Gene Targeting in NOD Mouse Embryos Using Zinc-Finger Nucleases

Studies in NOD mice have provided important insight into the genetics and pathogenesis of type 1 diabetes (T1D). Our goal was to further explore novel methods of genetic manipulation in this mouse model. We tested the feasibility of using zinc-finger nucleases (ZFNs) to knock out a gene directly in...

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Main Authors:	Chen, Yi-Guang, Forsberg, Matthew H., Khaja, Shamim, Ciecko, Ashley E., Hessner, Martin J., Geurts, Aron M.
Format:	Online
Language:	English
Published:	American Diabetes Association 2014
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3868049/

id	pubmed-3868049
recordtype	oai_dc
spelling	pubmed-38680492015-01-01 Gene Targeting in NOD Mouse Embryos Using Zinc-Finger Nucleases Chen, Yi-Guang Forsberg, Matthew H. Khaja, Shamim Ciecko, Ashley E. Hessner, Martin J. Geurts, Aron M. Technological Advances Studies in NOD mice have provided important insight into the genetics and pathogenesis of type 1 diabetes (T1D). Our goal was to further explore novel methods of genetic manipulation in this mouse model. We tested the feasibility of using zinc-finger nucleases (ZFNs) to knock out a gene directly in a pure NOD background, bypassing the need of embryonic stem cells. We report here the successful application of ZFN pairs to specifically and efficiently knock out Tnfrsf9 (encoding CD137/4–1BB) directly in the NOD mouse by embryo microinjection. Histology and T1D incidence studies indicated that CD137 was dispensable for the development of insulitis but played a role to promote progression to overt diabetes in NOD mice. We also demonstrated that CD137-deficient T-cells were less diabetogenic than their wild-type counterpart when adoptively transferred into NOD.Rag1−/− recipients, even when CD25+ cells were predepleted. In vitro assays suggested that CD137 deficiency had a limited effect on the suppressive function of CD4+CD25+ regulatory T-cells (Tregs). Therefore, CD137 deficiency predominately affected effector T-cells rather than Tregs. Our study demonstrates the ability to generate gene-targeted knockouts in a pure NOD background by using ZFNs without potential confounding factors introduced by contaminating genetic materials obtained from other strains. American Diabetes Association 2014-01 2013-12-13 /pmc/articles/PMC3868049/ /pubmed/23974926 http://dx.doi.org/10.2337/db13-0192 Text en © 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Chen, Yi-Guang Forsberg, Matthew H. Khaja, Shamim Ciecko, Ashley E. Hessner, Martin J. Geurts, Aron M.
spellingShingle	Chen, Yi-Guang Forsberg, Matthew H. Khaja, Shamim Ciecko, Ashley E. Hessner, Martin J. Geurts, Aron M. Gene Targeting in NOD Mouse Embryos Using Zinc-Finger Nucleases
author_facet	Chen, Yi-Guang Forsberg, Matthew H. Khaja, Shamim Ciecko, Ashley E. Hessner, Martin J. Geurts, Aron M.
author_sort	Chen, Yi-Guang
title	Gene Targeting in NOD Mouse Embryos Using Zinc-Finger Nucleases
title_short	Gene Targeting in NOD Mouse Embryos Using Zinc-Finger Nucleases
title_full	Gene Targeting in NOD Mouse Embryos Using Zinc-Finger Nucleases
title_fullStr	Gene Targeting in NOD Mouse Embryos Using Zinc-Finger Nucleases
title_full_unstemmed	Gene Targeting in NOD Mouse Embryos Using Zinc-Finger Nucleases
title_sort	gene targeting in nod mouse embryos using zinc-finger nucleases
description	Studies in NOD mice have provided important insight into the genetics and pathogenesis of type 1 diabetes (T1D). Our goal was to further explore novel methods of genetic manipulation in this mouse model. We tested the feasibility of using zinc-finger nucleases (ZFNs) to knock out a gene directly in a pure NOD background, bypassing the need of embryonic stem cells. We report here the successful application of ZFN pairs to specifically and efficiently knock out Tnfrsf9 (encoding CD137/4–1BB) directly in the NOD mouse by embryo microinjection. Histology and T1D incidence studies indicated that CD137 was dispensable for the development of insulitis but played a role to promote progression to overt diabetes in NOD mice. We also demonstrated that CD137-deficient T-cells were less diabetogenic than their wild-type counterpart when adoptively transferred into NOD.Rag1−/− recipients, even when CD25+ cells were predepleted. In vitro assays suggested that CD137 deficiency had a limited effect on the suppressive function of CD4+CD25+ regulatory T-cells (Tregs). Therefore, CD137 deficiency predominately affected effector T-cells rather than Tregs. Our study demonstrates the ability to generate gene-targeted knockouts in a pure NOD background by using ZFNs without potential confounding factors introduced by contaminating genetic materials obtained from other strains.
publisher	American Diabetes Association
publishDate	2014
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3868049/
_version_	1612039908802691072

Gene Targeting in NOD Mouse Embryos Using Zinc-Finger Nucleases

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