Tissue Collection for Genetic Identification of Rodents

PURPOSE:

The correct identification of the genotype of transgenic animals in a litter is a critical aspect of research projects using genetically modified animals.  The most common method for genotype identification involves the analysis of DNA extracted from the tissues of the mice using the Polymerase Chain Reaction (PCR). Sufficient DNA for PCR analysis can be obtained from a variety of tissue sources including blood, ear punch tissue samples, tail tip biopsies (tail snips), hair samples, stool, and oral swabs.  Investigators are encouraged to explore and use tissue collection methods that are less traumatizing than the tail biopsy (tail snip) procedure whenever possible when using PCR for genotyping. 

Determination of the genotype using Southern Blot analysis requires a greater amount of tissue than the PCR analysis procedure.  The use of the tail biopsy (tail snip) procedure, when properly performed, provides an adequate tissue sample with minimal or transient pain and distress.  

This document describes procedures and provides standard recommendations to PIs regarding the obtainment of tissue for genetic identification of mice.  The text under the section University of Kentucky DLAR Veterinary Recommendation may be copied and pasted into an animal use protocol description of the procedure.

APPLICATION:

The procedures described in this document are applicable to colony management personnel, researchers, and technicians who collect tissue samples for the genetic identification of mice.  The DNA prepared from tissue obtained all of the sites (blood, ear punch, fecal samples, oral swabs, hair, and tail biopsies) is suitable for analysis by PCR.  Southern Blot genotyping generally requires the use of tail biopsy (tail snip) to obtain an adequate tissue sample.

PROCEDURES:

• Ear Punching:
  Ear punching does not require anesthesia in rodents when performed by a skilled individual.  Ear punches are generally obtained at approximately 15-17 days of age, after the ear has “thinned.” Several tissue samples can be obtained using a commercially available rodent ear-punch. (http://www.harvardapparatus.com) and the punch pattern can be used for animal identification. 
  
  The ear punch procedure should be performed using clean gloves and a sterile ear-punch. Manually restrain the animal and place the punch device on the pinna of the ear (external ear) in a location where you want to mark the animal for identification. Press firmly to punch a circular hole through the ear. As you remove the punch, be careful not to rip the delicate membrane of the pinna. Gently separate the ear from the device and remove the sample for tissue sample. Bleeding after ear punching is uncommon and the animal can be released directly into the cage.
  
  If the analysis of the DNA is to be performed by PCR, great care should be taken to remove all tissue from the ear punch after each animal. Sterilizing the ear punch between animals using a hot-bead sterilizer will also minimize the potential of DNA cross contamination.

Suggested Wording for Animal Use Protocol:

• Fecal Sampling:
  Genetic analysis using feces is a completely non-invasive procedure with the potential of greatly reducing the effort required to collect samples for analysis. Feces contain sloughed intestinal epithelial cells which serve as a source of DNA for genetic analysis.  A fresh stool sample can be easily obtained directly from the animal (rodents routinely defecate when gently handled) or collected from the cage of singly housed rodents.  The genetic analysis procedures have been described in Lab Animal Magazine Vol41 No.4 April, 2012  Non-Invasive transgenic mouse genotyping using stool analysis by Broome, et.al. 
• Tail Biopsy:
  Obtaining tissue from a mouse or rat for DNA analysis via tail biopsy is a safe, effective and humane procedure. When performed properly it causes only minimal or transient pain and distress, and induces no more “physiological impact” (change in heart rate, body temperature, or activity level) than just restraining the animal for the procedure. DNA prepared from tail biopsies is suitable for analysis by either Southern Blot or PCR.
  
