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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Abstract

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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Materials

Basic Protocol 1: Maintenance of Specific‐pathogen‐free (SPF) Nod Mice

  Materials
  • 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)

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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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Figures

Videos

Literature Cited

   Ausubel, F.A., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.) 1997. Current Protocols in Molecular Biology. John Wiley & Sons, New York.
   Bach, J.‐F., Boitard, C., Yasunami, R., and Dardenne, M. 1990. Control of diabetes in NOD mice by suppressor cells. J. Autoimmun. 3:97‐100.
   Bendelac, A., Carnaud, C., Boitard, C., and Bach, J.F. 1987. Syngeneic transfer of autoimmune diabetes from diabetic NOD mice to healthy neonates: Requirement for both L3T4+ and Lyt‐2+ T cells. J. Exp. Med. 166:823‐832.
   Bowman, M.A., Leiter, E.H., and Atkinson, M.A. 1994. Autoimmune diabetes in NOD mice: A genetic programme interruptible by environmental manipulation. Immunol. Today 15:115‐120.
   Christianson, S.W., Shultz, L.D., and Leiter, E.H. 1993. Adoptive transfer of diabetes into immunodeficient NOD‐scid/scid mice: Relative contributions of CD4+ and CD8+ T lymphocytes from diabetic versus prediabetic NOD.NON‐Thy‐1a donors. Diabetes 42:44‐45.
   Coleman, D.L., Kuzava, J.E., and Leiter, E.H. 1990. Effect of diet on the incidence of diabetes in non‐obese diabetic (NOD) mice. Diabetes 39:432‐436.
   Dietrich, W.F., Miller, J., Steen, R., Merchant, M.A., Damronboles, D., Husain, Z., Dredge, R., Daly, M.J., Ingalls, K.A., O'Connor, T.J., Evans, C.A., Deangelis, M.M., Levinson, D.M., Kruglyak, L., Goodman, N., Copeland, N.G., Jenkins, N.A., Hawkins, T.L., Stein, L., Page, D.C., and Lander, E.S. 1996. A comprehensive genetic map of the mouse genome. Nature 380:149‐152.
   Elliott, J.I. and Altmann, D.M. 1996. Non‐obese diabetic mice hemizygous at the T cell receptor alpha locus are susceptible to diabetes and sialitis. Eur. J. Immunol. 26:953‐956.
   Gotoh, M., Maki, T., Kiyoizumi, T., Satomi, S., and Monaco, A.P. 1985. An improved method for the isolation of mouse pancreatic islets. Transplantation 40:437‐438.
   Hamaguchi, K., Gaskins, H.R., and Leiter, E.H. 1991. NIT‐1, a pancreatic β cell line established from a transgenic NOD/Lt mouse. Diabetes 40:842‐849.
   Hanson, M.S., Cetkovic‐Cvrlje, M., Ramiya, V.K., Atkinson, M.A., Maclaren, N.K., Singh, B., Elliot, J.F., Serreze, D.V., and Leiter, E.H. 1996. Quantitative thresholds of MHC class II I‐E expressed on hemopoietically derived antigen‐presenting cells in transgenic NOD/Lt mice determine level of diabetes resistance and indicate mechanism of protection. J. Immunol. 157:1279‐1287.
   Ho, Y., Wigglesworth, K., Eppig, J., and Schultz, R. 1995. Preimplantation development of mouse embryos in KSOM: Augmentation by amino acids and analysis of gene expression. Mol. Reprod. Dev. 41:232‐238.
