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

用于斑马鱼基因表达时空控制的可切换系统

• 批准号: 8004512
• 负责人: Alexander Deiters
• 金额: $ 14.27万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-08 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8004512
• 关键词: Binding; Biological; Catalytic RNA; Cells; Chemicals; Communities; Development; Down-Regulation; Embryo; Embryonic Development; Engineering; Functional RNA; Gene Expression; Gene Expression Regulation; Genes; Genetic; Goals; In Vitro; Individual; Isomerism; Light; Mediating; Methodology; Methods; Modeling; Oligonucleotides; Organic Synthesis; Organism; Plasmids; Protocols documentation; RNA; RNA Binding; RNA Interference; RNA Sequences; Receptor Cell; Recovery; Regulation; Research Personnel; Resolution; Switch Genes; System; Technology; Toxic effect; Transgenes; Transgenic Organisms; Ultraviolet Rays; Zebrafish; aptamer; base; biological systems; gene function; human disease; infancy; interest; irradiation; knock-down; novel; phosphodiester; programs; receptor; small molecule; spatiotemporal; tool; transgene expression; ultraviolet irradiation; web page

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.

描述（由申请人提供）：斑马鱼（Danio Rerio）已成为研究人类疾病和发育遗传学的有力模型。但是，目前尚无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，用于通过光照射通过光照射在斑马鱼中对基因和转基因表达的时空控制。具体意图是使用紫外线使用紫外线，利用小型的光活化（照相）和可切换有机化合物来严格调节核酶和核糖开关功能。这些基于RNA的方法将用于控制发展中斑马鱼胚胎中的基因和转基因表达。我们的策略基于：1）能够在体外选择适当的光活化和照片切换化合物，2）在体外选择变构核激素的能力，其活性依赖于特定的有机化合物的存在，3）能够选择短的RNA序列（RNA APATAMES）在体外粘结的能力，从而可以选择有机化的能力，从而可以选择有机化的能力，以组合有机化的能力，并结合有机化的能力，并组合4）。斑马鱼基因表达的激活和失活。具体目的是：1。开发用于RNA功能的光化学控制的系统。我们将合成无毒的，可渗透的，光子响应的小有机分子和体外选择的核酶，这些分子可以与紫外线一起激活。此外，RNA适体将以一种与这些分子结合的方式进化，可以用紫外线调节。 2。开发用于光化学介导的基因调节的系统。具体而言，我们将使用带有紫外线的辐射以不可逆的和可逆的方式来证明斑马鱼胚胎中基因敲低的时空控制。 3。开发用于光化学介导的转基因调节的系统。具体而言，我们将使用紫外线的照射以不可逆的和可逆的方式来证明斑马鱼胚胎中转基因表达的时空控制。所有开发的成分（例如光辅助小有机分子，变构核酶，RNA。适体，表达质粒等）和协议（例如，用于有机合成，紫外线照射等），可以通过专用的网页访问科学社区。