NC State IACUC Standard on NIH Guidelines for Tissue Collection for Genotyping Rodents
This standard describes tissue collection used for genotyping certain rodent strains bred in house. All persons involved with the care and/or use of animals in research and/or teaching at the University including, but not limited to Principal Investigators, researchers, animal handlers, and students.
Standard
The genotype is most often determined by analysis of DNA extracted from tissues of young rodents. Historically, tissue biopsies (e.g. ear, tail and toe clip) are the most common methods used, but biopsies must be carefully performed because they have the potential to result in some level of pain and/or distress. Researchers should use the least invasive method that is practical for their research and should collect the smallest sample necessary for reliable results. Prompt collection and analysis of tissue allows the desired mice/rats to be identified prior to weaning and will facilitate more efficient use of cage space.
The Principal Investigator must ensure sufficient training for individuals performing these technical procedures. The training must be documented. All tissue collection procedures must be described in either an approved IACUC protocol (AVAU) or referred to in an IACUC approved Standard Operating Procedure.
Biopsy Recommendations
Work space should be cleaned prior to procedure. Instruments should be sterilized to start and disinfected with 70% alcohol between each animal.
Ear (Pinna) Biopsy
Pinna biopsy or ear punch offers the advantage of having tissue collection and permanent identification completed in one procedure. In rodents, the ear is sufficiently developed around 14 days of age to allow suitable tissue collection. Pinna biopsy is considered similar to tagging the ear and results in minimal or transient associated pain and distress. A two (2) millimeter ear punch or marginal notch is recommended. If repeated biopsies are required, the use of the alternate pinna or an alternate method should be considered. Pinna biopsies performed as described do not require the use of anesthetics or analgesics.
Tail Biopsy
Tail biopsy is an effective and humane method of tissue collection analysis when performed correctly. Pain perception of mid-tail clamping in rats is reported to develop between 12 to 14 days of age, so performing tail biopsy as early as possible in rodents should minimize potential pain. Tail biopsy length should be limited to the smallest amount possible. In general, a biopsy of approximately 2 mm is sufficient to generate DNA for multiple PCR reactions. Initial biopsies of 2mm or less in young animals (<21 Days), likely prevents the cutting of ossified bone, a potentially painful procedure. If larger sample sizes are required at any age (>4mm), the justification should be described in the IACUC protocol.
For pre-weanling animals (<21 days of age), the use of anesthesia is recommended. For mice and rats 21 days of age or older, the use of anesthesia is required unless approved by the IACUC. General anesthesia with isoflurane is used safely in many programs for chemical restraint and procedural analgesia. A post procedural analgesia plan for animals after 21 days of age must be included in the IACUC protocol description. The need to provide an effective analgesic (e.g. an opioid such as buprenorphine) post-biopsy will increase with the age of the rodent post weaning, length of the biopsy or with repeated biopsies. Anesthetics and analgesics should be chosen in consultation with the University Attending Veterinarian.
Procedural steps for tail biopsies:
- Restrain the rodent manually or with a commercial restrainer.
- Starting with a sterile scalpel (recommended), razor blade, or scissors, cleanly excise the defined length of distal tail. If the analysis of the DNA is to be performed by PCR, great care should be taken to remove all tissue from the scalpel or scissors after each animal. Sanitize the scalpel or scissors between animals using an appropriate method (e.g. using detergent followed by 70% ethanol, bead sterilizer, etc.). If a scalpel is used, also sanitize the work surface on which the tail is placed between animals.
- The investigator must monitor the animals to assure hemostasis after the rodents are returned to the cage. If needed, apply digital pressure, heat cautery (briefly), silver nitrate, or some other means of hemostasis. If silver nitrate is used, the tissue must be washed free of the chemical with saline following hemostasis to neutralize the chemical reaction. Heat cautery should be used under local general anesthesia.
Toe (Distal Phalanx) Biopsy
Removal of a portion of a digit, distal phalanx biopsy (DPB), corresponding to the third phalanx, P3. DPB is used as a method of identifying small rodents by using a predetermined numbering code and may simultaneously be used as a method to obtain biopsy tissue for genotyping by polymerase chain reaction (PCR). DPB should only be used in altricial pre-weaning rodents (e.g. mice and rats, NOT guinea pigs) after the digits are no longer webbed (around 4-5 days) and before they reach seven days of age.
Studies in mice indicate that DPB produces no more acute pain or distress than other commonly used rodent identification procedures when performed from five to seven days of age. Every reasonable effort should be made to minimize pain or distress, including limiting the number of digits clipped to one digit per rodent. If more than one digit is required, it should be justified and rationale described in the AVAU. If possible, it is preferable to remove digits from a hind paw rather than a forepaw, especially if the animals will be used in studies that include grip strength testing. If the forepaw must be used, it is preferable to not cut the hallux (“dew claw” or “little toe” of the forepaw) as this may decrease the rodent’s grasping ability. To ensure pain and distress is minimized, small sharp scissors should be used and personnel performing the procedure should be trained and proficient.