   Hogan, B., Beddington, R., Costantini, F., and Lacy, E. 1994a. Recovery, culture, and transfer of embryos and germ cells. In Manipulating the Mouse Embryo: A Laboratory Manual (Section C). pp. 127‐188. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Hogan, B., Beddington, R., Costantini, F., and Lacy, E. 1994b. Production of transgenic mice. In Manipulating the Mouse Embryo: A Laboratory Manual, (Section E). pp. 217‐254. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Hoorfar, J., Buschard, K., and Dagnaes‐Hansen, F. 1993. Prophylactic nutritional modification of the incidence of diabetes in autoimmune non‐obese diabetic (NOD) mice. Br. J. Nutr. 69:597‐607.
   Ikegami, H., Eisenbarth, G.S., and Hattori, M. 1990. Major histocompatibility complex‐linked diabetogenic gene of the nonobese diabetic mouse. Analysis of genomic DNA amplified by the polymerase chain reaction. J. Clin. Invest. 85:18‐24.
   Kikutani, H. and Makino, S. 1992. The murine autoimmune diabetes model: NOD and related strains. In Adv. Immunol. (F. J. Dixon, ed.) pp. 285‐322. Academic Press, New York.
   Langmuir, P., Bridgett, M., Bothwell, A., and Crispe, I. 1993. Bone marrow abnormalities in the non‐obese diabetic mouse. Int. Immunol. 5:169‐177.
   Lawitts, J. and Biggers, J. 1993. Culture of preimplantation embryos. In Methods in Enzymology: Guide to Techniques in Mouse Development, (P. Wassarman and M. DePamphilis, eds.) pp. 153‐155. Academic Press, San Diego.
   Leiter, E.H. 1993. The nonobese diabetic mouse: A model for analyzing the interplay between heredity and environment in development of autoimmune disease. ILAR News 35:4‐14.
   Leiter, E.H. and Atkinson, M.A. 1998. NOD mice and related strains: Origin, husbandry, and biology. In NOD Mice and Related Strains: Research Applications in Diabetes, AIDS, Cancer, and Other Diseases. R.G. Landes, Austin, Tex.
   Leiter, E.H. and Serreze, D.V. 1991. Autoimmune diabetes in the nonobese diabetic mouse: Suppression of immune defects by bone marrow transplantation and implications for therapy. Clin. Immunol. Immunopathol. 59:323‐334.
   Leiter, E.H. and Serreze, D.V. 1992. Antigen presenting cells and the immunogenetics of autoimmune diabetes in NOD mice. Reg. Immunol. 4:263‐273.
   Leiter, E.H., Prochazka, M., Coleman, D.L., Serreze, D.V., and Shultz, L.D. 1986. Genetic factors predisposing to diabetes susceptibility in mice. In The Immunology of Diabetes Mellitus, (M.A. Jaworskil, G.D. Molnar, R.V. Rajotte, and B. Singh, eds.) pp. 29‐36. Elsevier/North‐Holland, Amsterdam.
   Liu, M. and Shapiro, M.E. 1995. New method for isolation of murine islets with markedly improved yields. Transplant Proc. 27:3208‐3210.
   Lund, T., O'Reilly, L., Hutchings, P., Kanagawa, O., Simpson, E.R.G., Chandler, P., Dyson, J., Picard, J.K., Edwards, A., Kioussis, D., and Cooke, A. 1990. Prevention of insulin‐dependent diabetes mellitus in non‐obese diabetic mice by transgenes encoding modified I‐A β‐chain or normal I‐E α‐chain. Nature 345:727‐729.
   McAleer, M.A., Reifsnyder, P., Palmer, S.M., Prochazka, M., Love, J.M., Copeman, J.B., Powell, E.E., Rodrigues, N.R., Prins, J.‐B., Serreze, D.V., DeLarto, N.H., Wicker, L.S., Peterson, L.B., Todd, J.A., and Leiter, E.H. 1995. Crosses of NOD mice with the related NON strain: A polygenic model for type I diabetes. Diabetes 44:1186‐1195.
