Creation of “Humanized” Mice to Study Human Immunity

Todd Pearson1, Dale L. Greiner2, Leonard D. Shultz2

1 Diabetes Division, University of Massachusetts Medical School, Worcester, Massachusetts, 2 The Jackson Laboratory, Bar Harbor, Maine
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 15.21
DOI:  10.1002/0471142735.im1521s81
Online Posting Date:  May, 2008
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Abstract

“Humanized” mice are a promising translational model for studying human hematopoiesis and immunity. Their utility has been enhanced by the development of new stocks of immunodeficient hosts, most notably mouse strains such as NOD‐scid IL2rγnull mice that lack the IL‐2 receptor common gamma chain. These stocks of mice lack adaptive immune function, display multiple defects in innate immunity, and support heightened levels of human hematolymphoid engraftment. Humanized mice can support studies in many areas of immunology, including autoimmunity, transplantation, infectious diseases, and cancer. These models are particularly valuable in experimentation where there is no appropriate small animal model of the human disease, as in the case of certain viral infections. This unit details the creation of humanized mice by engraftment of immunodeficient mice with hematopoietic stem cells or peripheral blood mononuclear cells, provides methods for evaluating engraftment, and discusses considerations for choosing the appropriate model system to meet specific goals. Curr. Protoc. Immunol. 81:15.21.1‐15.21.21. © 2008 by John Wiley & Sons, Inc.

Keywords: humanize mice; NOD‐SCID IL2rγnull; hu‐SRC‐SCID; hu‐PBL‐SCID; stem cell transplantation

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Hematopoietic Stem Cell Engraftment of Adult Immunodeficient Mice
  • Basic Protocol 2: Peripheral Blood Mononuclear Cell Engraftment of Adult Immunodeficient Mice
  • Alternate Protocol 1: Hematopoietic Stem Cell Engraftment of Newborn Immunodeficient Mice
  • Alternate Protocol 2: Peripheral Blood Mononuclear Cell Engraftment Via Intrasplenic Injection
  • Support Protocol 1: Flow Cytometric Analysis of Humanized Mice
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Hematopoietic Stem Cell Engraftment of Adult Immunodeficient Mice

  Materials
  • NOD/Lt‐scid IL2rγnull mice: males or females, 5 to 12 weeks old (official strain designation, NOD.Cg‐Prkdcscid Il2rgtm1Wjl/SzJ; The Jackson Laboratory, 005557; see Internet Resources), housed in microisolator cages
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 137Cs gamma irradiator and autoclaved, filtered, ventilated device for housing mice while in irradiator chamber (design varies, depending on model of irradiator used)
  • Laminar flow biocontainment hood
  • 1‐cc tuberculin syringes with 25‐G × ⅝‐in. needle
  • Additional reagents and equipment for preparing CD3 T cell–depleted HSC (Chapter 22) and restraining and injecting mice (units 1.3 and 1.6)

Basic Protocol 2: Peripheral Blood Mononuclear Cell Engraftment of Adult Immunodeficient Mice

  Materials
  • Ficoll‐isolated human PBMC: 20 × 106 cells per recipient mouse; freshly prepared from whole blood or single cell suspensions of splenocytes (see appendix 3F and unit 7.1)
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • NOD/Lt‐scid IL2rγnull mice: males or females, 5 to 12 weeks old (official strain designation, NOD.Cg‐Prkdcscid Il2rgtm1Wjl/SzJ; The Jackson Laboratory, 005557; see Internet Resources), housed in microisolater cages
  • 1‐cc tuberculin syringes with 25‐G × ⅝‐in. needles
  • Additional reagents and equipment for isolating and enumerating PBMC (unit 7.1) and restraining (unit 1.3) and injecting (unit 1.6) mice

Alternate Protocol 1: Hematopoietic Stem Cell Engraftment of Newborn Immunodeficient Mice