  The removal of tail tip of a mouse involves cutting skin, nervous tissue, vasculature and developing vertebrae. This procedure, while generally considered a minor procedure, carries a potential for acute and chronic pain. Careful consideration must be given to the timing of this procedure relative to the animal’s age and the length of tail tissue removed.  A review of the current literature (cited at the end of this document) will show that definitive guidelines have not been established concerning animal age at biopsy, length of biopsy tail tissue or anesthesia/analgesia.   Alternatives to tail biopsy involving the use of less invasive and more humane methods such as ear punches, blood samples, hair samples, stool, mucous epithelial cells or saliva swabs should be initially investigated prior to electing tail biopsy as the method for tissue collection for genetic analysis.                                                                                                                 
  Animals should be between 10 and 21 days old.  At this age, the tail tissue is still soft, the tail vertebrae have not yet calcified, and the yield of DNA is the highest. Prompt analysis allows mice to be genotyped prior to weaning, facilitating efficient cage use and housing cost savings. 
  • Anesthesia:
    For mice and rats 10-21 days of age: Because pain sensory development may be complete, and to further minimize any transient pain or distress, investigators are strongly encouraged to apply local anesthesia to the tail. Local anesthesia may be achieved by immersion of the tail in ice-cold isopropyl or ethyl alcohol for 10 seconds [Caution:  ethyl chloride, isopropyl alcohol, and ethyl alcohol are all flammable] or by the application of ethyl chloride (http://www.gebauerco.com/Default.asp) [see below for additional information].  Investigators may wish to consider the use of general anesthesia as an alternative to local anesthesia in specific cases.
    
    For mice and rats greater than 21 days of age: The use of a local or general anesthetic is required prior to collection of tissue.
    
    For rats greater than 35 days of age: The use of a general anesthetic is required.

• The tail biopsy procedure must be performed using clean gloves and a sterile sharp scalpel, scissors, or razor blade. Tail skin should be disinfected with alcohol prior to incising the tip (Do not use iodine solutions because they may interfere with DNA analysis.) Manually restrain the mouse or rat between thumb and forefinger. This is a convenient time to identify the animals using the appropriate method (i.e. ear punch, ear tag, transponder etc.). Make one clean cut through the tail. If the analysis of the DNA is to be performed by PCR, great care should be taken to remove all tissue from the scissors or scalpel after each animal. Sterilize the scalpel, scissors, or razor blade between animals. If a scalpel or razor blade is used, also disinfect the work surface on which the tail is placed between animals.
• The tissue taken should be as small as possible with 5 mm the maximum length of tail tip removed (skin can be pushed down toward the tip of the tail so that the vertebrae are avoided). Tail biopsy samples of greater than 5 mm in length will most likely damage the vertebrae and are not recommended.  If the proper procedures are followed, the yield of DNA from 5 mm of tail should exceed 50 micrograms, enough for multiple analyses. The amount of DNA obtained does not proportionally increase with samples of over 5 mm length and, in most cases; a 2 mm tail biopsy is more than adequate. 
• Following the biopsy procedure, bleeding should be controlled using local pressure and sterile gauze.  The use of a cotton-tipped applicator with 1% lidocaine with epinephrine may enhance hemostasis as well as providing post-procedural local analgesia. After releasing the animal back into the cage, it should be observed to make certain that the bleeding has stopped.  The presence of blood in the cage may cause aggression between cagemates. It may be necessary to use styptic powder, silver nitrate, or surgical glue in cases where bleeding is not controlled with digital pressure. 

Suggested Wording for Animal Use Protocol:

Using Ethyl Chloride for Local Hypothermic Anesthesia: 

• Hair Bulb Sampling:
  The use of hair bulbs in human forensic medicine is common and sufficient DNA can be extracted from a single hair bulb to perform genetic screening by PCR. The standard protocol for identification of transgenic animals is based on tissue samples and preparation of chromosomal DNA including proteinase K digestion and phenol/chloroform extraction. The hair bulb procedure is much simpler and faster method using alkaline lysis followed by PCR (Schmitteckert. et.al). Extension of these common procedures and protocols to the genetic identification of rodents reduces both the animal pain and distress and the technical effort required in comparison to the standard tail biopsy procedure. 
  
  The actual sample collection procedure requires the manual restraint of the animal and the removal of a tuft of hair from the ventral body using forceps. While there is no necessity for sterile instruments, the use of a new set of forceps for each animal minimizes the potential of cross contamination between animals. 
  
  Hair bulb sampling can be performed at any age and does not require any anesthesia or analgesia.  There are a number of commercially available DNA extraction kits for hair bulb samples.

Suggested Wording for Animal Use Protocol:

• Saliva Sampling:
  Saliva sampling is routinely used for genetic analysis in humans to determine paternity. A small amount of saliva contains sufficient oral epithelial cells to permit PCR analysis. Unlike the human situation where swabs are used to collect the epithelial cells, the collection technique in rodents involves the washing of the oral cavity of weanling animals using a plastic pipet tip (Irwin, et.al.)

