Construction of Human‐SCID Chimeric Mice

Maria Grazia Roncarolo1, José M. Carballido2

1 University of Turin, Turin, Italy, 2 Novartis Forschungsinstitut, Vienna, Austria
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 4.8
DOI:  10.1002/0471142735.im0408s25
Online Posting Date:  May, 2001
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Until recently, testing of new therapeutic agents has relied extensively upon the use of mice and nonhuman primates for in vivo preclinical studies. Unfortunately, these animal models do not always mimic the physiological and pathophysiological processes that occur in humans. The finding that C.B‐17 severe combined immunodeficiency (SCID) mice lack a competent immune system, and therefore are unable to mount effective cellular and humoral responses to foreign antigens, has led to their use as recipients for xenografts of human tissues. This unit is focused on the construction of human‐SCID chimeric through the surgical implantation of human fetal hematolymphoid tissues into SCID mice (SCID‐hu mice). The describes the surgical implantation of human fetal thymus and liver under the kidney capsules of SCID mice (SCID‐hu Thy/Liv model). Subcutaneous transplantation of human fetal bone marrow and thymus (SCID‐hu Bm/Thy mice) is described in the . Additional support protocols provide procedures to analyze human lymphocyte populations in the peripheral blood and grafted organs of SCID‐hu mice. The advantages and disadvantages of each protocol and potential applications are discussed in the Commentary.

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Table of Contents

  • Basic Protocol 1: SCID‐hu Thymus/Liver (Thy/Liv) Model
  • Support Protocol 1: Flow Cytometric Screening of Human T Cells in SCID‐hu Thy/Liv Chimeras
  • Alternate Protocol 1: SCID‐hu Bone Marrow/Thymus (Bm/Thy) Model
  • Support Protocol 2: Flow Cytometric Screening of Human Lymphocytes and ELISA of Immunoglobulins in SCID‐hu Bm/Thy Chimeras
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1: SCID‐hu Thymus/Liver (Thy/Liv) Model

  • Human fetal liver and fetal thymus of gestational ages ranging from 14 to 21 weeks
  • Dulbecco's phosphate‐buffered saline (PBS; appendix 2A) containing 50 µg/ml gentamicin
  • Complete RPMI‐10 ( appendix 2A), 4°C
  • RPMI 1640 medium with 50 µg/ml gentamicin
  • C.B‐17 scid/scid (SCID) or C.B‐17 scid/scid bg/bg (SCID.BG) mice, preferably male, 6 to 8 weeks old (Taconic Farms)
  • 100 mg/ml ketamine HCl (Ketaset; Fort Dodge Animal Health)
  • 20 mg/ml xylazine HCl (Rompum; Miles Labs)
  • Metofane (Schering‐Plough)
  • 5% betadine (Victor Medical)
  • 18‐G (1–in.) hypodermic needles (Popper and Sons)
  • Sandpaper (very fine, P400 grade)
  • 18‐G (3 1/2‐in.) spinal needle (Becton Dickinson Labware)
  • High‐vacuum grease (Dow Corning)
  • Stereomicroscope
  • Surgical instruments (sterilized by autoclaving before use):
    •  No. 21 scalpel blades
    •  No. 4 knife handle
    •  McPherson‐Vannas micro dissecting scissors, curved‐sharp
    •  Dumont tweezers, pattern no. 5 or 7
    •  Micro dissecting forceps
    •  Dressing forceps
    •  Operating scissors, straight, sharp‐sharp
    •  Micro dissecting scissors, straight‐sharp
    •  Olsen‐Hegar needle holder combined with suture scissors
    •  18‐G (3 1/2‐in.) spinal needle (Becton Dickinson Labware) or prepared transplantation needles (see step )
  • 60‐mm plastic petri dishes (Falcon), sterile
  • Balance suitable for weighing mice (Mettler‐Toledo or equivalent)
  • Steri‐drape towels (Baxter)
  • Electric clipper with size 40 blade
  • Operating board (Baxter or equivalent)
  • Sterile gauze pads
  • 10‐ml syringe and 18‐G needle
  • Reusable instant heat bags (Hood Thermo‐Pad Canada)
  • Autoclip wound clip applicator and 9‐mm autoclips
  • Autoclip‐removing forceps
  • 3‐0 Dexon “S” (polyglycolic acid), synthetic, sterile, absorbable suture (Sherwood‐Davis & Geck)
  • Additional reagents and equipment for mouse handling (units 1.1 1.5) and ELISA (unit 1.2; optional)

Support Protocol 1: Flow Cytometric Screening of Human T Cells in SCID‐hu Thy/Liv Chimeras

  • recipePBS/EDTA (see recipe)
  • SCID‐hu Thy/Liv mice (see protocol 1)
  • Two untransplanted SCID mice
  • recipeLysing buffer (Tris‐buffered ammonium chloride; see recipe)
  • Human peripheral blood lymphocytes (unit 7.1)
  • Normal mouse serum (Sigma)
  • PE‐ and FITC‐conjugated mouse anti‐human MAbs to CD3 and CD45 (Becton Dickinson Immunocytometry or equivalent)
  • 2 µg/ml propidium iodide in PBS
  • Disposable 12 × 75–mm conical tubes
  • Sorvall RT6000B centrifuge with H1000B rotor (Du Pont), or equivalent
  • V‐bottom 96‐well microtiter plates (Costar)
  • Multichannel pipettor (Labsystems)
  • Additional reagents and equipment for blood collection (unit 1.7) and flow cytometry (e.g., unit 5.4)

Alternate Protocol 1: SCID‐hu Bone Marrow/Thymus (Bm/Thy) Model

  • Human fetal long bones (femur, tibia, humerus) and thymuses, ranging from 18 to 24 gestational weeks

Support Protocol 2: Flow Cytometric Screening of Human Lymphocytes and ELISA of Immunoglobulins in SCID‐hu Bm/Thy Chimeras

  • PE‐conjugated anti‐CD3 and FITC‐conjugated anti‐CD19 mouse antihuman MAbs (e.g., Becton Dickinson Immunocytometry)
  • PercP‐ (Becton Dickinson Immunocytometry) or Tricolor‐ (Caltag) anti‐CD45 MAbs (optional)
  • Capillary blood collection tubes with serum separator (Terumo T‐MG or equivalent)
  • Additional reagents and equipment for blood collection (unit 1.7), flow cytometry (unit 5.4), and sandwich ELISA (units 2.1 & 7.12)
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Literature Cited

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Key Reference
   Roncarolo et al., 1995. See above.
  Comprehensive and easy‐to‐read overview on the different humanized SCID models.
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