A Mouse Model for Cytomegalovirus Infection

Wolfram Brune1, Hartmut Hengel1, Ulrich H. Koszinowski1

1 University of Munich, Max von Pettenkofer Institute, Munich, Germany
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 19.7
DOI:  10.1002/0471142735.im1907s43
Online Posting Date:  August, 2001
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Abstract

This unit describes procedures for infecting newborn and adult mice with murine cytomegalovirus (mCMV). Methods are included for propagating mCMV in cell cultures and for preparing a more virulent form of mCMV from salivary glands of infected mice. A plaque‚Äźforming cell (PFC) assay is provided for measuring mCMV titers of infected tissues or virus stocks. In addition, a method is described for preparing the murine embryonic fibroblasts used for propagating mCMV and for the PFC assay.

     
 
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Table of Contents

  • Basic Protocol 1: Infection of Mice with Cytomegalovirus
  • Support Protocol 1: Preparation of mCMV Stocks
  • Support Protocol 2: Preparation of Salivary Gland Virus
  • Support Protocol 3: Determination of CMV Titer Using Plaque‐Forming Cell Assay
  • Support Protocol 4: Preparation of Primary Murine Embryonic Fibroblasts
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Infection of Mice with Cytomegalovirus

  Materials
  • BALB/c mice (newborn or adult) or other strain
  • Purified murine cytomegalovirus (mCMV; see protocol 2) or salivary gland virus (SGV, see protocol 3)
  • recipeComplete medium (see recipe)
  • Stainless steel wire mesh (0.45‐mm grid size)
  • Additional reagents and equipment for parenteral injection (unit 1.6), euthanasia (unit 1.8), removal of lymphoid organs (unit 1.9), removal of salivary glands (unit 1.11), and determining viral titer in tissues (see protocol 4)

Support Protocol 1: Preparation of mCMV Stocks

  Materials
  • Murine embryonic fibroblasts (MEFs; see protocol 5) or other suitable cells (e.g., NIH 3T3, ATCC #CRL‐1658; BALB/c 3T3, ATCC #CCL‐163; M2‐10B4, ATCC #CRL‐1972)
  • recipeComplete medium (see recipe)
  • Murine cytomegalovirus (mCMV): Smith strain (ATCC #VR‐194, VR‐1399) or K181 strain
  • recipe15% (w/v) sucrose/VSB (see recipe)
  • 165‐cm2 tissue culture dishes
  • Disposable cell scraper
  • Centrifuge and rotors (e.g., Beckman J2‐21, rotors JA‐10, JA‐14; or Sorvall RC‐5, rotors GS3, GSA)
  • 500‐ml and 250‐ml centrifuge buckets, sterile
  • Dounce homogenizer with tight‐fitting pestle (Kontes or Wheaton)
  • Ultracentrifuge with swing‐out rotor (Beckman SW28 or equivalent) and 20‐ml ultracentrifuge tubes
  • Sterile 0.45‐µm filter (optional)
  • Additional reagents and equipment for determining viral titer (see protocol 4)

Support Protocol 2: Preparation of Salivary Gland Virus

  • BALB/c mice (4 to 6 weeks old)
  • PBS ( appendix 2A)

Support Protocol 3: Determination of CMV Titer Using Plaque‐Forming Cell Assay

  Materials
  • Murine embryonic fibroblasts (MEFs; see protocol 5) or other suitable cell type (e.g., NIH 3T3, ATCC #CRL‐1658; BALB/c 3T3, ATCC #CCL‐163; M2‐10B4, ATCC #CRL‐1972)
  • Test sample: organ homogenate from mCMV‐infected mice (see protocol 1), mCMV stock (unpurified or purified; see protocol 2), or SGV (see protocol 3)
  • recipeComplete medium (see recipe)
  • recipeViscous medium (see recipe)
  • Inverted microscope
  • 48‐well tissue culture plates

Support Protocol 4: Preparation of Primary Murine Embryonic Fibroblasts

  Materials
  • Pregnant mouse
  • PBS without calcium and magnesium ( appendix 2A)
  • Trypsin solution: 0.25% (w/v) trypsin/1 mM EDTA in PBS
  • recipeComplete medium (see recipe)
  • Sterile surgical instruments: scissors, scalpel, forceps
  • 2‐ to 4‐mm sterile glass beads
  • Sterile gauze or stainless steel wire mesh (0.45‐mm grid size)
  • 165‐cm2 tissue culture dishes or 175‐cm2 tissue culture flasks
  • Additional reagents and equipment for euthanasia (unit 1.8) and counting cells ( appendix 3B)
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Figures

Videos

Literature Cited

Literature Cited
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Key References
   Mocarski, 1996. See above.
  A systematic and comprehensive overview of all virological aspects of CMV.
   Mocarski, E.S. and Kemble, G.W. 1996. Recombinant cytomegaloviruses for study of replication and pathogenesis. Intervirology 39:320‐330.
  A review of methods for the construction and application of recombinant human and murine CMV.
   Reddehase and Lucin, 1993. See above.
  An overview of the immune responses to hCMV and mCMV infection.
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