Tissue Sampling Policy for Rodents and Zebrafish Genotyping

IACUC Policy: 014-07, Effective Date: 02/25/2005, Last Revision: 03/18/2025

Overview/Purpose

Analysis of DNA may be necessary to verify the genetic composition of individual animals. In rodents, ear punch/biopsy is a common site for tissue collection beginning at 14 days of age and can serve as a means of identification in addition to providing tissue for genotyping. Tail biopsy is an alternative site for tissue collection, though care must be taken due to risk of bone exposure as the distal tail ossifies with age. In zebrafish, a small portion of the caudal fin is frequently clipped to obtain tissue for genotyping. For both rodents and zebrafish, minimally invasive methods such as fecal pellet, buccal swabs, and skin swabs (zebrafish) are becoming increasingly successful and should be considered.

Requirements

Rodents

Tissue sampling for genotyping should be done as early as possible in the lifespan of the animal to minimize the potential for pain/distress. Tissue sampling must be performed using sharp devices to avoid tissue crushing. Instruments must be cleaned between individual animals to minimize cross-contamination of genetic material. Animals must not be returned to the home cage until hemostasis is achieved.

• Ear Punch/Notch Procedure: This technique can be performed beginning as early as 14 days of age in most strains, allowing collection of a ≤2mm biopsy. Manual restraint without anesthesia is acceptable. The ear punch/notch can also serve as a method of individual animal identification.
• Tail-Snip/Biopsy Procedure: Ideally collection of tail tissue should be performed at 14-17 days of age, prior to ossification of the distal coccygeal vertebrae for optimal animal welfare as well as DNA yield. When tail tissue (<2mm) is collected in mice up to and including 21 days of age, anesthesia is not required.  If removing greater than 2 mm of tissue (regardless of age) or if performing a tail-snip/biopsy in rodents 22 days or older general anesthesia is required, and analgesia should be provided. In all cases, hemostasis must be accomplished using digital pressure or styptic powder (Kwik-Stop ®). Note: less invasive methods for genotyping should be considered for mice 22 days and older. Tail-snip/biopsy of mice ≥22 days of age is discouraged as other less invasive methods (i.e. ear punch/notch) are available. When tail-snip/biopsy is performed in mice of ≥22 days of age, or if collection of >2mm of tissue is required it must be described as a protocol activity and approved by the IACUC.

• Age	Sample Size	Anesthesia/Analgesia
  ≤ 21 days	≤ 2 mm	Not Required
  > 21 days	≤ 2 mm	Required
  Any Age	> 2mm, up to 5mm max	Required

• If other tissues or blood are being collected and used for genotyping, this must be described as a protocol activity and be approved by the IACUC.

Zebrafish

• Caudal Fin Clip / Biopsy: Removal of a small portion of the distal aspect of the caudal fin is a common collection site to obtain tissues for genotyping of adult zebrafish. Care should be taken to collect the minimum amount of tissue necessary and should not exceed 20% of the fin area.  Anesthesia and analgesia are recommended when performing fin clips. 
• Less invasive methods of genotyping such as skin swabbing have been validated for use in adult zebrafish and should be considered.
• All tissue collection methods for genotyping in zebrafish must be described as a protocol activity and be approved by the IACUC.

Additional Resources 

• Boivin et al, 2016. A highly efficient strategy to determine genotypes of geneticallyengineered mice using genomic DNA purified from hair roots http://journals.sagepub.com/doi/abs/10.1177/0023677216646088
• Bonaparte D, Cinelli P, Douni E, Herault Y, Maas M, Pakarinen P, Poutanen M, Lafuente MS, Scavizzi F. 2013. FELASA guidelines for the refinement of methods for genotyping genetically-modified rodents: a report of the Federation of European Laboratory Animal Science Associations Working Group. Lab Anim. Jul;47(3): 134-45.
• Boivin, GP, et al. 2013. Genotyping DNA Isolated Using Cross-Linked Iminodiacetate Sytrene Divinylbenzene Copolymer Beads. J Am Assoc Lab Anim Sci 52:682.
• Braden-Weiss, G.C., Brice, A.K., Hankenson, F.C. 2011. Minimizing the impact of tail biopsy in preweanling laboratory mice: inhaled isoflurane compared with topical anesthetics. J Am Assoc Lab Anim Sci 50: 736-737.
• Broome, R. L., L. Feng, Q. Zhou, A. Smith, N. Hahn, S. M. Matsui, and M. B. Omary. 1999. Non-invasive transgenic mouse genotyping using stool analysis. FEBS Letters 462:159-60.
• Burkhart, C. A., M. D. Norris, and M. Haber. 2002. A simple method for the isolation of genomic DNA from mouse tail free of real-time PCR inhibitors. Journal of Biochemical
  and Biophysical Methods 52:145-9.
• Cinelli, P., A. Rettich, B. Seifert, K. Burki, and M. Arras. 2007. Comparative analysis and physiological impact of different tissue biopsy methodologies used for the genotyping of laboratory mice. Laboratory Animals 41:174-84.
• Gaire K, Maquart G, Merlin JF. 2017.Animal-friendly mouse genotyping using direct PCR. Thermoscientific White Paper.
• Garzel LM , Hankenson FC, Combs J, Hankenson KD. 2010. Use of quantitative polymerase chain reaction analysis to compare quantity and stability of isolated murine DNA. Lab Anim (NY), 39(9): 283-289.
• Hankenson, F.C., Braden-Weiss, G., Blendy, J.A. 2011. Behavioral and activity assessment of laboratory mice (Mus musculus) after tail biopsy under isoflurane anesthesia. J Am Assoc Lab Anim Sci 50:686-94.
• Hankenson, F. C., L. M. Garzel, D. D. Fischer, B. Nolan, and K. D. Hankenson. 2008. Evaluation of tail biopsy collection in laboratory mice (Mus musculus): vertebral ossification, DNA quantity, and acute behavioral responses. J Am Assoc Lab Anim Sci 47:10-8.
• Jones CP, Carver S, Kendall LV. 2012 . Evaluation of common anesthetic and analgesic techniques for tail biopsy in mice. J Am Assoc Lab Anim Sci. Nov;51(6):808-14.
• Okada M, Miller TC, Roediger J, Shi Y, Schech J. 2017. An Efficient, Simple, and Noninvasive Procedure for Genotyping Aquatic and Nonaquatic Laboratory Animals. JAALAS Vol56(5): 570-573.
• Paluch, LR, Lieggii, CC, Dumont, M, Monette, S, Riedel, E, Lipman, NS. 2014. Developmental and Behavioral Effects of Toe Clipping on Neonatal and Preweanling Mice with and without Vapocoolant Anesthesia. JAALAS Vol 53(2): 132-140.