Introduction to Basic Mouse Handling Techniques

John Donovan1, Patricia Brown2, J.P. Reeves3, P.A. Reeves3

1 Rhone‐Poulenc Rorer, Collegeville, Pennsylvania, 2 National Cancer Institute, Bethesda, Maryland, 3 Food and Drug Administration, Bethesda, Maryland
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Appendix 3L
DOI:  10.1002/0471142905.hga03ls36
Online Posting Date:  May, 2003
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Purification of DNA from fixed, paraffin‐embedded tissue (PET) requires the removal of paraffin, proteins, and, in some cases, heavy metal ions. The resulting DNA is not generally suitable for Southern blot analysis. However, the polymerase chain reaction (PCR) can be used to amplify short regions of this DNA. In this appendix, the basic protocol can be performed rapidly and involves only a small number of manipulations, thus minimizing the possibility of contamination. An alternate protocol involves additional steps including extractions with xylenes and phenol/chloroform. This procedure is recommended as the primary approach when tissue has not been fixed in an optimal manner or as a secondary approach when the basic protocol has failed to produce satisfactory DNA.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Mouse Handling and Manual Restraint
  • Alternate Protocol 1: Rodent Restrainers
  • Basic Protocol 2: Injectable Anesthesia for the Mouse
  • Basic Protocol 3: Inhalant Anesthesia using Methoxyflurane for the Mouse
  • Injection Methods for Mouse
  • Basic Protocol 4: Intramuscular Injection of the Mouse
  • Basic Protocol 5: Intradermal Injection of the Mouse
  • Basic Protocol 6: Subcutaneous Injection of the Mouse
  • Basic Protocol 7: Intravenous Injection of the Mouse
  • Basic Protocol 8: Intraperitoneal Injection of the Mouse
  • Basic Protocol 9: Footpad Injection of the Mouse
  • Blood Collection from Mouse
  • Basic Protocol 10: Blood Collection from Orbital Sinus or Plexus of the Mouse
  • Basic Protocol 11: Blood Collection from Tail Vein of the Mouse Using Microhematocrit Tube
  • Alternate Protocol 2: Blood Collection from Tail Vein of the Mouse Using Centrifuge Tube
  • Basic Protocol 12: Blood Collection from Axillary Plexus of Mouse
  • Basic Protocol 13: Cardiac Puncture of the Mouse
  • Methods of Euthanasia for Mouse
  • Basic Protocol 14: Carbon Dioxide Asphyxiation
  • Alternate Protocol 3: Anesthesia with Exsanguination
  • Alternate Protocol 4: Cervical Dislocation of Mouse
  • Basic Protocol 15: Removal of Mouse Lymphoid Organs
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Mouse Handling and Manual Restraint

  Materials
  • Animal
  • Anesthetic of choice (Table 3.0.1)
  • Laboratory balance for weighing the animal
  • 1‐ or 3‐ml syringe with 22‐G needle
  • Additional reagents and equipment for handling and restraint (see protocol 1 or protocol 2) and intraperitoneal injection (see protocol 8)
    Table 0.l.1   MaterialsGuidelines for i.p. Injection of Anesthetics in Rodents

    Agent or combination Species Dosage (mg/kg) Duration (min)
    Ketamine + xylazine a Mouse 50–150 15–20
    Rat 50–150 15–20
    Hamster 50–150 15–20
    Tribromoethanol b Mouse 125–160 15–30
    Rat 300 15–20

     aAdd 1.0 ml of xylazine (20 mg/ml) to 10 ml ketamine (100 mg/ml) and dose as if mixture were 100 mg/ml. Dilution 1/10 with sterile saline is recommended for accuracy in dosing mice, hamsters, and young rats. This mixture is based on the use of commercially available sterile stock solutions of the two anesthetics (available from veterinary supply companies; see Table 3.0.2), not reagent‐grade compounds. Use of nonsterile dry compounds for making this mixture is contraindicated because of the risk of introducing bacteria or other contaminants during the preparation process and because the dry forms of these drugs do not dissolve readily. It is recommended that the veterinarian member of the IACUC be contacted to determine how to obtain these anesthetics legally for use in research. Some institutions maintain institutional research licenses with the U.S. Drug Enforcement Administration (DEA) for use of these or other veterinary drugs; at other institutions, each individual researcher must have a DEA license.
     bMake a 100% (w/v) stock solution by dissolving 5 g of 2,2,2‐tribromoethanol in 5 ml 2‐methyl‐2‐butanol (tert‐amyl alcohol). Gentle heating may be required. A good working solution is a 1/250 dilution (4 mg/ml) in normal saline; this is stable 4 to 6 months at 4°C in a dark bottle.

Alternate Protocol 1: Rodent Restrainers

  Materials
  • Methoxyflurane
  • Animal
  • Gauze or cotton sponges
  • Closed glass container with raised floor
  • Empty syringe case or 50‐ml centrifuge tube (optional)
CAUTION: All procedures with methoxyflurane should be conducted in a fume hood or safety cabinet that continuously exhausts anesthetic gases away from personnel.

