Name: Identification of Pre-weanling 7-Day-Old Mice with Pinna Edge Biopsies (Video) | JoVE Journal
Uploaded: 2025-05-23T14:45:00.000+00:00
Duration: 4 min 6 s
Description: Learn pinna edge biopsy for early mouse identification and genotyping using 7-day-old pups. This method enables simultaneous tissue collection for genetics research in developmental biology.

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5 minutes

The authors would like to thank Drs. Lori S Palley and Donna M Jarrell, the authors of the original paper in this work, for their contributions.

All procedures outlined in the protocol were performed in compliance with the NIH Guide for the Care and Use of Laboratory Animals6and approved by the Massachusetts General Hospital (MGH) Institutional Animal Care and Use Committee (IACUC). The identification method described below has been tested in C57BL/6NCrl mice of both sexes5.&#160;This method has not been used in other strains, stocks, or transgenics. Pinna biopsy identification may not be suitable for mice that have an increased healing capacity (e.g., MRL mouse strain)7.
1. Mouse pup restraint
<ol>
	<li>Ensure the mouse is PND 7 or older. If the mouse is younger than PND 7, this PEB method may not be feasible.</li>
	<li>Gently restrain the mouse in a manner that allows for visualization and access to the ears. Pick the pup up by the base of the tail with the user&#39;s non-dominate hand and transfer it to a stable surface.</li>
	<li>Apply gentle pressure along the pup&#39;s back with the thumb and forefinger of the non-dominant hand until the loose skin between the ears can be pinched. Pinch the skin tightly enough so that the head does not move freely, but ensure the animal is breathing normally (Figure 1).</li>
	<li>If the animal is being restrained with the left hand, ensure the pup is in a position that makes the right ear pinna accessible (i.e., the pup&#39;s nose is facing away from the hander&#39;s body). To access the left ear pinna, supinate the hand grasping so that the left side of the animal faces the handler.</li>
	<li>If the animal is being restrained with the right hand, ensure the pup is in a position that makes the left ear pinna readily accessible (i.e., the pup&#39;s nose is facing away from the handler&#39;s body). To access the right ear pinna, supinate the hand grasping the animal so that the right side of the animal faces the handler.</li>
</ol>
2. Selection of pinna biopsy pattern
<ol>
	<li>Use a total of nine patterns with eight PEB patterns and one no-biopsy pattern (Figure 2). Select and record a PEB pattern.</li>
</ol>
3. Pinna edge biopsy
<ol>
	<li>Use clean instruments for each biopsy. Small dissection scissors or McPherson-Vannas scissors are recommended (see Table of Materials). Spray the instruments with disinfectants and wipe clean with 70% isopropyl alcohol after each use.</li>
	<li>Hold the scissors with the dominant hand.</li>
	<li>To create a vertical PEB, trim the lateral edge of the pinna in a vertical direction so that approximately 1/4th to 1/3rd of the total pinnae (i.e., about 0.8 mm x 3 mm tissue) is removed from the side of the ear (Figure 3G).</li>
	<li>To create a horizontal PEB, trim the dorsal edge of the pinna so that approximately 1/4th to 1/3rd of the total pinna (i.e., about 0.8 mm x 3 mm tissue) is removed (Figure 3H). Make the cut at a 45&#176; angle instead of a straight line (Figure 4). The angle accounts for ear development and growth over time and assures the pattern remains horizontal in nature.
	<ol>
		<li>The eight pinna patterns are a combination of the vertical PEB and horizontal PEB on either the left, right, or both pinnae. Make the following eight pinna biopsy patterns: left vertical (LV), right vertical (RV), left horizontal (LH), right horizontal (RH), left vertical and right horizontal (LVRH), left and right vertical (LVRV), left and right horizontal (LHRH), and left horizontal and right vertical (LHRV).</li>
	</ol>
	</li>
	<li>Use fine forceps to carefully deposit the excised pinna tissue into a sample container to be used for genotyping.
	<ol>
		<li>If in the unlikely event that blood pools from the pinna biopsy site, with the dominant hand and while the animal is still restrained, use a piece of clean gauze or a cotton tip applicator to place pressure on the site of bleeding until the bleeding stops.</li>
	</ol>
	</li>
	<li>Place the pup gently back in the cage with the dam. Observe pups for 1-2 min after placement back into the cage to assess for any further bleeding from the PEB site.</li>
</ol>

