Stabile Research Building Training Presentation

Tail Snips, Tattoos and Identification Procedures - Training Presentation University of South Florida (July 2005) Course Objectives This presentation will outline common methods used to obtain tissues for DNA/RNA sampling, as well as prevalent identification methods. Tissues commonly used for assessment of DNA and RNA in mice are the tail and ear. Simple identification methods include ear punching, ear notching, placement of ear tags, and tattooing of the tail or foot pad. (NOTE: Any tissue collection over the age of 21 days and/or over 5cm requires anesthesia; use of local anesthetic agents is suggested for animals under 21 days of age). Ear Punches/Notches In order to establish which is the right or left of the animal - position it so that the belly is down and the head is facing away from the you. Restrain, as appropriate, to facilitate performing the punch or notch. Ear Punches/Notches A single hole or notch may be made using an ear punch tool. Notches are accomplished by positioning the instrument at the edge of the ear, resulting in a hole that is only partially surrounded by tissue. Notches may also be made by removing a small wedge shaped piece(s) of tissue with small scissors Ear Punches Tissues removed during this process are generally ample Ear Notches enough to allow for genetic testing, especially when PCR is utilized. Sterile instruments and tubes are used to obtain and manipulate the tissue after collection, and should be sterilized between animals. Multiple punches may be made to collect enough tissue for analysis – each punch is approximately 0.5mm in diameter. Ear Punch/Notch Code Right ear notch or hole indicates a single digit. Left ear notch or hole indicates a multiple of ten. One hole located in the middle/center of the Left ear = 100 One hole located in the middle/center of the Right ear = 200 Ear Punch/Notch Code Holes/notches begin at the top of the animal’s ear and progress along the outside edge of the ear to the bottom portion of the ear. There are three basic positions on the outer portion of each ear and the numbers progress as illustrated. Tail Snipping Anesthesia is required for mice over 21 days of age! Sample should be < 0.5cm DNA does not proportionally increase if tail fragments larger than .05cm are used. Scissors or scalpel are both acceptable - both must be sterilized between mice. Scalpel use is preferred by Comparative Medicine Staff. Tail Snipping Sterilization between animals: Use of the bead sterilizer provides a two for one punch: Hemostatsis and cautery. Complete sterilization between animals. Tail Snipping Sterilization between animals is important ! • You don't want to cross contaminate DNA samples ! • Wiping with alcohol will not prevent this from occurring (contact time is too short and may not remove or destroy all DNA/RNA). Tail Snipping Tattooing should be completed first, if applicable. Insert the scalpel blade into the beads ~15sec. Just the blade itself - don't introduce past the tip, or it will heat the handle (could burn yourself) Tail Snipping To perform the snip: Press the blade through the tissue. Hold the flat surface of the blade against the tip - the residual heat will cauterize to the area. Tail Snipping Use of a hard surface is recommended to facilitate asepsis & cleaning. It also provides a suitable surface to push the blade into with no damage to underlying surfaces. (Note the use of hard Plexiglas or a glass Petri dish - can be used with or without disposable pad) Tail Snipping Many labs use VetBond /glue for hemostasis Can cause an exothermic reaction that often makes the mouse rip off the scab once placed back within the home cage. Use of silver nitrate sticks is another popular choice (Note: don't 'roll' the tip around, but just sort of 'plug' the stick into the end of the tail). Ear Tagging The animal is positioned, as with the ear punch method, belly down, facing away from the handler. The ear tag is pre-loaded into a crimping device. The mouse is restrained. The tag is applied to the left ear so that the self piercing portion is firmly attached through the interior portion of the ear tissue. The tag should be applied so the number is visible to the outside of the ear. Ear Tagging Many animals will scratch or pull out tags. Care must be taken to position the tag so that it does not extend too far past the ear edge, but not so close as to wrinkle the ear. Tail Tattooing The animal should be properly restrained or anesthetized, in accordance with existing SOPs and IACUC guidelines, before attempting to tattoo. The most common instrument used on rodents is the tattoo pen. Tattooing is the preferred method of identification for animals being utilized on long-term projects, while tagging is commonly used on animals involved in short term models. The numbers used will be determined by the user. Tail Tattooing The tattoo pen is normally used on the tail, but can also be used on the foot pads for long term identification. Several practice attempts should be completed to learn proper technique with the tattoo pen, since legibility is a major factor. Tail Tattooing The area to be tattooed is gently cleansed prior to tattooing. The area can be sprayed with a numbing agent to alleviate potential distress – these sprays provide transient effects only, and the tattoo should proceed quickly following prepping of the area. A thin layer of tattooing oil will be applied in an even manner across the entire area. The area prepped should extend just beyond the tattoo pattern. The pen is positioned so that the needles just pierce the tissue. Tail Tattooing The numbers will be arranged so that they progress down the tail beginning at the base and extending to the tip. Care must be taken to not tattoo directly over any visible underlying vasculature. Once the tattoo is completed, any excess ink is wiped away. Tail Tattooing • All tattoo equipment (especially needles) should be cleaned thoroughly at the end of the session. OR as directed by the PI: • between animals • between cages • between strains • between rooms and protocol assignments AUTOCLAVE needles weekly at a minimum! Special Thanks Monica Torres – SRB Quarantine Manager Jim Gildea – SRB Facility Manager Amanda Rockhill and Omar Guevarra SRB staff members And the MICE who so graciously “posed” for us! .

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