Guidelines for Genotyping Laboratory Mice and Rats

Revised: 2/28/22

Laboratory Animal Resources

Laboratory Animal Resources Guidelines

Guidelines for Genotyping Laboratory Mice and Rats

I. Purpose- Researchers must consider all sources of DNA to perform genotype analysis, including

alternatives to invasive procedures such as tail biopsy. As with any procedure, the specific method

of tissue collection must be detailed in the approved IACUC protocol. This document serves to

describe examples of procedures that could be used to safely and appropriately collect samples to

genotype rodents used in laboratory animal research at Indiana University Bloomington.

II. Definitions:

Genotyping- The process through which an animal’s genetic make-up is determined using a tissue

sample.

Identification- The process of uniquely marking an animal so it can be differentiated from other

animals within a group.

III. Responsibility:

The laboratory staff is responsible for implementing the IACUC approved genotyping methods

with oversight and assistance from LAR staff, as needed.

The Principal Investigator (PI) is responsible for ensuring that all methods of animal genetic

sampling are explicitly listed and approved by the Institutional Animal Care and Use Committee

(IACUC) in the applicable animal use protocol and that laboratory staff are trained to perform these

procedures.

IV. Acceptable Sources of Animal Genetic Material for Genotyping

Determining the genotype in a rodent litter that has been genetically engineered is critically

important. The genotype is most often determined by analysis of DNA extracted from tissues of

young rodents. Analysis by the Polymerase Chain Reaction (PCR) requires the least amount of

DNA. Small amounts of DNA can be obtained from ear punches, tail biopsies, or various non-

invasive alternatives. Larger amounts of DNA required for determination of genotype by Southern

Blot are usually obtained from tail biopsies. Depending on the requirements of the study,

investigators are urged to consider noninvasive alternatives such as hair, fecal or oral samples.

1. Ear Punching/Notching- application of a specific combination of small hole punches or

notches to the outside edges of a rodent’s ear (see Fig. 1a).

a. Ear notching/punching can provide tissue for genotyping and as a means of permanent

identification.

b. The procedure should be performed after 14 days of age which is when the pinnae

(ears) are generally large enough to punch/notch.

c. Ear punch or notching instruments are disinfected with alcohol or a hot bead sterilizer

between animals to avoid sample contamination.

d. Ear punches should be no larger than 2 mm.

e. Mouse Ear-punching (see Figure 1a)

i. The mouse is restrained by the scruff (see Fig. 1b) and an ear puncher (see Fig. 1c)

is used to make holes and/or notches in the ears following an identification chart.

Laboratory Animal Resources

Guidelines for Genotyping Laboratory Mice and Rats

Revised: 2/28/22

ii. A small amount of bleeding is expected and can be controlled by gentle, constant

pressure. Animals should be checked several hours post-procedure to ensure proper

hemostasis.

Fig. 1a Mouse ear punch Fig. 1b Mouse restraint Fig. 1c Mouse ear punches

f. Rat Ear-punching (see Fig. 2)

i. Firmly restrain the animal. Restraint methods that can be used include:

 The two-handed technique: first place the rat on your arm, holding the base of

the tail with your hand. Hug the rat at the shoulders and push in gently at the

elbows to cross the front legs. Still maintaining your hold at the base of the tail,

stretch out the abdomen. Grasp the rear legs and immobilize (see Fig. 2a).

 If more restraint is needed, the rat may need to be scruffed by grasping the

loose skin behind the neck.

 A restraining device, which allows access to the head while still immobilizing the

animal, may also be used. An ear puncher is then used to make holes and/or

notches in the ears (a second person will be needed to do this).

ii. Ear puncher (see Fig. 2b) is used to make holes and/or notches in the ears following

an identification chart. (Fig. 2c)

iii. Hemostasis of the ear notch/punch site can be achieved by compression.

Fig. 2a Rat restraint Fig. 2b Collecting punch Fig. 2c Rat ear punches

2. Tail Biopsy

a. The tail clip or biopsy procedure is momentary, but involves bone or cartilage, blood

vessels, nervous tissue and skin. There is potential for pain from this procedure. Analgesics

may be used if prolonged pain is anticipated. The smallest possible section of tail is removed

and adequate hemostasis is achieved. Current literature shows that 5mm is sufficient tissue

to extract DNA for genotyping procedures. No more than 5 mm can be removed at one

time without first securing IACUC approval.

Laboratory Animal Resources

Guidelines for Genotyping Laboratory Mice and Rats

Revised: 2/28/22

b. In the mouse, the distal tail is completely ossified and innervated between 17-21 days of

age. Thus, tail sampling is recommended in mice and rats less than 3 weeks of age to avoid

undue stress and discomfort to the animals. The optimum age at which to perform

biopsy is between 12-17 days, before the distal tail completely ossifies.

