Characterization of retinal function and morphology in GNAT1-edited Xenopus laevis
department or school: Medicine, Faculty of
department or school: Medicine, Department of
data provider: DSpace
degree campus: UBCV
creator: Skitsko, Dominic
issued: 2024-05-21T16:18:45Z
issued: 2024
related degree: Master of Science - MSc
degree grantor: University of British Columbia
description: GNAT1 is the α-subunit of a heterotrimeric G-protein present in rod photoreceptors that plays a critical role in phototransduction. Photoactivated rhodopsin binds GNAT1, which stimulates phosphodiesterase to hydrolyze cGMP, closing ion channels and hyperpolarizing the cell. Mutations in human GNAT1 cause Congenital Stationary Night Blindness. Experiments in knock-out GNAT1-/- mice have observed an absent rod ERG with largely unaffected morphology (Calvert et al., 2000). We sought to generate GNAT1-KO Xenopus laevis, taking advantage of their genetic similarity to humans and large photoreceptor size to study anatomically normal but non-functional photoreceptors. We have previously generated F0 GNAT1-KO animals with CRISPR technology, but these animals are genetically chimeric. Therefore, we interbred these F0 animals to produce F1 animals of defined genotypes. Investigation of F1 animals via western blot, histology, electroretinography (ERG), and DNA sequencing has identified true and functional knockouts, a relationship between GNAT1 expression and phototransduction kinetics, and mutations that alter trafficking. X. laevis tadpoles with nonsense mutations in all four GNAT1 alleles presented absent scotopic ERGs. Histology and western blot confirmed the absence of GNAT1 protein, indicating full knockout. Reduction in the level of GNAT1 protein led to a suppression of the ERG, however the relationship was not as direct as expected. We also noted a delay in the timing of the A and B-waves. Greater reduction in GNAT1 protein was associated with greater suppression and delay of the ERG, indicating a second functional role of GNAT1 in addition to signal transduction. We identified two distinct mutations tied to unexpected phenotypes. GNAT1.L-Q184L conferred an ERG phenotype similar to full knockout animals but with minimally reduced GNAT1 protein. A three base pair deletion (T183,Q184→K183) in GNAT1.S caused abnormal rod morphology at the connecting cilium, diminished ERG…
aggregated CHO: https://circle.library.ubc.ca/rest/handle/2429/88339
note: Characterization of Retinal Function and Morphology in GNAT1-Edited Xenopus laevis by Dominic Skitsko B.Sc., University of Alberta, 2020 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) May 2024 © Dominic Skitsko, 2024 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Characterization of Retinal Function and Morphology in GNAT1-Edited Xenopus laevis submitted by Dominic Skitsko in partial fulfilment of the requirements for the degree of Master of Science in Experimental Medicine Examining Committee: Dr Orson Moritz, Professor, Department of Ophthalmology and Visual Sciences, UBC Supervisor Dr Robert Molday, Professor, Department of Biochemistry and Molecular Biology, UBC Supervisory Committee Member Dr Hakima Moukhles, Associate Professor, Department of Cellular and Physiological Sciences, UBC Additional Examiner Additional Supervisory Committee Members: Dr Myeong Jin Ju, Assistant Professor, Department of Ophthalmology and Visual Sciences and School of Biomedical Engineering, UBC Supervisory Committee Member iii Abstract GNAT1 is the a-subunit of a heterotrimeric G-protein present in rod photoreceptors that plays a critical role in phototransduction. Photoactivated rhodopsin binds GNAT1, which stimulates phosphodiesterase to hydrolyze cGMP, closing ion channels and hyperpolarizing the cell. Mutations in human GNAT1 cause Congenital Stationary Night Blindness. Experiments in knock-out GNAT1-/- mice have observed an absent rod ERG with largely unaffected morphology (Calvert et al., 2000). We sought to generate GNAT1-KO Xenopus laevis, taking advantage of their genetic similarity to humans and large photoreceptor size to study anatomically normal but non-functional photoreceptors. We have p…
has type: Thesis/Dissertation
date issued: 2024-11
is shown at: 10.14288/1.0443570
language: eng
degree discipline: Experimental Medicine
provider: Vancouver : University of British Columbia Library
publisher: University of British Columbia
rights: Attribution-NonCommercial-NoDerivatives 4.0 International
rights URI: http://creativecommons.org/licenses/by-nc-nd/4.0/
scholar level: Graduate
contributor: Moritz, Orson L.
type: Text
identifier URI: http://hdl.handle.net/2429/88339