The NOD Mouse: A Model for Insulin‐Dependent Diabetes Mellitus

Edward H. Leiter1

1 The Jackson Laboratory, Bar Harbor, Maine
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 15.9
DOI:  10.1002/0471142735.im1509s24
Online Posting Date:  May, 2001
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Nonobese diabetic (NOD) mice spontaneously develop autoimmune T cell‐mediated insulin‐dependent diabetes mellitus (IDDM). This unit presents a protocol for maintaining NOD mice under conditions permissive to full expression of their autoimmune potential. Methods are also described for diagnosing IDDM on the basis of glycosuria and glycosemia as well as for the semiquantitation of insulitis, a valuable subphenotype diagnostic of prediabetes in these mice, including a procedure for aldehyde fuchsin staining to identify β granules in β islet cells for diagnostic purposes. An adoptive‐transfer method is also included in which leukocytes, purified T cells, or T cell infiltrates obtained from the insulitic pancreas tissue of NOD mice are injected into prediabetic NOD or diabetes‐resistant F1 mice, which then develop disease in an accelerated fashion. This protocol also includes alternative steps in which bone‐marrow cells from NOD mice are transferred to syngeneic, irradiated NOD mice, allowing for reconsistution with a diabetogenic immune system. Steps for isolating pancreatic islet cells, which can then be used for a variety of purposes (e.g., as a source of islet antigens to establish and maintain autoreactive T cell lines) are included. Finally, steps are outlined that can be used to introduce transgenes into NOD mice. This protocol also discusses important considerations for introduction of targeted mutations produced in embryonic stem cells derived from other inbred strains, or introduction of other genes from non‐diabetes‐prone strains.

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Table of Contents

  • Basic Protocol 1: Maintenance of Specific‐pathogen‐free (SPF) Nod Mice
  • Basic Protocol 2: Diagnosis of Insulin‐dependent Diabetes Mellitus (IDDM)
  • Alternate Protocol 1: Semiquantitation of Insulitis as a Subclinical Phenotype of Progression to Insulin‐dependent Diabetes Mellitus
  • Basic Protocol 3: Adoptive Transfer of IDDM into Nod and Nod/ltsz‐scid/scid MICE
  • Support Protocol 1: Isolation of Pancreatic Islet Cells
  • Basic Protocol 4: Production of Transgenic Nod Stocks
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Maintenance of Specific‐pathogen‐free (SPF) Nod Mice

  • NOD and control mice (see Table 15.9.1 for control strains)
  • Appropriate SPF vivarium (see ; also see unit 1.2)
  • Autoclaved white pine shavings or cellulose paper bedding (Alpha‐Dri, Shepard Specialty Papers)
  • Autoclaved mouse food (Purina 5001, Purina NIH‐31, or equivalent)
  • Acidified water (pH 2.8 to 3.2, adjusted using HCl) or hyperchlorinated water containing 10 ppm sodium hypochlorite
  • Additional reagents and equipment for diagnosis of IDDM (see protocol 2 and protocol 3; also see unit 15.3)

Basic Protocol 2: Diagnosis of Insulin‐dependent Diabetes Mellitus (IDDM)

  • NOD mice (see protocol 1)
  • recipeModified Bouin's fixative (see recipe)
  • 50%, 70%, 80%, 95%, and 100% ethanol
  • 1:1 (v/v) 100% xylene/100% ethanol
  • Xylene
  • recipeAldehyde fuchsin stain (see recipe)
  • Mayer's hematoxylin (e.g., Sigma)
  • 1% (w/v) eosin Y in 80% ethanol
  • Mounting medium (e.g., Permount, HSR, or Coverbond)
  • Dissecting instruments
  • Histology or pathology laboratory equipped for paraffin embedding and sectioning
  • Glass microscope slides
  • Staining dishes
  • Coverslips
  • Additional reagents and equipment for euthanasia of mice by carbon dioxide asphyxiation (unit 1.8)

Alternate Protocol 1: Semiquantitation of Insulitis as a Subclinical Phenotype of Progression to Insulin‐dependent Diabetes Mellitus

  • Donor strain: diabetic or prediabetic NOD or NOD/LtSz‐scid/scid mice (see protocol 1)
  • Recipient strain: NOD, NOD/LtSz‐scid/scid, or IDDM‐resistant F1 mice (choice of strain is at the discretion of the investigator)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 137Cs γ irradiator (e.g., Gammacell 1000, Nordion)
  • Additional reagents and equipment for preparing spleen or lymph node cells (unit 3.1), enrichmentunit 3.2 or ), T cell fractionation (unit 1.6), intravenous injection (unit 6.4), preparation of bone‐marrow cells (unit 3.1), and diagnosis of IDDM (see protocol 2 and protocol 3)

Basic Protocol 3: Adoptive Transfer of IDDM into Nod and Nod/ltsz‐scid/scid MICE

  • NOD mice or controls (see protocol 1)
  • 70% ethanol
  • recipeDNase/collagenase P working solution (see recipe), freshly prepared
  • recipeSupplemented HBSS (see recipe)
  • Dissecting microscope with reflected lighting
  • Artery clamp
  • 30‐G, 0.5‐in. hypodermic needle
  • 10‐ml syringe
  • 60‐mm glass petri dish, sterile
  • Silanized glass micropipet: hand‐pulled from silanized soft‐glass tubing (PROSIL28 from PCR) according to manufacturer's instructions
  • Tabletop centrifuge
  • Amber rubber tubing connected to gentle vacuum, preferably applied by turning a micrometer‐operated suction device
  • Additional reagents and equipment for euthanasia of mice (unit 1.8), T cell enrichment (unit 3.2 or unit 3.3), and T cell fractionation (unit 3.5)

Support Protocol 1: Isolation of Pancreatic Islet Cells

  • DNA construct of interest
  • TE buffer, pH 7.5 ( appendix 2A)
  • NOD mice (females, 10‐ to 12‐weeks old, and “stud” males, usually 10‐ to 12‐weeks old)
  • recipeSupplemented MEM (see recipe)
  • Nonalbino pseudopregnant females: e.g., (C57BL/6J × BALB/cByJ)F1 or (C57BL/6J × SJL/J)F1 females
  • Kit for purifying DNA construct: e.g., QiaQuick (Qiagen) or GeneClean (Bio 101)
  • Equipment for microinjection (see Hogan et al., )
NOTE: It is critical to use the mostly highly purified water available (18 MΩ obtained using Mill‐Q still or equivalent) for all reagents. The specialist operating the transgenic facility will normally supply this, or it can be purchased from Life Technologies).
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Key References
   Kikutani and Makino, 1989. See above.
   Summarizes immunology and immunopathology of NOD mice.
   Wicker et al., 1995. See above.
   Summarizes immunogenetics of NOD mice.
   Leiter and Atkinson, 1998. See above.
   Covers husbandry, related strains, immunogenetics, T cell biology, antigen‐presenting cell defects, immunopathology, and multiple uses of NOD‐scid/scid mice.
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