   Miller, B.J., Appel, M.C., O'Neil, J.J., and Wicker, L.S. 1988. Both the Lyt‐2+ and L3T4+ T cell subsets are required for the transfer of diabetes in nonobese diabetic mice. J. Immunol. 140:52‐58.
   Miller, J.C., Dietrich, W.F., Steen, R.G., Joyce, D.C., Merchant, M.A., Wessel, M.T., Damron, D.M., Nahf, R.W., Stein, L.D., Dredge, R.D., Marquis, A.L., Daly, M.J., Reeve, M.P., Goodman, N., Lord, C.J., Montague, C.T., Prins, J.B., Todd, J.A., and Lander, E.S. 1996. SSLP/microsatellite genetic linkage map of the mouse. In Genetic Variants and Strains of the Laboratory Mouse, 3rd ed., (M. Lyons, S. Rastan, and S. Brown, eds.) pp. 1671‐1755. Oxford University Press, Oxford.
   Ohsugi, T. and Kurosawa, T. 1994. Increased incidence of diabetes mellitus in specific pathogen–eliminated offspring produced by embryo transfer in NOD mice with low incidence of the disease. Lab. Anim. Sci. 44:386‐388.
   Pozzilli, P., Signore, A., Williams, A., and Beales, P. 1993. NOD mouse colonies around the world: Recent facts and figures. Immunol. Today 14:193‐196.
   Prochazka, M., Serreze, D.V., Frankel, W.N., and Leiter, E.H. 1992a. NOR/Lt; MHC‐matched diabetes‐resistant control strain for NOD mice. Diabetes 41:98‐106.
   Prochazka, M., Gaskins, H.R., Shultz, L.D., and Leiter, E.H. 1992b. The NOD‐scid mouse: A model for spontaneous thymomagenesis associated with immunodeficiency. Proc. Natl. Acad. Sci., U.S.A. 89:3290‐3294.
   Rapoport, M., Zipris, D., Lazarus, A., Jaramillo, A., Serreze, D., Leiter, E., Cyopick, P., and Delovitch, T. 1993. IL‐4 reverses thymic T cell anergy and prevents the onset of diabetes in NOD mice. J. Exp. Med. 178:87‐99.
   Serreze, D.V. and Leiter, E.H. 1988. Defective activation of T suppressor cell function in nonobese diabetic mice. Potential relation to cytokine deficiencies. J. Immunol. 140:3801‐3807.
   Serreze, D.V. and Leiter, E.H. 1991. Development of diabetogenic T cells from NOD/Lt marrow is blocked when an allo‐H2 haplotype is expressed on cells of hematopoietic origin but not on thymic epithelium. J. Immunol. 147:1222‐1229.
   Serreze, D.V. and Leiter, E.H. 1994. Genetic and pathogenic basis for autoimmune diabetes in NOD mice. Curr. Opin. Immunol. 6:900‐906.
   Serreze, D.V. and Leiter, E. H. 1995. Insulin dependent diabetes mellitus (IDDM) in NOD mice and BB rats: Origins in hematopoietic stem cell defects and implications for therapy. In Lessons from Animal Diabetes. V (E. Shafrir, ed.) pp. 59‐73. Smith‐Gordon, London.
   Serreze, D.V., Hamaguchi, K., and Leiter, E.H. 1990. Immunostimulation circumvents diabetes in NOD/Lt mice. J. Autoimmun. 2:759‐776.
   Serreze, D.V., Gaskins, H.R., and Leiter, E.H. 1993. Defects in the differentiation and function of antigen presenting cells in NOD/Lt mice. J. Immunol. 150:2534‐2543.
   Serreze, D.V., Prochazka, M., Reifsnyder, P.C., Bridgett, M., and Leiter, E. 1994a. Use of recombinant congenic and congenic strains of NOD mice to identify a new insulin dependent diabetes resistance gene. J. Exp. Med. 180:1553‐1558.
   Serreze, D.V., Leiter, E.H., Christianson, G.J., Greiner, D., and Roopenian, D.C. 1994b. MHC class I deficient NOD‐B2mnull mice are diabetes and insulitis resistant. Diabetes 43:505‐509.
