Technical Guidelines for Use

Prepared By:  Dr. Grant MacGregor
(Last updated 5/20/2001)

Materials

1. Squirt bottle with 70% ethanol

2. Surgical scissors (e.g. Fine Science Tools (FST, (http://www.finescience.com) ) catalog # 14090-11; Fine Iris Scissors)

3. Watchmaker forceps, Dumont # 5 FST catalog # 11252-20  (Biology tip).

4. Suitable non-porous work surface – e.g. resin bench top or stainless steel surface in hood.

5. Appropriate disinfectant

6. Small Kimwipe

7. Fresh cage bottom with bedding

8. Toe-clipping ID chart

9. 1.5ml Eppendorf tubes in rack

10. Glass bead sterilizer (e.g. FST cat # 18000-45; FST 250 Portable hot bead sterilizer)

Notes

2. Anesthesia should not be used

3. Mice should be no more than 12 days old where the day of birth is defined as day 0. Experience suggests that the optimum time for this procedure is between 8 and 12 days of age.  Rats, hamsters and voles should be 7 days old or less.  Note, if eyes are open, toes and tails should not be cut. In such cases, animals should be weaned, then toe and tail biopsies conducted at 4 weeks of age as described in the “Biopsy for ID and Genotyping of Weaned Animals:  Technical Guidelines for Use” instructions.

4. Prior to use, thoroughly clean and disinfect the work surface where the procedure is to be performed by washing with an appropriate disinfectant. Protective mask, gown and gloves should be worn.

5. Use a fine pair of surgical scissors (e.g. FST catalog # 14090-11) to perform the toe biospies. Experience suggests that it is worthwhile to use quality scissors that will cut cleanly and not require frequent sharpening. Prior to use on the first animal, the scissors can be sterilized by placing the tips in a glass bead sterilizer (e.g. FST cat # 18000-45) for 20 seconds. Between animals, the scissors may be cleaned using a fresh Kimwipe soaked in 70% ethanol. This serves to disinfect and remove traces of tissue from the previous animal, which could serve as a source of DNA contamination. It is unnecessary to sterilize the scissors in the glass bead unit between animals.

6. It is unnecessary to cut more than two digits per limb. In practice, investigators often revert to ID # 1 at around number 500. Thus, the thousand series is seldom used for conventional ID purposes and can be utilized to discriminate animals under special circumstances.

7. No more than one half of each digit should be amputated. This is sufficient to permit identification.

8. Minimal bleeding is normal. At this age, the wounds heal very quickly.

9. Experience indicates that this is a very safe and reliable procedure. In the MacGregor lab, no mortality or morbidity (e.g. infection) has been experienced when using this method in over 25,000 procedures performed between 1993 and 2000.

10. The IACUC Guidelines for Biopsy Procedures to Facilitate Identification and DNA-based Molecular Genotyping of Rodents can be found at:  http://www.emory.edu/IACUC/pdfs/BiopsyPolicy.pdf

Method

2. Count the number of pups on which biopsies are to be performed. Label this number of Eppendorf tubes using a permanent sharpie. It is advisable to label the top and side of each tube.

3. Carefully remove all pups from home cage and place together in a clean cage. While removing the pups, sex them by looking for presence of nipples in females or by relative ano-genital distance (larger in males). Record the number of males and females. By convention, male pups are numbered first in a litter. To minimize distress to the parents, return the pups to the home cage as soon as possible.

4. Assuming you are right handed, using the thumb and first finger of your left hand, lift the pup firmly but carefully by the skin at the nape of the neck. Position the pup so that it is facing you. The pup can be supported by placing your third and fourth fingers of your left hand underneath its hindquarters. At this point the pup will often extend its limbs.

5. Working quickly, but carefully, refer to the toe-ID chart and clip half of each digit required to provide an ID number for the pup. Allow the clipped digits to fall to the clean bench surface. In some cases, the clipped digit may adhere to the scissors.

6. If using toes as a source of DNA for genotyping, carefully use the forceps to place two toes in an Eppendorf, while discarding the remainder. Note, to maintain consistency in the amount of input template DNA used for each PCR reaction, only two toes are used per animal. Note, if using toes for genotyping, the bench top must be wiped with a Kimwipe soaked in ethanol between animals. However, if DNA is to be extracted from tail biopsy, ignore toes and proceed to next step.

7. After the toes have been clipped, hold the animal’s tail over Eppendorf tube and carefully cut approximately 0.5cm. Allow the tail tip to fall into the tube.

8. Immediately replace the pup in the home cage. When the pup is replaced outside of the nest, the mother will frequently retrieve the pup to the nest. Invariably, the parents continue to care for the pups and will clean the wounds. Minimal bleeding is normal. Infection has never been observed.

9. Wipe the forceps and scissors with a fresh Kimwipe, soaked in ethanol between each animal.

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