1) Tail snips in animals < 17 days of age, 2-5 mm tail tip sample is obtained

without the need for anesthetics or analgesics.

2) Tail snips in animals 18-21 days of age, 2-5 mm tail tip sample is obtained

with local and systemic analgesics

recommended

. For local analgesia, use

Bupivacaine 0.75% and immerse the tail in the bupivacaine for 10 sec. after

biopsy. Systemic analgesic agents such as Meloxicam or Buprenorphine are

encouraged

but not required.

3) Tail snips in animals > 21 days old, 2-5 mm tail tip sample is obtained using

the required local analgesic bupivacaine 0.75% and immersing the tail in

the bupivacaine for 10 sec. after biopsy. Tail biopsy must be performed with

general anesthesia (i.e. Isoflurane) followed by analgesic such as

Meloxicam or Buprenorphine (see LAR

Guidelines for Anesthesia and

Analgesia in Mice or Rats

for recommendations). Any situation where general

anesthesia cannot be used for tail biopsies in mice or rats over 21 days of age

must be scientifically justified in the animal use protocol and approved by IACUC.

4) If multiple tail biopsies must be performed because of inconclusive PCR or lost

samples, local analgesia with bupivacaine 0.75% is recommended as long as 2-5

mm tail sample is taken and the animal is <21 days old. If animals are >21 days,

2-5 mm tail tip sample is obtained while the animal is under isoflurane

anesthesia using bupivacaine as a local analgesic and Meloxicam or

Buprenorphine as systemic analgesics.

c. Mouse Procedure:

 Gently but securely restrain the rodent. (Fig. 3a)

 Swab the tail with alcohol (povidone iodine or chlorhexidine may interfere with the DNA

identification tests).

 Sterile tools for this procedure are recommended. Disposable scalpel blades or razor

blades can be used as a sterile method as long as a new, sterile blade is used for each

mouse or rat. Alternatively, scissors can be sterilized using a hot bead sterilizer between

animals. Reuse of a scalpel blade or not sterilizing scissors can lead to contamination of

samples and invalidation of results.

 Snip the skin sample that is < 5 mm and place in sample container with label. (Fig. 3b)

 Hemostasis of the tail biopsy site can be achieved using compression of the tail,

application of silver nitrate or styptic pencils or cautery, and possibly the use of tissue

adhesives (e.g. Nexaband

) to close the wound if needed. (Fig. 3c) 

 Observe rodent for bleeding or abnormal behavior; check daily to ensure tip is healing.

d. If the mouse or rat is anesthetized using general anesthesia, the animal is then recovered

individually in a clean cage after the biopsy is completed. The mouse or rat must be fully

ambulatory before it is returned to the original cage or co-housed with other rodents.

e. ALL animals (regardless of age) must be monitored for 5 minutes after

returning to their cage for any signs of bleeding from the site.

Laboratory Animal Resources

Guidelines for Genotyping Laboratory Mice and Rats

Revised: 2/28/22

Fig. 3a Measure tail Fig. 3b Cut tail Fig. 3c Hemostasis materials

3. Toe Amputation/Toe Clipping

a. Toetip clipping (toe amputation) involves the removal of the toe at the most distal

joint. According to the Guide for the Care and Use of Laboratory Animals (

Guide

“toe clipping should be used only when no other individual identification method is

feasible. It may be the preferred method for neonatal mice up to 7 days of age as it

appears to have few adverse effects on behavior and well-being at this

age…especially if toe clipping and genotyping can be combined”.

b. Toe clipping in rodents greater than 7 days old is a stressful procedure that should

only be performed if no other identification and genotyping options are feasible. If

toe clipping is requested, it should be used for genotyping and identification

purposes.

c. Conditions for Approval: Toe clipping will only be permitted when the following

conditions are met:

1. The Principal Investigator (PI) provides a scientific justification with

documentation to the IACUC explaining why each of the alternative identification

methods listed in the

Policy for Identification of Mice and Rats

are not feasible.

Cost alone is NOT considered an adequate justification.

2. Once the procedure is approved, all laboratory personnel designated to perform

the procedure must undergo training and/or demonstrate proficiency to the PI or

LAR trainer.

3. Toe clipping can only be performed on animals thirteen (13) days of age

or younger. Neonatal mice 5-7 days of age may be toe clipped for

genotyping and identification purposes without the use of anesthesia.

4. Mice 8-13 days of age at the time of the procedure may be toe clipped ONLY if

the toe tissue is also used for genetic analysis. This would be considered a

refinement by making it unnecessary to perform tail biopsies for tissue sampling.