   Serreze, D.V., Leiter, E.H., Hanson, M.S., Christianson, S.W., Shultz, L.D., Hesselton, R.A.M., and Greiner, D.L. 1995. Emv30null NOD‐scid mice: An improved host for adoptive transfer of autoimmune diabetes and growth of human lymphohematopoietic cells. Diabetes 44:1392‐1398.
   Serreze, D.V., Gallichin, W.S., Snider, D.P., Croituru, K., Rosenthal, K.L., Leiter, E.H., Christianson, G.J., Dudley, M.E., and Roopenian, D.C. 1996. MHC class I—mediated antigen presentation and induction of CD8+ cytotoxic T lymphocyte responses in autoimmune diabetes—prone NOD mice. Diabetes 45:902‐908.
   Serreze, D.V., Chapman, H.C., Varnum, D.S., Gerling, I., Leiter, E.H., and Shultz, L.D. 1997. Initiation of autoimmune diabetes in NOD/Lt mice is MHC class ‐dependent. J. Immunol. 158:3958‐3986.
   Shultz, L.D., Schweitzer, P.A., Christianson, S.W., Gott, B., Birdsall‐Maller, I., Tennent, B., McKenna, S., Mobraaten, L., Rajan, T.V., Greiner, D.L., and Leiter, E.H. 1995. Multiple defects in innate and adaptive immunological function in NOD/LtSZ‐scid mice. J. Immunol. 154:180‐191.
   Todd, J.A., Aitman, T.J., Cornall, R.J., Ghosh, S., Hall, J.R.S., Hearne, C.M., Knight, A.M., Love, J.M., McAleer, M.A., Prins, J.‐B., Rodrigues, N., Lathrop, M., Pressey, A., DeLarato, N.H., Peterson, L.B., and Wicker, L.S. 1991. Genetic analysis of autoimmune type 1 diabetes mellitus in mice. Nature 351:542‐547.
   Uehira, M., Uno, M., Kurner, T., Kikutani, H., Mori, K., Inomoto, K., Uede, T., Miyazaki, J., Nishimoto, H., Kishimoto, T., and Yamamura, K. 1989. Development of autoimmune insulitis is prevented in Eαd but not in Aβk NOD transgenic mice. Int. Immunol. 1:209‐213.
   Varlamov, O., Fricker, L., Furukawa, H., Steiner, D., Langley, S., and Leiter, E.H. 1997. β cell lines derived from transgenic Cpefat/Cpefat mice are defective in carboxypeptidase E and proinsulin processing. Endocrinology 148:4883‐4888.
   Wicker, L.S., Appel, M.C., Dotta, F., Pressey, A., Miller, B.J., DeLarato, N.H., Fischer, P.A., Boltz, R.C., and Peterson, L.B. 1992. Autoimmune syndromes in major histocompatibility complex (MHC) congenic strains of nonobese diabetic (NOD) mice. The NOD MHC is dominant for insulitis and cyclophosphamide‐induced diabetes. J. Exp. Med. 176:67‐77.
   Wicker, L.S., Leiter, E.H., Todd, J.A., Renjilian, R.J., Peterson, E., Fischer, P.A., Podolin, P.L., Zijlstra, M., Jaenisch, R., and Peterson, L.B. 1994. β2 microglobulin‐deficient NOD mice do not develop insulitis or diabetes. Diabetes 53:500‐504.
   Wicker, L.S., Todd, J.A., and Peterson, L.B. 1995. Genetic control of autoimmune diabetes in the NOD mouse. Annu. Rev. Immunol. 13:179‐200.
   Wong, S., Guerder, S., Visintin, I., Reich, E.P., Swenson, K.E., Flavell, R.A., and Janeway, C.A. 1995. Expression of the co‐stimulator molecule B7‐1 in pancreatic beta‐cells accelerates diabetes in the NOD mouse. Diabetes 44:326‐328.
   Zeller, R. 1989. Fixation, embedding, and sectioning of tissues, embryos, and single cells. In Currents in Molecular Biology (Ausubel, F., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K., eds.) pp. 14.1.1‐14.1.8. John Wiley & Sons, New York.
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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