Animal Models Using Lymphocytic Choriomeningitis Virus

Matthias von Herrath1, J. Lindsay Whitton1

1 The Scripps Research Institute, La Jolla, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 19.10
DOI:  10.1002/0471142735.im1910s36
Online Posting Date:  May, 2001
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Abstract

This unit includes protocols for inducing systemic infection and persistent infection of mice with lymphocytic choriomeningitis virus (LCMV). Methods used to measure T cell responses to LCMV are then described. A protocol to assess anti‐LCMV immunity in vivo is also included. Support protocols for preparing LCMV stocks and measuring LCMV titers using a plaque assay are also included. Finally, a support protocol for detecting anti‐LCMV antibodies by ELISA is presented.

     
 
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Table of Contents

  • Biosafety Considerations
  • Basic Protocol 1: Systemic Infection of Mice with LCMV
  • Basic Protocol 2: Persistent Infection of Mice with LCMV
  • Basic Protocol 3: Measurement of T Cell Responses to LCMV
  • Basic Protocol 4: In Vivo Methods to Measure LCMV Vaccine Efficacy
  • Support Protocol 1: Preparation of LCMV Stocks
  • Support Protocol 2: Plaque Assay to Measure LCMV Titers
  • Support Protocol 3: Detection of Anti‐LCMV Antibodies by ELISA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Systemic Infection of Mice with LCMV

  Materials
  • LCMV stock of known titer (see Support Protocols protocol 51 and protocol 62)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Mice of appropriate strain(s), in sufficient numbers to ensure statistical significance of resulting experimental data.
  • Anesthetic (e.g., ketamine/xylazine; unit 1.4)
  • 1‐ml luer‐lock syringes and 25‐ to 27‐G needles (do not use syringes with built‐in needles)
  • Additional reagents and equipment for intraperitoneal, intravenous, subcutaneous, footpad, or intracranial injections (unit 1.6), and anesthesia of mice (unit 1.4)

Basic Protocol 2: Persistent Infection of Mice with LCMV

  Materials
  • LCMV stock of known titer (see Support Protocols protocol 51 and protocol 62)
  • Phosphate buffered saline (PBS; appendix 2A)
  • Pregnant mice
  • 1‐ml luer‐lock syringes and 27‐G needles (do not use syringes with built‐in needles)
  • Additional reagents and equipment for injecting mice with LCMV (see protocol 1)

Basic Protocol 3: Measurement of T Cell Responses to LCMV

  Materials
  • Stock of LCMV, of known LD 50 titer for the mouse strain being used.
  • Mice (control and LSMV‐immunized; see protocol 1)
  • Additional reagents and equipment for intracranial injection (see protocol 1) and measuring LCMV titers ( protocol 6; optional)

Basic Protocol 4: In Vivo Methods to Measure LCMV Vaccine Efficacy

  Materials
  • BHK‐21 cells (ATCC #CCL‐10)
  • Complete DMEM/10% FBS medium ( appendix 2A)
  • Seed stock of LCMV (available from Dr. M. von Herrath, Scripps Research Institute; )
  • Polyethylene glycol 6000 (PEG‐6000)
  • Sodium chloride (solid)
  • recipeTNE buffer (see recipe)
  • Renografin‐76 (Sterling Medical)
  • 162‐cm2 tissue culture flasks
  • 50‐ml centrifuge tubes
  • 1‐ml cryotubes, for storing aliquots of virus stock.
  • Sorvall centrifuge with GS‐3 rotor (or equivalent)
  • Beckman ultracentrifuge with SW 41 rotor (or equivalent)
  • Two‐chamber gradient former (e.g., Jule Biotechnologies; http://hometown.aol.com/precastgel/formers1.htm)
  • Additional reagents and equipment for measurement of LCMV titers (see protocol 6)

Support Protocol 1: Preparation of LCMV Stocks

  Materials
  • Vero 76 cells (ATCC #CRL‐1587).
  • Complete DMEM/10% FBS medium ( appendix 2A)
  • LCMV stock to be titered (see protocol 5)
  • 1% agarose (Seakem ME, FMC Bioproducts): dissolve in Milli‐Q‐grade H 2O and autoclave
  • recipeComplete 2× medium 199/10% FBS (see recipe)
  • 25% (v/v) formaldehyde: dilute formalin (37% v/v formaldehyde) to 25% formaldehyde final
  • recipeCell staining solution (see recipe)
  • 6‐well tissue culture plates
  • 50°C water bath

Support Protocol 2: Plaque Assay to Measure LCMV Titers

  Materials
  • High‐titer, Renografin‐gradient‐purified LCMV (see protocol 5)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Mouse serum (or MAb) to be tested for anti‐LCMV antibody
  • Secondary antibody [e.g., rat anti‐(mouse Ig)] labeled with horseradish peroxidase
  • Additional reagents and equipment for ELISA (unit 2.1)
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Literature Cited

Literature Cited
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