Collection of Tissues for Genotyping

Introduction

The use of tail biopsy (also known as tail tip amputation, tail snip, or tailing) for genetic analysis of mice must be scientifically justified. Administration of anesthesia is required prior to performance of tail biopsy in mice greater than 3 weeks of age.

Background

Analysis of the DNA of individual mice is necessary for the identification of genetically modified animals that may serve as valuable models for research. The nature and quantity of mouse tissue required for this analysis are based on the type of DNA test being performed. The massive amplification inherent in the polymerase chain reaction (PCR) test permits identification of the gene of interest with a very small sample of DNA. Samples of peripheral blood (which contains only a small amount of DNA), hair, saliva, and ear punch biopsy snippets are often sufficient for PCR testing. Most often, several milligrams of solid tissue are needed to identify the presence or absence of a segment of DNA in an animal. The mouse tail biopsy procedure is the most typically used procedure to obtain tissue for a Southern blot test.  The tail of a mouse is composed of bone, cartilage, blood vessels, and nerves. In a young mouse < 3 weeks of age the tissue near the tip of the tail is soft and the bones have not completely mineralized. Therefore, amputation of the tail tip of a young mouse is judged to cause only momentary pain for the animal. As the mouse reaches 3-4 weeks of age, tail tissue maturation includes mineralization of the bone and increased vascularity. Tail tip amputation performed on an older animal is likely to involve more than momentary pain, and the procedure has the potential to cause significant hemorrhage and must be done using appropriate anesthesia.

Guidelines

  1. Tail biopsy for genetic analysis of mice must be performed only when scientifically justified. If PCR testing is available, it is likely that sufficient material for genotyping mice can be obtained from samples collected by less invasive methods such as ear biopsy. Ear punch biopsy has the advantage of offering simultaneous sampling and mouse identification.  Toe clipping is not a standard procedure and must be justified in the IACUC application form. It is recommended that tail snip be performed at 2 – 2.5 weeks of age. Negative mice can then be culled when mice are weaned at 21 days of age.
  2. Tail biopsy in mice greater than 3 weeks of age is categorized as a potentially painful procedure. Anesthesia must be provided. Because of this classification, the proposal to perform tail snip in mice over 21 days of age must be accompanied by a search for alternatives to this potentially painful procedure.
  3. The total amount of tail tissue removed should be kept to the minimum required for the test to be performed. The excised length of tail should not exceed 0.5 cm (about 3/16 inch). Mice use their tails in thermoregulation and balance. The tail of the mouse is also an important site for research procedures such as blood collection, intravenous injection, and restraint.
  4. Hemostasis must be assured before returning the tail-snipped mouse to its cage. When tails are clipped at the recommended age of less than 3 weeks, hemostasis is easily achieved by direct manual pressure around the end of the tail. Light, direct pressure minimizes tissue damage and the subsequent risk of mouse-inflicted trauma to the tail end. Use of heat to cauterize the amputated end of the tail is discouraged unless bleeding problems are severe. A medical-grade, non-toxic styptic powder may be used if necessary. Silver nitrate is toxic and is not recommended for this purpose as mice may ingest the agent. Application of a tourniquet to the tail of a mouse is strictly prohibited. Contact the Veterinary Staff for advice if problems with hemostasis are encountered or expected (e.g., mice with blood clotting disorders).
  5. Repeated tail amputations on a single mouse are discouraged. If an additional sample from a mouse may be needed at a later date, cut the original tissue biopsy in half and preserve the extra piece at -20°- -80o F.

Recommended Procedures

Please follow these steps when performing tail snipping:

Instruments

Tail snips in mice at any age must be made as cleanly as possible. The blade or instrument used must be very sharp and either sterile or thoroughly disinfected.  This can be accomplished by using disposable scalpel blades or razor blades; a fresh blade must be used for each mouse.  Alternatively, scissors can be disinfected through the use of a hot bead sterilizer in between animals; the use alchohol is not considered an appropriate disinfectant for instruments.  The cross-sectional cut should be made perpendicular to the long axis of the tail to minimize the surface area of traumatized tissue.


Anesthesia

For mice older than 3 weeks isoflurane is the recommended anesthesia.  This is most appropriate since the procedure is very brief.

Analgesia

In mice older than 3 weeks buprenorphine 0.05-0.1 mg/kg SC may be administered BEFORE the procedure.  However, this is not mandatory and may be administered at the PI’s discretion.

Hemostasis

Styptic powder (Kwik-Stop® or Clotisol®) is applied to the tail to stop bleeding.

Sampling

  • Use sterile, sharp scissors or blade
  • Apply pressure to tail after snipping, or use styptic pencil
  • Do not take more than 0.5 cm of tissue
  • If ear-punching mice for identification, the section of ear removed by the punch tool should be used for genotyping.

REFERENCES

1.  Forman, L.A.,  2000; Rodent Surgery guidelines, Northwestern University, Chicago, IL.

2.  Slatter, D., 1993; Textbook of Small Animal Surgery, Philadelphia, W.B. Saunders and Co.

3. Ryden E. and Larsen D.  2004.  Comparative Medicine Resources, New Jersey Medical School, UMDNJ Newark Campus

4.  IACUC Guidelines, University of California at San Francisco, 2005.

5.  UCUCA Guidelines, University of Michigan, 2005.

BU IACUC Approved October 2008