Topical anesthesia is required and can be accomplished by immersing the toe in

bupivacaine 0.75% for 10 sec. immediately after biopsy.

5. The numbering system should be designed to minimize the total number of toes

clipped per animal. Similarly, a given foot should have as few toes clipped as

possible.

6. The following must be followed:

 No more than two toes are amputated per foot, and two feet per animal may

be clipped.

 Avoid clipping toes on forepaws, if possible.

 DO NOT clip the 1

digit (i.e. thumb) on either forepaw.

Laboratory Animal Resources

Guidelines for Genotyping Laboratory Mice and Rats

Revised: 2/28/22

 Remove the 3

phalanx (i.e. toe-nail bearing, last bone of digit); cutting the

very distal portion of the 2

phalanx to remove the complete nail bed.

7. If animal genotyping is required, the tissue yielded from toe clipping is used. A

second method of tissue collection (e.g. tail biopsy) will only be accepted if tissue

is lost or a second genotyping is required.

d. Appropriate Method for Toe Clipping

1. Instrument preparation- instrument must be disinfected between animals.

Instruments should be sharpened regularly to ensure minimization of tissue

injury.

2. Skin preparation- Gross debris, if present, will be cleaned from the foot. Alcohol

may be used to wipe down the foot and digits prior to the amputation.

3. Neonatal mice are placed on a soft padding of some kind (e.g. blue diaper pad,

towel). Using gentle pressure, restrain the mouse and extend the leg. Rodents

are restrained only by hand for this procedure, and not by a restraint device.

Taking care to hold a mouse properly is the most important aspect of ensuring

its comfort and safety.

4. With a sharp pair of dissecting scissors or scalpel, remove the toe at the most

distal joint and into the distal tip of the 2

phalanx.

5. After removing the digit, apply a piece of gauze to the distal portion of the digit

with finger pressure to ensure hemostasis. ALL animals (regardless of age)

must be monitored for 5 minutes after returning to their cage for any signs

of bleeding from the site.

6. Place the pups that have been identified into a new cage to distinguish them

from their littermates. When all of the pups have been identified, return the pups

to their mother’s cage.

a. Day 5-7 and 8-13: No more than 2 toes per foot should be removed,

with a maximum of 4 total clipped toes per animal.

N=nail

P3= phalanx 3

P2= phalanx 2

P1= phalanx 1

Line indicates where cut of digit should occur

4. Other

a. Blood- can be used for genotyping but not identification; noninvasive.

b. Hair follicles- noninvasive; used for genotyping, high risk of contamination

c. Colonic and Rectal cells- samples rectal swab or scrape or fecal pellets;

genotyping but not identification; feces collected within 24 hours.

Laboratory Animal Resources

Guidelines for Genotyping Laboratory Mice and Rats

Revised: 2/28/22

d. Cells from the Oral Mucosa- oral cavity scraped or swabbed; low amount of

DNA obtained.

Table 1. Methods for Genotyping Rodents

Method

Age

Anesthesia

Requirements

Additional Information

Ear-punching

14 days or older

No anesthesia is required when

performed by trained personnel



Punch devices should be disinfected

between animals



Tissue can also be used for genotyping



Punched tissue may re-seal; must be

rechecked periodically and punching

may need to be repeated



after 2 weeks of age

Tail Biopsy

12-17 days

old

No anesthesia is required when

performed by trained personnel



2-5 mm tail can be biopsied



Restrain animal



Swab tail with alcohol



Use sterile scalpel blade, razor or sharp

scissors (clean with alcohol between or

hot bead sterilizer)



Hemostasis of tail by compression



Monitor for at least 5 min. after biopsy

18-21 days old

Local and systemic analgesia is

recommended but not required

>21 days old

General anesthesia (isoflurane)

required

Local analgesic Bupivicaine 10 sec.

required

Systemic analgesics (Meloxicam or

Buprenorphine) required

Toe-clipping

Rats from 5-7

days old

Mice from 5-13

days old

No anesthesia is required P 5-7

when performed by trained

personnel

Topical/local anesthesia is required

for P 8-13 animals



Equipment and site should be

disinfected prior to clipping to minimize

risk of infection



Ensure only distal bone (P3) and nail

bed are removed including distal portion

of P2



May impair grip strength



Tissue can also be used for genotyping



Day 5-7: no more than

2 toes/foot

(up

to 4 toes total) can be removed



Day 8-13: No more than 2 toes total can

be removed.

Non-permanent

marking

Any age

Only for dying



Inexpensive but not permanent



Use nontoxic dyes or markers

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Laboratory Animal Resources

Guidelines for Genotyping Laboratory Mice and Rats

Revised: 2/28/22

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