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

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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.