  Materials
  • NOD/Lt‐scid IL2rγnull breeder pairs: as many as are required to produce sufficient newborn mice for experimental needs (official strain designation, NOD.Cg‐Prkdcscid Il2rgtm1Wjl/SzJ; The Jackson Laboratory, 005557; see Internet Resources)
  • Phosphate‐buffered saline (PBS), sterile
  • Topical petrolatum‐based nasal decongestant (e.g., Vicks Vaporub)
  • 6‐well tissue culture plate
  • 100‐mm2 petri dishes (∼1 dish per litter to be injected)
  • 137Cs gamma irradiator and autoclaved, filtered, ventilated device for housing mice while in irradiator chamber (design varies, depending on model of irradiator used)
  • Ice bucket with ice
  • Disposable weigh boats or sterile gauze
  • 1‐cc tuberculin syringes (BD)
  • 27‐G × ½‐in. winged infusion set with 8‐in. tubing (Terumo, SV*27EL)
  • Heating pad or warming lamp
  • Additional reagents and equipment for preparing CD34+ T cell–depleted umbilical cord blood (UCB) hematopoietic stem cells (see Chapter 22)

Alternate Protocol 2: Peripheral Blood Mononuclear Cell Engraftment Via Intrasplenic Injection

  Materials
  • NOD/Lt‐scid IL2rγnull mice: males or females, 5 to 12 weeks old (official strain designation, NOD.Cg‐Prkdcscid Il2rgtm1Wjl/SzJ; The Jackson Laboratory, 005557; see Internet Resources)
  • 20/4 mg/ml ketamine/xylazine in PBS (e.g., ketaset/zylaject, Webster Veterinary Supply)
  • Sterile petrolatum ophthalmic ointment (e.g., Purelube veterinary ointment, Webster Veterinary Supply)
  • Ficoll‐isolated human PBMC, 20 × 106 cells per recipient mouse: freshly prepared from whole blood (see appendix 3F and unit 7.1)
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • 0.01 mg/ml buprenorphine (see recipe)
  • Electric clippers
  • 70% (v/v) ethanol
  • Betadine
  • Sterile surgical instruments: iris scissors, forceps, needle holder
  • 1‐cc syringes with 27‐G × ½‐in. needle
  • Sterile cotton swabs
  • 3‐0 coated vicryl suture
  • Wound clips
  • Sterile gauze
  • Warming pad or warming lamp

Support Protocol 1: Flow Cytometric Analysis of Humanized Mice

  Materials
  • Commonly used antibodies (additional formats available), labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinine chlorophyll‐a protein (PerCP), allophycocyanin (APC):
    • Mouse pan‐CD45, clone 30‐F11, BD Biosciences catalog numbers: 553079 (FITC), 553081 (PE), 557235 (PerCP), 559864 (APC); isotype control, clone A95‐1 (rat IgG2 b, k), BD Biosciences 552991 (PerCP)
    • Human CD45, clone HI30, BD Biosciences 555485 (APC); isotype control, clone MOPC‐21 (mouse IgG1, k), BD Biosciences 555751 (APC)
    • Human CD3, clone UCHT1, BD Biosciences 555333 (PE); isotype control, clone MOPC‐21 (mouse IgG1, k), BD Biosciences 555749 (PE)
    • Human CD20, clone 2H7, BD Biosciences 555622 (FITC); isotype control, clone 27‐35 (mouse IgG2 b, k), BD Biosciences 555742 (FITC)
    • Anti‐FcγRI (Fc Block), clone 2.4G2, BD Biosciences (purified)
  • FACS buffer (see unit 5.3)
  • BD FACS lysing solution (BD), prepared according to manufacturer's instructions
  • 12 × 75–mm round bottom tubes for flow cytometry
  • Flow cytometer capable of supporting four‐color cytometry (e.g., FACScalibur, BD; see unit 5.4)
  • Additional reagents and equipment for collecting blood from mice (unit 1.7)
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Figures