References:

1.    Hofstetter JR, Zhang A, Mayeda AR, Guscar, T, Nurnberger JI and Lahiri DK. Genomic DNA from Mice: A Comparison of Recovery Methods and Tissue Sources. Biochem Mol Med 1997 Dec; 62(2):197-202.

2.    Dennis, MB. IACUC Review of Genetic Engineering. Lab Animal 2000 Mar; 29(3):34-37

3.    Irwin MH, Moffatt RJ and Pinkert CA. Identification of Transgenic Mice by PCR Analysis of Saliva. Nat Biotechnol 1996 Sep;14(9): 1146-8.

4.    Schmitteckert EM, Prokop CM and Hedrich HJ. DNA Detection in Hair of Transgenic Mice - A Simple Technique Minimizing the Distress on the Animals. Laboratory Animals 1999; 33/4: 385-389.

5.    Couse JF, Davis VL, Tally WC and Korach KS. An Improved Method of Genomic DNA Extraction for Screening Transgenic Mice. National Institute of Environmental Health Sciences, National Institutes of Health. BioTechniques 1994; 17:1030-1032.

6.    Malumbres M, Mangues R, Ferrer N, Lu S and Pellicer A. Isolation of High Molecular Weight DNA for Reliable Genotyping of Transgenic Mice. BioTechniques1997; 22/6:1114-1119.

7.    Broome RL, Feng L, Zhou Q, Smith A, Hahn N, Matsui SM, Omary MB. Non-invasive Transgenic Mouse Genotyping Using Stool Analysis. FEBS Lett 1999; 462:159-160.

8.    Pinkert CA. Transgenic Animal Technology: Alternatives in Genotyping and Phenotyping. Comp Med 2003; 53/2:126-139.

9.    Meldgaard M, Bollen PJA, Finsen B. Non-invasive method for sampling and extraction of mouse DNA for PCR. Laboratory Animals 2004; 38:413-417.

10.  Shinohara H. The Musculature of the Mouse Tail is Characterized by Metameric Arrangements of Bicipital Muscles. Okajimas Folia Anat Jpn 1999; 76-157-169

11.  Cinelli P., et.al. Comparative Analysis and Physiological Impact of Different Tissue Biopsy Methodologies Used for the Genotyping of Laboratory Mice. Lab Animals 2007; 41: 174-184.

12.  Baumans V, Burgere H, et al. 1994.  Pain and Distress in Laboratory Rodents and Lagomorphs.   Report of the Federation of European Laboratory Animal Science Association Working Group on Pain and Distress. Laboratory Animals (28): 97-112.

13.  Refinement and reduction in production of genetically modified mice.  Sixth report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement. 2003. Laboratory Animals (37):supplement 1 , S1:27-S1:33.

14.  Hankenson CF, Garzel LM, Fischer DD, Nolan B, Hankenson KD. 2008. Mice (Mus musculus): Vertebral Ossification, DNA Quantity, and Acute Behavioral Responses. Journal of the American Association of Laboratory Animal Science. Nov 47(6):10-18.

15.  Williams WO,  Riskin DK, Mott KM. 2008. Ultrasonic Sound as an Indicator of Acute Pain in Laboratory Mice.  Journal of the American Association of Laboratory Animal Science 47 (1):8-10.

16.  DeMarco G ,Pascoe P, Meyer R, Fish R, Brunson D, Heavner J, Cooper D. 2008.  Anatomy, Physiology, and Pharmacology.  In: Anesthesia and Analgesia in Laboratory Animals, 2^nd Edition.  American College of Laboratory Animal Series.  P3-87.

17.  Guidelines for the Genotyping of Mice and Rats, ARAC Guidelines, Office of Animal Care and Use, National Institutes of Health, Revised 9/12/07 (http://oacu.od.nih.gov/ARAC/documents/Rodent_Genotyping.pdf)

18.  Boston University http://www.bu.edu/research/compliance/oversight-committee/iacuc/policies/mouse-tail-biopsy-guidelines.shtml

19.  University of Minnesota http://www.ahc.umn.edu/rar/tailsnip.html

20.  Indiana University School of Medicine Accessed from: http://www.iupui.edu/~somiacuc/worddoc/Tail%20snip%20guidelines%20Feb%2003.doc