Date: April 2006
Reference: Administrative Panel on Laboratory Animal Care

DIRECTIONS: Review the following material. Keep copies of guidelines with applicable protocols. You may find it helpful to post a copy of these guidelines in your laboratory. Questions should be forwarded to the A-PLAC office, 723-4550.

TRAINING: Training in these techniques and the humane treatment of laboratory animals during the procedures is taught by the Veterinary Service Center (VSC) staff. All new personnel who will be performing these techniques should contact VSC staff for training (723-6735).

These guidelines have been written to assist Protocol Directors in obtaining blood/tissue samples from mice in a safe, effective and humane manner.

DNA analysis of transgenic animals is necessary to monitor production of the desired genotype.  Tissue or blood samples must be obtained for analysis by PCR, Southern Blot or other method.

In the mouse, ossification of caudal (tail) vertebrae occurs between 2 and 4 weeks of age.  Therefore, removing the tail tip of a young mouse probably amounts to minimal pain and can be performed without anesthetic.  As the animal ages, tissue maturation results in mineralization of bone and increased vascularity.  Tail tip sampling performed on an older animal (>21 days) is likely to cause more than momentary pain and distress as well as the potential for significant bleeding.  Therefore, animals over 3 weeks of age (>21 days) should be first anesthetized with a short acting anesthetic (e.g., isoflurane).

Tail tip removal should be performed at as young an age as possible.  In animals ≤ 21 days of age clipping of the tail can be performed without general anesthesia.  Sampling should be performed using sharp, sterile scalpel blades or scissors.  If tail biopsies are performed on multiple mice, instruments must be disinfected appropriately (e.g. Alcide) between animals.  Additionally, instruments must be replaced (scalpel blades) or sharpened (scissors) regularly to minimize tissue trauma caused by blunted instruments. The smallest possible section should be removed (3mm is recommended), but no more than one (1) cm may be taken at any age without the use of anesthesia.  Some DNA kits may recommend larger samples be taken but in our experience this is not required1. It is understood that sampling may occasionally have to be repeated. Under these guidelines, tail clips can be performed up to three (3) times without a specific justification in the approved protocol for repetitive sampling.  If four (4) or more tail clips are required the rationale must be justified, within the relevant protocol, submitted to and approved by the A-PLAC prior to performing the procedure.  If possible, samples should be frozen to reduce the number of tail clips required.  More than one tail clip requires the use of anesthesia.  Regardless of the size of the sample, bleeding must be controlled by applying direct pressure to the wound or through the application of heat (cautery), silver nitrate, or tissue adhesive.  The animal should be monitored until it is fully recovered from the procedure/anesthesia.

The Veterinary Service Center staff can assist with blood collection for genotyping at a reasonable cost on a fee-for-service basis.  Persons interested in learning more about this option should contact the VSC at x3-3876.

References:

1. Campbell, D.B., Hess, E.J. Rapid genotyping of mutant mice using dried blood spots for polymerase chain reaction (PCR) analysis. Brain Research Protocols, 1:117-123, 1997.

 2. Ren S, et al. A Simplified Method to Prepare PCR Template DNA for Screening of Transgenic and Knockout Mice. Contemporary Topics in Laboratory Animal Medicine, 40 (2): 27- 30, March 2001.