Videos

Literature Cited

   Banuelos, S.J., Shultz, L.D., Greiner, D.L., Burzenski, L.M., Gott, B., Lyons, B.L., Rossini, A.A., and Appel, M.C. 2004. Rejection of human islets and human HLA‐A2.1 transgenic mouse islets by alloreactive human lymphocytes in immunodeficient NOD‐scid and NOD‐Rag1(null)Prf1(null) mice. Clin. Immunol. 112: 273‐283.
   Bente, D.A., Melkus, M.W., Garcia, J.V., and Rico‐Hesse, R. 2005. Dengue fever in humanized NOD/SCID mice. J. Virol. 79: 13797‐13799.
   Bosma, G.C., Custer, R.P., and Bosma, M.J. 1983. A severe combined immunodeficiency mutation in the mouse. Nature 301: 527‐530.
   Cao, X., Shores, E.W., Hu‐Li, J., Anver, M.R., Kelsall, B.L., Russell, S.M., Drago, J., Noguchi, M., Grinberg, A., Bloom, E.T., Paul, W.E., Katz, S.I., Love, P.E., and Leonard, W.J. 1995. Defective lymphoid development in mice lacking expression of the common cytokine receptor gamma chain. Immunity 2: 223‐238.
   Fujii, H., Trudeau, J.D., Teachey, D., Fish, J.D., Grupp, S.A., Schultz, K.R., and Reid, G.S. 2007. In vivo control of acute lymphoblastic leukemia by immunostimulatory CpG oligonucleotides. Blood 109: 2008‐2013.
   Gimeno, R., Weijer, K., Voordouw, A., Uittenbogaart, C.H., Legrand, N., Alves, N.L., Wijnands, E., Blom, B., and Spits, H. 2004. Monitoring the effect of gene silencing by RNA interference in human CD34+ cells injected into newborn RAG2−/− gammac−/− mice: Functional inactivation of p53 in developing T cells. Blood 104: 3886‐3893.
   Hesselton, R.M., Greiner, D.L., Mordes, J.P., Rajan, T.V., Sullivan, J.L., and Shultz, L.D. 1995. High levels of human peripheral blood mononuclear cell engraftment and enhanced susceptibility to human immunodeficiency virus type 1 infection in NOD/LtSz‐scid/scid mice. J. Infect. Dis. 172: 974‐982.
   Holyoake, T.L., Nicolini, F.E., and Eaves, C.J. 1999. Functional differences between transplantable human hematopoietic stem cells from fetal liver, cord blood, and adult marrow. Exp. Hematol. 27: 1418‐1427.
   ILAR. 2003. Institute for Laboratory Animal Research (U.S.) Committee on Guidelines for the Use of Animals in Neuroscience and Behavioral Research: Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. The National Academies Press, Washington, D.C.
   Ishikawa, F., Yasukawa, M., Lyons, B., Yoshida, S., Miyamoto, T., Yoshimoto, G., Watanabe, T., Akashi, K., Shultz, L.D., and Harada, M. 2005. Development of functional human blood and immune systems in NOD/SCID/IL2 receptor {gamma} chain (null) mice. Blood 106: 1565‐1573.
   Ishikawa, F., Shimazu, H., Shultz, L.D., Fukata, M., Nakamura, R., Lyons, B., Shimoda, K., Shimoda, S., Kanemaru, T., Nakamura, K., Ito, H., Kaji, Y., Perry, A.C., and Harada, M. 2006. Purified human hematopoietic stem cells contribute to the generation of cardiomyocytes through cell fusion. FAESB J. 20: 950‐952.
   Ito, M., Hiramatsu, H., Kobayashi, K., Suzue, K., Kawahata, M., Hioki, K., Ueyama, Y., Koyanagi, Y., Sugamura, K., Tsuji, K., Heike, T., and Nakahata, T. 2002. NOD/SCID/gamma(c)(null) mouse: An excellent recipient mouse model for engraftment of human cells. Blood 100: 3175‐3182.
   King, M., Pearson, T., Shultz, L.D., Leif, J., Bottino, R., Trucco, M., Atkinson, M.A., Wasserfall, C., Herold, K.C., Woodland, R.T., Schmidt, M.R., Woda, B.A., Thompson, M.J., Rossini, A.A., and Greiner, D.L. 2008. A new Hu‐PBL model for the study of human islet alloreactivity based on NOD‐scid mice bearing a targeted mutation in the IL‐2 receptor gamma chain gene. Clin. Immunol. 126: 303‐314.
   Lim, W.H., Kireta, S., Russ, G.R., and Coates, P.T. 2007. Human plasmacytoid dendritic cells regulate immune responses to Epstein‐Barr virus (EBV) infection and delay EBV‐related mortality in humanized NOD‐SCID mice. Blood 109: 1043‐1050.
