For the production of genetically altered rodents, it is often necessary to sample tissue for DNA analysis. Usually, the end of the tail is sampled. However, alternatives to tail biopsies are available. The procedures below are listed in increasing order of their relative degree of invasiveness. They should be considered in this order.

Procedures for DNA extraction
Invasiveness Anesthesia Needed References
Fecal No No 1, 3
Rectal Swab Minimal No 2, 11
Buccal Swab Minimal No 4, 5, 6, 7, 9, 11
Hair Minimal No 8, 11
Blood Yes Yes, depending on method 3, 13
Tail Snip Yes Over 3 weeks of age 10, 11
Ear Punch/Snip Yes Not for mice; yes for rats over 3 weeks of age 11

In a 2007 article in Laboratory Animal by Cinelli et al, all methods - ear punch, tail snip, hair, mouth swab, rectal swab - were evaluated. [11]

various dna extractions

Figure 1:
Representative picture of DNA extracts tested for the presence of the LacZ-transgene (8 animals out of 40). DNA was extracted from ear punch, tail biopsy, hairs, mouth, and rectum swabs of the same animals. DNA from the negative and the positive animals was extracted and processed in parallel. The expected transgene PCR fragment is 394 base pairs long. [11]

Fecal Pellets/Rectal Swabs

  • Non-invasive procedure can be used on rodents of any age.
  • Fresh stool pellets can be collected directly from the animal or from the cage (if singly housed). [1]
  • Rectal swabs also give DNA yield sufficient for PCR. No purification was needed in this paper. [2]
  • 7ug DNA/pellet. [3]

Buccal Swabs

  • Minimally invasive method can be used on rodents of any age.
  • A method for collecting buccal swabs from pups as young as 1 day old was developed at UCLA. [4-7]
  • 2-5ul of saliva comparable to 100ng tail on PCR. [6]
  • 20ug DNA from buccal swab. [9]

Hair Bulbs

  • Minimally invasive procedure in which a tuft of hair is plucked using forceps.
  • Use separate forceps for each animal or sterilize in between use; fur can cling to forceps.
  • Gave same DNA yield as tail. [8]

Blood

  • Anesthesia required depending on method; see ARC Policy on Blood Collection From Laboratory Animals for guidelines.
  • A 2mm spot of dried blood on Whatman GF/C filter paper gives enough DNA for PCR. [13]

Tail tissue collection

  • 30ug/2-3mm.
  • Recommended for mice less than 3 weeks of age due to ossification of tail between 2-4 weeks.
  • Anesthesia required for mice over 3 wks of age, preferably using a short acting anesthetic (e.g. inhaled Isoflurane), but recommended for mice of all ages.
  • No more than 5mm may be removed (larger samples require ARC approval and use of anesthesia).
  • Adequate hemostasis (blood-clotting) must be achieved via styptic powder (anesthesia is required for electrocautery).
  • Resampling must be approved by DLAM veterinarian.

Ear Punching/Snipping

  • Small circle (about 1 mm in diameter) of ear tissue for genetic analysis is collected ("punched out") with a special tool ("ear punch").
  • Ear snipping can also be done. A small portion (2-3 mm) of the ear pinna is sliced off with sharp scissors to obtain tissue, avoiding ear blood vessels.
  • Anesthesia is not required for mice at any age, but is required for rats over 3 weeks of age.

Instruments

Ear punches and scissors must be well-maintained to stay sharp; they must be cleaned and disinfected between animals to minimized infection and DNA contamination of the samples. This can be done by wiping off any blood or debris adhered to the instrument using a new sterile alcohol pad for each animal, soaking in Clidox® or 70% alcohol for at least 5 minutes, using a hot bead sterilizer, or autoclaving the punching instruments. (Note that ear punches rust easily when autoclaved.) Ear punches or scissors placed in a glass bead sterilizer must be allowed to cool down to body temperature before they are used on a subsequent mouse. It is recommended that the use of blades be limited to no more than 5 tails.

Considerations

  • There is some evidence to indicate that tail tip amputation may affect certain behavioral tests, such as tail flick assay and hot plate response, later in life [10]. This should be kept in mind when choosing what approach to use for DNA collection.
  • All methods equally raised body temperature and heart rate as much as handling alone. This emphasizes the importance of good technique and proper restraint. [11]
  • PCR takes approximately 1 day to complete while Southern blot takes 3 days. Because of its high sensitivity, PCR is prone to false positive results. Although most papers used PCR, PCR takes less DNA than Southern blot and so most methods should also work for Southern Blot. It is important to remember that success also lies in your techniques: the methods of digestion, the characteristics of the target sequence, primer selection, and minimizing contamination. [12]

References

  1. Broom, RL, Feng L, Zhou Q, Smith A, Hahn N, Matsui SM, Omary MB. Non-invasive transgenic mouse genotyping using stool analysis. FEBS Letters. November 26, 1999. 462(1-2): 159-160.
  2. Lahm H, Hoeflich A, Rieger N, Wanke R, Wolf E. (1998) Identification of transgenic mice by direct PCR analysis of lysates of epithelial cells obtained from the inner surface of the rectum. Transgenic Research 7, 131.4.
  3. Kalippke, K. DNA analysis from stool samples: a fast and reliable method avoiding invasive sampling methods in mouse models of bleeding disorders. Lab Animals 2009, 1-4.
  4. Zimmermann, K., H.P. Schwarz, and P.L. Turecek. Deoxyribonucieic Acid Preparation in Polymerase Chain Reaction Genotyping of Transgenic Mice. Comparative Medicine (2000) 50(3), 314-316 10.
  5. Y.-H. Zhang, B.-L. Huang, K. Eastman, L.L. McCabe, N.K. MacLennan, and E.R.B. McCabe. Mouth cell collection device for newborn mice. Molecular Genetics and Metabolism 89 (2006) 164.167.
  6. Irwin, M.H.; Mofatt, R.J.; Pinkert, C.A. Identification of Transgenic Mice by PCR Analysis of Saliva. Nature Biotechnology (1996) 14, 1146-1148.
  7. Meldgaard, M., P.J.A. Bollen, and B. Finsen. Non-invasive method for sampling and extraction of mouse DNA for PCR. Laboratory Animals (2004) 38, 413-417.
  8. Schmitteckert EM, Prokop CM, Hedrich HJ. DNA detection in hair of transgenic mice – a simple technique minimizing the distress on the animals. Lab Animal. October 1999. 33(4): 385-9.
  9. Mitrecic, D. et al. Mice Genotyping Using Buccal Swab Samples: An Improved Method. Biochem Genetics (2008) 46:105-112.
  10. Zhuo, Min. NMDA receptor-dependent long term hyperalgesia after tail amputation in mice. European Journal of Pharmacology 1998. vol. 349, pp. 211-220.
  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. Schneider, M. and E. Wolf. Genotyping of transgenic mice: Old principles and recent developments. Analytical Biochemistry 344 (2005) 1-7.
  13. Campbell DB, and EJ Hess. Rapid genotyping of mutant mice using dried blood spots for PCR analysis. Brain Research Protocols (1997) 1: 117-123.

Approved 11/4/13

Replaces ARC Policy on Tissue Collection for DNA Extraction for Genotyping in Mice 6/29/09