in vitro that bind selectively organic compounds, and 4) the ability to combine 1-3 to create light-responsive switches for activation and deactivation of gene expression in zebrafish. The Specific Aims are to: 1. Develop systems for photochemical control of RNA function. We will synthesize non-toxic, cell permeable, photo-responsive small organic molecules and in vitro select ribozymes that can be activated by these molecules in conjunction with UV light. Moreover, RNA aptamers will be evolved in a way that their binding to these molecules can be modulated with UV light. 2. Develop systems for photochemically mediated gene regulation. Specifically we will demonstrate spatio- temporal control of gene knock-down in zebrafish embryos in an irreversible and a reversible manner using irradiation with UV light. 3. Develop systems for photochemically mediated transgene regulation. Specifically, we will demonstrate spatio-temporal control of transgene expression in zebrafish embryos in an irreversible and a reversible manner using irradiation with UV light. All developed components (e.g. photoresponsive small organic molecules, allosteric ribozymes, RNA. aptamers, expression plasmids, etc.) and protocols (e.g. for organic synthesis, UV irradiation, etc.) will be made accessible to the scientific community via a dedicated web-page.