title: Gene targeting in NOD mouse embryos using zinc-finger nucleases.
abstract: 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.
authors: Max McGee National Research Center for Juvenile Diabetes, Medical College of Wisconsin, Milwaukee, WI.
authors: Yi-Guang
authors: YG
authors: Chen
authors: Matthew H
authors: MH
authors: Forsberg
authors: Shamim
authors: S
authors: Khaja
authors: Ashley E
authors: AE
authors: Ciecko
authors: Martin J
authors: MJ
authors: Hessner
authors: Aron M
authors: AM
authors: Geurts
mesh_terms: D000818:Animals; D003922:Diabetes Mellitus, Type 1 / Q000235:genetics* / Q000276:immunology; D055786:Gene Knockout Techniques / Q000379:methods*; D051379:Mice; D016688:Mice, Inbred NOD; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9 / Q000235:genetics / Q000276:immunology; D016335:Zinc Fingers / Q000235:genetics* / Q000276:immunology
chemical_list: D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9
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