A conventional procedure for genotyping transgenic animals entails cutting of the distal portion of the animal’s tail (tail tip biopsy). From this tissue, template DNA may be extracted to ascertain the presence or absence of a specific transgene using PCR or other DNA analysis techniques. Tail biopsy is safe and humane when performed correctly.
The PHS Policy1 requires minimization of pain and distress, consistent with the study’s aims, irregardless of the procedure being performed.
- Tail tip biopsy must be limited to a maximum of two times, since the tail is important in body temperature control and balance in rodents.
- The minimum amount of tissue required for analysis should be obtained, with 5mm (0.5 cm) being the maximum allowed without specific prior approval by the IACUC.
- In rodents less than 21 days of age, tail tip biopsy may be performed without use of analgesia or anesthesia.
- Appropriate local analgesics (spray on or topically applied agents) or general anesthetics must be used in rodents weighing 50 grams or more and in animals 21 days of age and older, except in extreme circumstances, when up to 25 days of age can be allowed. The IACUC must approve the extreme circumstance prior to performing tail tip biopsy without the use of analgesics or anesthetic
The IACUC must approve any research protocol where tail tip biopsy procedures are used.
Recommended age for performing tail tip biopsy in rodents is between 8 to 21 days of age. In rodents, the terminal vertebrae ossify between 2 and 3 weeks of age. Thusly, tail tip sampling is recommended in animals less than 3 weeks of age. The tails of mice contain a variety of tissues, including bone, cartilage, blood vessels and nerves. In pre-weanling animals, the caudal tail is soft, and the bones have not completely mineralized; therefore, amputation of the tip in animals less than 21 days old likely results in momentary pain for the animal. As the animal ages, tissue maturation occurs, which includes mineralization of the tail verterbrae, increased vascularity, and maturation of the nervous system. Tail tip amputation performed on animals greater than 21 days of age is likely to involve more than momentary pain and distress, as well as the potential for significant hemorrhage.
The purpose of this policy is to describe the requirements and restrictions for protocols that request murine tail tip biopsy procedures and provide consistency for IACUC protocol review.
Murine tail tip biopsies should be conducted using sterile, sharp instruments such as a stainless steel surgical blade or sharp scissor. The instrument should be decontaminated between animals with 70% Isopropyl alcohol, or an appropriate disinfecting agent such as Nolvasan (chlorhexide diacetate). Alternately, instruments can be heat sterilized using a hot bead sterilizer or flame. If a chemical disinfectant is used, the instrument must be rinsed with sterile water or saline between animals. The instrument must be changed when dullness of the cutting edge is noted.
Since bleeding may occur after tail biopsy, the biopsy site must be observed for active bleeding, and hemostasis must be achieved before returning the animal to the home cage. Hemostasis can be achieved by digital pressure, cautery, tissue adhesives, coagulation powder or coagulation sticks.
Appropriate local analgesia or general anesthetics must be used on animals over 21 days old unless justified in writing and approved in advance by the IACUC. In rodents less than 21 days of age, use of analgesics/anesthetics is optional.
Specific justification to the IACUC and prior approval must be obtained, prior to completing tail tip biopsies on rodents weighing more than 50 grams.
The size of the tail biopsy must be less than 5mm (0.5cm). A maximum of two biopsies can be performed on an individual animal. In the event that more than two biopsies or more than 5mm needs to be removed at one time, the investigator must:
- Provide justification to the ACUC for review and approval; AND
- Local analgesics or general anesthetics must be used for the second biopsy, regardless of the animal’s age.
- No more than 5mm (0.5 cm) of tail tissue may be removed from any one animal during a biopsy procedure.
- No more than two tail biopsies are permitted per animal.
- Animals should not be returned to their home cage until hemostasis and complete recovery from anesthesia has been obtained. Hypothermia should be prevented at all times during the procedure.
- Disposable gloves and other personnel protective equipment, as needed.
- Sharp scissors or surgical blade
- Appropriate collection vessel
- Isopropyl alcohol or appropriate disinfectant (sterile saline/water for rinsing instruments)
- Equipment to achieve hemostasis (cautery, tissue adhesives, coagulation powder, coagulation sticks, or digital pressure)
Collection tubes should be labeled with relevant transgene name, animal number, date of birth and cage number (if applicable) This ensures that if the rack is tipped over, tubes can still be sorted out.
- Restrain animals (if less than 21 days old) or use appropriate analgesia/anesthesia in over animals over 21 days old
- Disinfect the tail tip
- Clip less than 5 mm off the tip of the tail with a sharp instrument
- Apply hemostasis, and recover the animal in a warm environment
- Return animal to its home cage after full recover
Personnel in the Office of Laboratory Animal care will be happy to assist and train personnel in the above techniques.
**Extreme Circumstances that would allow tail biopsy from 21-25 days without the use of analgesics and/or anesthetics are defined as lines of animals whose offspring exhibit a failure to thrive, have developmental or growth delays, or a genetically induced disorder that causes fragile health. These extreme circumstances must be described in the original protocol or must be approved as an amendment to the protocol. The IACUC must review and approve the extreme circumstances prior to the performance of the tail tip biopsy.
1Public Health Service Policy on Humane Care and Use of Laboratory Animals, PHS, 1986.