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An efficient method for generation of bi-allelic null mutant mouse embryonic stem cells and its application for investigating epigenetic modifiers.

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Title: An efficient method for generation of bi-allelic null mutant mouse embryonic stem cells and its application for investigating epigenetic modifiers.
Authors: Fisher, CL
Marks, H
Cho, LT-Y
Andrews, R
Wormald, S
Carroll, T
Iyer, V
Tate, P
Rosen, B
Stunnenberg, HG
Fisher, AG
Skarnes, WC
Item Type: Journal Article
Abstract: Mouse embryonic stem (ES) cells are a popular model system to study biological processes, though uncovering recessive phenotypes requires inactivating both alleles. Building upon resources from the International Knockout Mouse Consortium (IKMC), we developed a targeting vector for second allele inactivation in conditional-ready IKMC 'knockout-first' ES cell lines. We applied our technology to several epigenetic regulators, recovering bi-allelic targeted clones with a high efficiency of 60% and used Flp recombinase to restore expression in two null cell lines to demonstrate how our system confirms causality through mutant phenotype reversion. We designed our strategy to select against re-targeting the 'knockout-first' allele and identify essential genes in ES cells, including the histone methyltransferase Setdb1. For confirmation, we exploited the flexibility of our system, enabling tamoxifen inducible conditional gene ablation while controlling for genetic background and tamoxifen effects. Setdb1 ablated ES cells exhibit severe growth inhibition, which is not rescued by exogenous Nanog expression or culturing in naive pluripotency '2i' media, suggesting that the self-renewal defect is mediated through pluripotency network independent pathways. Our strategy to generate null mutant mouse ES cells is applicable to thousands of genes and repurposes existing IKMC Intermediate Vectors.
Issue Date: 13-Sep-2017
Date of Acceptance: 4-Sep-2017
URI: http://hdl.handle.net/10044/1/52092
DOI: https://dx.doi.org/10.1093/nar/gkx811
ISSN: 0305-1048
Publisher: Oxford University Press
Start Page: e174
End Page: e174
Journal / Book Title: Nucleic Acids Research
Volume: 45
Issue: 21
Copyright Statement: © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Keywords: 05 Environmental Sciences
06 Biological Sciences
08 Information And Computing Sciences
Developmental Biology
Publication Status: Published online
Open Access location: https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/gkx811
Appears in Collections:Clinical Sciences
Molecular Sciences
Faculty of Medicine



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