<ol><li>Bonaparte, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FELASA+guidelines+for+the+refinement+of+methods+for+genotyping+genetically-modified+rodents%3A+a+report+of+the+Federation+of+European+Laboratory+Animal+Science+Associations+Working+Group%5BTitle%5D)+AND+%22Lab+Anim%22%5BJournal%5D)">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.</a> Lab Anim. 47 (3), 134-145 (2013).</li>
<li>Dahlborn, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Report+of+the+federation+of+European+laboratory+animal+science+associations+working+group+on+animal+identification%5BTitle%5D)+AND+%22Lab+Anim%22%5BJournal%5D)">Report of the federation of European laboratory animal science associations working group on animal identification.</a> Lab Anim. 47 (1), 2-11 (2013).</li>
<li>Castelhano-Carlos, M. J., Sousa, N., Ohl, F., Baumans, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+methods+in+newborn+C57BL%2F6+mice%3A+a+developmental+and+behavioural+evaluation%5BTitle%5D)+AND+%22Lab+Anim%22%5BJournal%5D)">Identification methods in newborn C57BL/6 mice: a developmental and behavioural evaluation.</a> Lab Anim. 44 (2), 88-103 (2010).</li>
<li>Chen, M., Kan, L., Ledford, B. T., He, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tattooing+various+combinations+of+ears%2C+tail%2C+and+toes+to+identify+mice+reliably+and+permanently%5BTitle%5D)+AND+%22J+Am+Assoc+Lab+Anim+Sci%22%5BJournal%5D)">Tattooing various combinations of ears, tail, and toes to identify mice reliably and permanently.</a> J Am Assoc Lab Anim Sci. 55 (2), 189-198 (2016).</li>
<li>Chen, D. D., Molk, D. M., Palley, L. S., Jarrell, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pinna+Edge+Biopsy+of+7+and+21+Day+Old+C57BL%2F6+Mice+as+a+Method+for+Identification+and+Genotyping%5BTitle%5D)+AND+%22J+Am+Assoc+Lab+Anim+Sci%22%5BJournal%5D)">Pinna Edge Biopsy of 7 and 21 Day Old C57BL/6 Mice as a Method for Identification and Genotyping.</a> J Am Assoc Lab Anim Sci. 62 (5), 438-448 (2023).</li>
<li>Institute for the Laboratory Animal Research. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Guide+for+the+care+and+use+of+laboratory+animals%22">Guide for the care and use of laboratory animals</a>. , 8th ed, National Academies Press. Washington, DC. (2011).</li>
<li>Rajnoch, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regeneration+of+the+ear+after+wounding+in+different+mouse+strains+is+dependent+on+the+severity+of+wound+trauma%5BTitle%5D)+AND+%22Dev+Dyn%22%5BJournal%5D)">Regeneration of the ear after wounding in different mouse strains is dependent on the severity of wound trauma.</a> Dev Dyn. 226 (2), 388-397 (2003).</li>
<li>Han, M., Yang, X., Lee, J., Allan, C. H., Muneoka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+regeneration+of+the+neonatal+digit+tip+in+mice%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Development and regeneration of the neonatal digit tip in mice.</a> Dev Biol. 315 (1), 125-135 (2008).</li>
<li>Schaefer, D. C., Asner, I. N., Seifert, B., Bürki, K., Cinelli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+physiological+and+behavioural+parameters+in+mice+after+toe+clipping+as+newborns%5BTitle%5D)+AND+%22Lab+Anim%22%5BJournal%5D)">Analysis of physiological and behavioural parameters in mice after toe clipping as newborns.</a> Lab Anim. 44 (1), 7-13 (2010).</li>
<li>Frezel, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Does+toe+clipping+for+genotyping+interfere+with+later-in-life+nociception+in+mice%5BTitle%5D)+AND+%22Pain+Rep%22%5BJournal%5D)">Does toe clipping for genotyping interfere with later-in-life nociception in mice.</a> Pain Rep. 4 (3), e740(2019).</li>
</ol>

The authors do not have anything to disclose.

Significance with respect to existing methods 
Previous articles have stated that for PND 14 animals and younger, their pinna may not be suitable for manipulation because of the small size2,3.&#160;While existing ear identification systems such as ear punch biopsies, ear notching, or ear tags can perform both identification and genotyping, they may require larger pinnae and, thus, older mice for identification. This novel method of PEB offer...

In certain scientific studies, the method to identify and simultaneously genotype a mouse can be crucial. When using a mouse model, there are several commonly used methods that have been developed to genotype and identify animals at the same time (e.g., ear punching, ear tagging), and other methods that only identify animals (e.g., tattooing, subcutaneous transponder implantation, marker or dye application) or only genotype animals (e.g., tail biopsy)1,2.&#160;Most of these techniques are intended to be performed at the time of weaning. Techniques to simultaneously identify and genotype in younger, pre-weanling animals are fewer1,2,3,4.
To assist work that requires identification and genotyping of mice at younger ages, a study was performed to validate PEB as a reliable identification and genotyping method for PND 7 pups and compare it to the use of PEB in mice weaned at PND 215. As this study details, horizontal and vertical pinna slices were selected because a prior study using PND 7 pup carcasses found pinna patterns created using PEB were clear, quick, provided a variety of pattern combinations, identifiable, and the tissue was easy to remove5. Other methods, such as ear punches and ear notches, were also explored, but there was not enough surface area on PND 7 pups for the creation of complete circular punches or notches. This preliminary work led the referenced study to test these patterns in live PND 7 mice5.&#160;This study showed that the tissue size retrieved from the biopsy of the ear pinna can be used for successful genotyping. The PEB patterns were also intact as mice aged to PND 63, regardless of when the pinna was biopsied at PND 7 or PND 215.
The goal for the development of this technique is to provide a simultaneous identification and genotyping method for animals aged PND 7 to 14 that, according to current literature, do not have a method available. The current article, accompanied by the guided video, illustrates how to perform an ear biopsy at the pinna edge of a 7-day mouse&#39;s ear to simultaneously identify and retrieve a sufficient quantity of tissue for PCR genotyping. There are eight different biopsy combinations and one with no biopsy for a total of nine pinna patterns available for use to identify individual mice. Tips on performing pinna biopsies are further discussed to maximize success for the clear identification of the various pinna patterns in PND 7 pups.

