Gene Targeting in NOD Mouse Embryos Using Zinc-Finger Nucleases

  1. Aron M. Geurts2,5,6
  1. 1Max McGee National Research Center for Juvenile Diabetes, Medical College of Wisconsin, Milwaukee, WI
  2. 2Human and Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, WI
  3. 3Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI
  4. 4Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI
  5. 5Cardiovascular Center, Medical College of Wisconsin, Milwaukee, WI
  6. 6Department of Physiology, Medical College of Wisconsin, Milwaukee, WI
  1. Corresponding author: Yi-Guang Chen, yichen{at}mcw.edu.

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.

  • Received February 4, 2013.
  • Accepted August 19, 2013.

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  1. Diabetes vol. 63 no. 1 68-74
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