Rector & Visitors of The University of Virginia
Grant: $158,877 - National Institutes of Health - Jul. 17, 2009
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Award Description: Gene manipulation in vivo using transgenic or targeted gene deletion can result in embryonic lethality, developmental defects or compensation by other gene products, all of which confound interpretation of normal gene function. Alternate strategies have been developped that allow for ear specific gene deletion or expression but do not allow for on/off control of gene expression. The goal ofthis project is to design a novel system that permits precise spatiotemporal control of gene expression which can be used to examine the function of any gene of interest specifically in hair cells. We are constructing a hair cell-specific, inducible gene expression system based on the lac operatorrepressor regulatory system. In the lac system, regulated gene expression results from binding/unbinding ofLacI protein to lac operator sequences introduced in the promoter sequence to flank the transcriptional start site. In the repressed state LacI is bound and transcription is blocked. In the derepressed state, exposure to the inducer a lactose analogue (IPTG) releases LacI from the operators, which turns on transcription. Since the regulation is reversible, addition or removal of IPTG can be used to tum on or off gene expression at any point during development or in the mature animal. We have modified the hair cell specific myosin 7A promoter by inserting lac operators (MYO-lacO) and successfully controled gene expression in vitro. We now propose to use this promoter to produce a transgenic model in which we will reversibly express a gene that we can monitor in hair cells and whose functions are of interest for the field of auditory neuroscience. For these reasons, we now propose to make a MY07A lacO-EGFP::ACTB transgenic mouse. We will analyze expression ofGFP labeled Actin in repressed and derepressed conditions (with or without IPTG) in vitro and in vivo. Doing so we will be able to determine protocols to follow to optimize the use of our transgenic model. We will also be able to analyze incorporation and renewal of actin into stereocilia and study stereocilia turnover and length regulation. Once validated and characterized, the MY07A-lacO transgenic model will be made available to the scientific community. Hair cell-specific, regulatable gene expression will be a valuable tool for understanding gene function during development and in age-related hearing loss. RELEVANCE: We are constructing a novel trangenic mouse model which will allow hair cell-specific, regulatable gene expression. This model will be made available to the scientific community once validated. It will become a valuable tool used to understand gene function during development and age-related hearing loss.
Project Description: See Award Description; Recruitment for a research fellow is underway as well as preparation of the final construct that will be used to make the transgenic mouse and will soon be sent to the UVA transgenic core facility.
Jobs Summary: None (Total jobs reported: 0)
Project Status: Less Than 50% Completed
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