Basic Protocol 2: Injectable Anesthesia for the Mouse

  Materials
  • Animal
  • 70% ethanol
  • Injectate
  • Clippers with no. 40 blade
  • Gauze sponge or swab
  • 1‐ to 3‐ml syringe with 25‐ to 30‐G needle
  • Additional reagents and equipment for handling and restraint (see protocol 1 or protocol 2) and anesthesia (see protocol 3 or protocol 43)

Basic Protocol 3: Inhalant Anesthesia using Methoxyflurane for the Mouse

  Materials
  • Injectate
  • Animal
  • 70% ethanol
  • 1‐ml syringe with 25‐ to 30‐G needle
  • Heat lamp or beaker containing warm water
  • Gauze sponge or swab
  • Additional reagents and equipment for using a restrainer (see protocol 2)

Basic Protocol 4: Intramuscular Injection of the Mouse

  Materials
  • Animal
  • Sterile saline or phosphate‐buffered saline (PBS; appendix 2D)
  • Microhematocrit tube
  • Gauze sponge or swab
  • Additional reagents and equipment for handling and restraint (see protocol 1 or protocol 2) and anesthesia (see protocol 3 and protocol 43)

Basic Protocol 5: Intradermal Injection of the Mouse

  Materials
  • Animal
  • Heat lamp or beaker containing warm water
  • 25‐ to 30‐G needle
  • Microhematocrit tube
  • Gauze sponge
  • Additional reagents and equipment for handling and restraint (see protocol 1 or protocol 2)

Basic Protocol 6: Subcutaneous Injection of the Mouse

  • No. 10 scalpel blade
  • 12‐ to 50‐ml plastic centrifuge tube

Basic Protocol 7: Intravenous Injection of the Mouse

  Materials
  • Animal
  • Surgical board and pins
  • Scalpel or razor blade
  • Thumb forceps
  • Surgical scissors
  • Additional reagents and equipment for anesthesia (see Basic Protocols protocol 32 or protocol 43) and cervical dislocation (see protocol 18)

Basic Protocol 8: Intraperitoneal Injection of the Mouse

  Materials
  • 60% to 100% CO 2 (tank or house carbon dioxide) or dry ice
  • Impervious container with raised floor and lid or CO 2 chamber with lid

Basic Protocol 9: Footpad Injection of the Mouse

  Materials
  • Animal
  • 70% ethanol
  • Hanks balanced salt solution (HBSS; appendix 2D) or tissue culture medium
  • Iris scissors, straight or angled
  • Iris forceps, curved
  • Tissue culture plate
  • Additional reagents and equipment for euthanasia (see Methods of Euthanasia for Mouse)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •   FigureFigure a0.3L.1 Mouse handling and manual restraint. (A) Apply slight, rearward traction on the tail. (B) Grasp skin behind ears with thumb and index finger. (C) Transfer the tail from the preferred hand to beneath the little finger of the hand holding the scruff of the neck.
  •   FigureFigure a0.3L.2 Rodent restrainers. With animal under control as described for handling and manual restraint (see ), place the head at opening of the box while maintaining tension on the tail. Allow animal to crawl in and place the securing block appropriately.
  •   FigureFigure a0.3L.3 Nose cone for supplementation of methoxyflurane anesthesia.
  •   FigureFigure a0.3L.4 Intramuscular injection of the mouse.
  •   FigureFigure a0.3L.5 Subcutaneous injection of the mouse.
  •   FigureFigure a0.3L.6 Intravenous injection of the mouse in the tail vein.
  •   FigureFigure a0.3L.7 Intraperitoneal injection of the mouse.
  •   FigureFigure a0.3L.8 Blood collection from the orbital sinus or plexus of the mouse and rat.
  •   FigureFigure a0.3L.9 Blood collection from the tail vein of the mouse and rat.
  •   FigureFigure a0.3L.10 Blood collection from the axillary plexus of the mouse.
  •   FigureFigure a0.3L.11 Cardiac puncture of the mouse and rat.
  •   FigureFigure a0.3L.12 Midline skin incision for removal of lymphoid organs.
  •   FigureFigure a0.3L.13 The lymph nodes and spleen of the mouse.

Videos

Literature Cited

   Andrews, E.J. 1993. 1993 report of the American Veterinary Medical Association panel on euthanasia. J. Am. Vet. Med. Assoc. 202:229‐249.
   Scobie‐Trumper, P. 1987. Animal handling and manipulations. In Laboratory Animals: An Introduction for New Experimenters (A.A. Tuffery, ed.) pp. 153‐170. John Wiley & Sons, Chichester, England.
   Tuffery, A.A. 1987a. Laboratory Animals: An Introduction for New Experimenters, pp. 225‐226. John Wiley & Sons, Chichester, England.
   Tuffery, A.A. 1987b. Lab Animals: An Introduction for New Experimenters, p. 248. John Wiley & Sons, Chichester, England.
Key References
   Fox, J.G., Cohen, B.J., and Loew, F.M. (eds.) 1984. Laboratory Animal Medicine. Academic Press, Orlando, Fla.
  Provides additional details for injection methods.
   Gay, W.I. 1986. Methods of Animal Experimentation, Vol. VII, pp. 31‐65. Academic Press, San Diego, Calif.
   Green, C.J. 1987. Anesthesia and analgesia. In Laboratory Animals: An Introduction for New Experimenters (A.A. Tuffery, ed.) pp. 261‐301. John Wiley & Sons, Chichester, England.
   White, W.J. and Field, K.J. 1987. Anesthesia and surgery of laboratory animals. In The Veterinary Clinics of North America, Small Animal Practice, Exotic Pet Medicine, 17:5 (J.E. Harkness, ed.) pp. 989‐1017. W.B. Saunders, Philadelphia, PA.
   Wixson, S.K. and Smiler, K.L. 1997. Anesthesia and analgesia in rodents. In Anesthesia and Analgesia in Laboratory Animals (D.F. Kohn, S.K. Wixson, W.J. White, and G.J. Benson, eds.) pp. 165‐200. Academic Press, San Diego, Calif.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library