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

描述（由申请人提供）：斑马鱼（Danio rerio）已经成为研究人类疾病和发育遗传学的强大模型；然而，目前还没有一种机制能够以一种可切换的、时空的方式解剖胚胎基因功能。本项目的总体目标是通过光照射开发斑马鱼基因和转基因表达的时空控制的多种机制。具体目的是利用小的光激活（光笼）和光开关有机化合物，利用紫外线严格调节核酶和核开关功能。这些基于RNA的方法将用于控制发育中的斑马鱼胚胎中的基因和转基因表达。我们的策略是基于：1)合成光激活和光切换化合物的能力，2)选择体外变构核酶的能力，其活性依赖于特定有机化合物的存在，3)选择体外选择性结合有机化合物的短RNA序列（RNA适体）的能力，以及4)结合1-3的能力来创建光敏开关，用于斑马鱼基因表达的激活和失活。具体目标是：1。开发RNA功能光化学控制系统。我们将合成无毒、细胞可渗透、光响应的有机小分子，并在体外选择可以被这些分子与紫外线一起激活的核酶。此外，RNA适体将进化成一种与这些分子的结合可以用紫外线调节的方式。2. 开发光化学介导的基因调控系统。具体来说，我们将展示斑马鱼胚胎基因敲除的时空控制，以不可逆和可逆的方式使用紫外线照射。3. 开发光化学介导的转基因调控系统。具体来说，我们将通过紫外线照射，以不可逆和可逆的方式展示斑马鱼胚胎中转基因表达的时空控制。所有已开发的成分（如光反应小有机分子、变构核酶、RNA）。适配体，表达质粒等)和方案（例如有机合成，紫外线照射等）将通过专门的网页向科学界开放。

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.