   Nagy, A., 2003. Manipulating the mouse embryo: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Pearson, T., Greiner, D.L., and Shultz, L.D. 2008. Humanized SCID mouse models for biomedical research. In Current Topics in Microbiology and Immunology, Vol. 324. (T. Nomura, T. Watanabe, and S. Habu, eds.) pp. 25‐52. Springer, Berlin.
   Ponomaryov, T., Peled, A., Petit, I., Taichman, R.S., Habler, L., Sandbank, J., Arenzana‐Seisdedos, F., Magerus, A., Caruz, A., Fujii, N., Nagler, A., Lahav, M., Szyper‐Kravitz, M., Zipori, D., and Lapidot, T. 2000. Induction of the chemokine stromal‐derived factor‐1 following DNA damage improves human stem cell function. J. Clin. Invest. 106: 1331‐1339.
   Shultz, L.D., Lyons, B.L., Burzenski, L.M., Gott, B., Chen, X., Chaleff, S., Kotb, M., Gillies, S.D., King, M., Mangada, J., Greiner, D.L., and Handgretinger, R. 2005. Human lymphoid and myeloid cell development in NOD/LtSz‐scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. J. Immunol. 174: 6477‐6489.
   Shultz, L.D., Ishikawa, F., and Greiner, D.L. 2007. Humanized mice in translational biomedical research. Nat. Rev. Immunol. 7: 118‐130.
   Traggiai, E., Chicha, L., Mazzucchelli, L., Bronz, L., Piffaretti, J.C., Lanzavecchia, A., and Manz, M.G. 2004. Development of a human adaptive immune system in cord blood cell–transplanted mice. Science 304: 104‐107.
   Turgeon, N.A., Banuelos, S.J., Shultz, L.D., Lyons, B.L., Iwakoshi, N., Greiner, D.L., Mordes, J.P., Rossini, A.A., and Appel, M.C. 2003. Alloimmune injury and rejection of human skin grafts on human peripheral blood lymphocyte‐reconstituted nonobese diabetic severe combined immunodeficient beta2‐microglobulin‐null mice. Exp. Biol. Med. 228: 1096‐1104.
   Watanabe, S., Terashima, K., Ohta, S., Horibata, S., Yajima, M., Shiozawa, Y., Dewan, M.Z., Yu, Z., Ito, M., Morio, T., Shimizu, N., Honda, M., and Yamamoto, N. 2006. Hematopoietic stem cell‐engrafted NOD/SCID/IL2R{gamma}null mice develop human lymphoid system and induce long‐lasting HIV‐1 infection with specific humoral immune responses. Blood 109: 212‐218.
Internet Resources
   http://www.jax.org
  The Jackson Laboratory. This Web site provides detailed information on nomenclature, genotyping protocols and phenotypes for several of the immunodeficient strains described in this unit, and is the sole repository and provider of the NOD.Cg‐PrkdcscidIl2rgtm1Wjll/SzJ (NOD‐scid IL2rγnull) stock.
   http://jaxmice.jax.org/library/notes/501a.html
  Choosing an Immunodeficient Mouse Model, Jax Notes, Spring 2006
   http://www.taconic.com
  Taconic Farm Web sites. A comprehensive overview of immunodeficient hosts for HSC engraftment, including salient features of each strain, availability, and references. Taconic Farms is another supplier of immunodeficient mice, including CB17‐scid, NOD‐scid, BALB/c‐Rag2null, and “C57BL/6‐Rag2null IL2rgnull (C57BL/6J × C57BL/10SgSnAi)‐[KO]γc‐[KO]Rag2”
   http://www.ndriresource.org
  National Disease Research Exchange (NDRI) is a nonprofit resource providing access (through an application process) to human biomaterials for investigators’ research studies.
   http://www.miltenyibiotec.com/en/NN_625_Hematopoietic_stem_and_progenito_cells.aspx
  Miltenyi Biotec is a commercial supplier of cell separation technologies to isolate/enrich for human hematopoietic stem cells.
   https://www.stemcell.com/productcatalog/huprog.asp
  Stem Cell Technologies is a commercial supplier of cell separation technologies to isolate/enrich for human hematopoietic stem cells.
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