Genotyping Mice and Rats, Guidelines for

Details

Identification of individual genotypes for animals in a litter is critical for research and in reducing the number of animals used in a protocol. Genotype can be determined by analysis of DNA extracted from the tissues of young rodents. Analysis by the Polymerase Chain Reaction (PCR) requires the least amount of DNA which is easily obtained from ear punches or tail biopsy (1-7) although alternative methods are available (4,6,7).

Obtaining tissue from a mouse or rat for DNA analysis by ear punch or tail biopsy is safe, effective, and humane. When performed properly, these techniques cause only minimal or transient pain and distress, and induce no more “physiological impact” (change in heart rate, body temperature, or activity level) than just restraining the animal for the procedure. This document is based on the NIH Intramural Animal Care and Use Guidelines for Genotyping (8).

Guidelines for Tail Biopsy

The procedures for tail biopsy for DNA analysis or genotyping must be described in an approved IACUC protocol.
(a) Ideally, mice and rats should be less than 21 days old for genotyping. At this age, the tail tissue is soft (vertebra are not yet calcified) and the yield of DNA is high. Prompt analysis of tail tissue allows the desired mice and rats to be identified prior to weaning which can facilitate efficient use of cage space. (b) For mice and rats 21-35 days of age: Because sensory development is complete, the use of a local or general anesthetic is required prior to collection of tissue. An appropriate agent should be recommended by the attending veterinarian. (c) For rats older than 35 days of age: The use of a general anesthetic is required. An appropriate agent should be recommended by the attending veterinarian.
Manually restrain the mouse or rat between thumb and forefinger. This is a convenient time to identify the animals using the appropriate method (i.e., ear punch, ear tag, transponder, etc.).
With a sterile scalpel, razor blade, or scissors cleanly excise the distal 2 mm (maximum 5 mm) of the tail. If the proper procedures are followed, the yield of DNA from 5 mm of the tail should exceed 50 micrograms, enough for multiple analyses. The yield of DNA does not proportionally increase as tail fragments larger than 5 mm are used. If small amounts of DNA are required, investigators should take only 2 mm of the tail. If analysis of DNA is to be done by PCR, particular care should be taken to remove all tissue from the scissors or scalpel after each animal. Disinfect the scalpel or scissors between animals. If a scalpel or razor blade is used, also clean and disinfect the work surface on which the tail is placed between animals.
The investigator must monitor the animals to assure hemostasis after the animals are returned to the cage. If needed, as indicated by bleeding greater than 10-20 microliters or multiple streaks of blood in the cage, apply digital pressure, silver nitrate, or other means of hemostasis. Monitoring is easier if animals are placed in a clean cage.
If additional DNA is needed for retesting alternatives to a second tail biopsy should be considered. Repeat tail biopsies require anesthesia and must be justified in the IACUC protocol. Post-procedural analgesia should be used.

References

Cinelli P., et.al. Comparative Analysis and Physiological Impact of Different Tissue Biopsy Methodologies Used for the Genotyping of Laboratory Mice. Lab Animals 2007; 41: 174-184
Hofstetter JR, Zhang A, Mayeda AR, Guscar, T, Nurnberger JI and Lahiri DK. Genomic DNA from Mice: A Comparison of Recovery Methods and Tissue Sources. Biochem Mol Med 1997 Dec; 62(2):197-202. [tissue comparison]
Malumbres M, Mangues R, Ferrer N, Lu S and Pellicer A. Isolation of High Molecular Weight DNA for Reliable Genotyping of Transgenic Mice. BioTechniques 1997; 22/6:1114-1119.
Meldgaard M, Bollen PJA, Finsen B. Non-invasive method for sampling and extraction of mouse DNA for PCR. Laboratory Animals 2004; 38:413-417. [alternates]
Pinkert CA. Transgenic Animal Technology: Alternatives in Genotyping and Phenotyping. Comp Med 2003; 53/2:126-139.
Schneider MR, Wolf E. Anal Biochem. Genotyping of transgenic mice: old principles and recent developments 2005 Sep 1;344(1):1-7.
Phenotyping of Genetically Engineered Mice ILAR Journal Volume 47(2) [multiple articles].
Guidelines for the Genotyping of Mice and Rats Intramural Animal Care and Use (ACU) program of the National Institutes of Health (NIH).

Approved Date

05/09/2009

Revised Date