<table><tbody><tr><td>Dissection scissors</td><td>Kent Scientific</td><td>INS600393-G</td><td>10 cm long</td></tr><tr><td>McPherson-Vannas Scissors</td><td>Kent Scientific</td><td>INS600124</td><td>8 cm long</td></tr></tbody></table>

identification of pre-weanling 7-day-old mice with pinna edge biopsies

Identification of Pre-weanling 7-Day-Old Mice with Pinna Edge Biopsies

Identification and genotyping of mice is often an essential part of many in vivo scientific studies. Several methods have been described for identification and genotyping in weaned mice; however, to date, there are far fewer techniques described for pre-weanling mice. Pinna Edge Biopsy (PEB) is a method of identification with subsequent use of tissue for genotyping. This article describes and provides video instruction on a stepwise approach to PEB on post-natal day (PND) 7 mice. Eight different patterns of pinna biopsy location and technique are outlined which have been shown to sustain into adulthood for identification. This article also discusses ways to optimize the PEB and shows the benign effect this technique has on the growth and development of the pups, with all mice showing appropriate weight gain without any morbidity or mortality throughout the study. This technique will provide investigators with a method for mouse identification at an age where there are few identification and genotyping methods available.

Figure 1 illustrates a manual method of gently restraining PND 7 mice so their ears are visible for manipulation. The restraint uses the thumb and index finger to grasp the excess skin of the scruff. The amount of skin grasped depends on the animal's age, with less tissue available in smaller animals.
The PEB patterns listed in Figure 2 have been tested to be identifiable up to PND 63 and anecdotally have been seen to last for longer ...

The protocol presents a novel stepwise approach to producing a pinna-edge biopsy (PEB) in post-natal day (PND) 7 mice for the purpose of identification and simultaneous genotyping.

Identification and genotyping of mice is often an essential part of many in vivo scientific studies. Several methods have been described for ...

The scope of this research is to discover, develop, and then verify a novel method of simultaneous identification and genotyping for pups between the ages of 7 and 14 days old. This protocol demonstrates a novel method to identify and genotype mice at 7 days old, enabling earlier research access than current methods typically performed at 14 days or later. The current PEB method provides nine ID patterns, sufficient for litters up to nine pups.For larger litters, new PEB combinations must be developed to expand identification options. To begin, gently restrain a mouse pup in a position that permits visualization and access to both ears. Using the non-dominant hand, pick up the pup by the base of the tail and place it on a stable surface on top of the cage bars.Use the thumb and forefinger of the non-dominant hand to apply gentle pressure along the pup's back until the loose skin between the ears can be pinched. Then pinch the skin securely to restrict head movement, ensuring the animal breathes normally. If restraining with the left hand, orient the pup so that its nose points away from the handler's body, making the right ear pinna accessible.To access the left ear pinna, rotate the restraining hand so that the left side of the pup faces the handler. If restraining with the right hand, orient the pup's nose away from the handler to expose the left ear pinna. To access the right ear pinna, supinate the hand grasping the animal until the right side of the pup faces the handler.Use a total of nine patterns for the pinna edge biopsy, or PEB, which includes eight biopsy patterns and one no biopsy option. Use clean, small dissection scissors, or McPherson-Vannas scissors to perform each biopsy. Disinfect the instruments by either spraying them with disinfectant and/or 70%isopropyl alcohol or wiping with alcohol wipes after every use.Hold the scissors using the dominant hand. To create a vertical PEB, trim the lateral edge of the ear vertically, removing approximately one-third of the pinna. To create a horizontal PEB trim the dorsal edge of the pinna at a 45-degree angle, removing about one-third of the pinna.Create one of eight designated PEB patterns. Use fine forceps to carefully place the excised tissue into a sample container for genotyping. If bleeding occurs at the biopsy site, apply gentle pressure using clean gauze or a cotton tip applicator while the animal remains restrained.Gently return the pup to its cage with the dam. Then observe the animal for one to two minutes to check for continued bleeding from the biopsy site. Pinna biopsy patterns remained visibly identifiable up to postnatal day 63 in mice initially marked on postnatal day 7.The right horizontal pinna biopsy created at a 45-degree dorsal angle appeared more horizontal by postnatal day 63 as the ear matured. Both male and female mice showed a significant increase in body weight from postnatal day 7 to 63, with males weighing more than females from postnatal day 30 onwards.

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Learn pinna edge biopsy for early mouse identification and genotyping using 7-day-old pups. This method enables simultaneous tissue collection for genetics research in developmental biology.

Identification of Pre-weanling 7-Day-Old Mice with Pinna Edge Biopsies

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