Auxin-inducible protein degradation as a novel approach for protein depletion and reverse genetic discoveries in mammalian oocytes

Auxin-inducible protein degradation as a novel approach for protein depletion and reverse genetic... Abstract The disruption of protein expression is a major approach used for investigating protein function in mammalian oocytes. This is often achieved with RNAi/morpholino-mediated knockdown or gene knockout, leading to long-term loss of proteins of interest. However, these methods have noteworthy limitations, including (a) slow protein turnover can prohibit use of these approaches; (b) essential roles in early events precludes characterization of functions in subsequent events; (c) extended protein loss can allow time for compensatory mechanisms and other unanticipated events that confound interpretation of results. The work presented here examines the use of auxin-inducible degradation, a powerful new approach that overcomes these limitations through the depletion of one's protein of interest through controllable ubiquitin-mediated degradation. This method has been employed in yeast and mammalian cell lines, and here we demonstrate the utility of auxin-inducible degradation in mouse oocytes at multiple stages of meiosis, through degradation of exogenously expressed EGFP. We also evaluate important parameters for experimental design for use of this system in oocytes. This study thus expands the toolkit of researchers in oocyte biology, establishing the use of this unique and versatile approach for depleting proteins in oocytes, and providing researchers with valuable information to make use of this system. Notes Grant Support: Work was funded by NIH grant HD087561 to JPE. Conference Presentation: Presented in part at the 51th Annual Meeting of the Society for the Study of Reproduction, July 10–13, 2018, New Orleans, Louisiana, USA. Author notes Current address: Department of Biological Sciences, Purdue University, West Lafayette, IN. 47907 © The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biology of Reproduction Oxford University Press

Auxin-inducible protein degradation as a novel approach for protein depletion and reverse genetic discoveries in mammalian oocytes

Biology of Reproduction, Volume Advance Article – Jan 9, 18

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Publisher
Oxford University Press
Copyright
© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.
ISSN
0006-3363
eISSN
1529-7268
D.O.I.
10.1093/biolre/ioz113
Publisher site
See Article on Publisher Site

Abstract

Abstract The disruption of protein expression is a major approach used for investigating protein function in mammalian oocytes. This is often achieved with RNAi/morpholino-mediated knockdown or gene knockout, leading to long-term loss of proteins of interest. However, these methods have noteworthy limitations, including (a) slow protein turnover can prohibit use of these approaches; (b) essential roles in early events precludes characterization of functions in subsequent events; (c) extended protein loss can allow time for compensatory mechanisms and other unanticipated events that confound interpretation of results. The work presented here examines the use of auxin-inducible degradation, a powerful new approach that overcomes these limitations through the depletion of one's protein of interest through controllable ubiquitin-mediated degradation. This method has been employed in yeast and mammalian cell lines, and here we demonstrate the utility of auxin-inducible degradation in mouse oocytes at multiple stages of meiosis, through degradation of exogenously expressed EGFP. We also evaluate important parameters for experimental design for use of this system in oocytes. This study thus expands the toolkit of researchers in oocyte biology, establishing the use of this unique and versatile approach for depleting proteins in oocytes, and providing researchers with valuable information to make use of this system. Notes Grant Support: Work was funded by NIH grant HD087561 to JPE. Conference Presentation: Presented in part at the 51th Annual Meeting of the Society for the Study of Reproduction, July 10–13, 2018, New Orleans, Louisiana, USA. Author notes Current address: Department of Biological Sciences, Purdue University, West Lafayette, IN. 47907 © The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Journal

Biology of ReproductionOxford University Press

Published: Jan 9, 18

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