Name: Author Spotlight: Refining Xenopus laevis Marking Techniques for Biomedical Studies
Uploaded: 2024-06-28T15:00:00
Description: Animal models expand the scope of biomedical research, furthering our understanding of developmental, molecular, and cellular biology and enabling ...

We thank Dartmouth College&#39;s Center for Comparative Medicine and Research for providing daily husbandry for the animals used in this protocol. We also thank Leah Jacob and Adwaita Bose for their help in testing the protocol and photographing animals. Lastly, we thank Ann Miller&#39;s lab for training in the practice of tattooing. This work was supported by NIH grant R00 GM147826 to J.L.

All animal procedures described were approved by Dartmouth College&#39;s Institutional Animal Care and Use Committee.
1. Equipment setup
NOTE: A workflow for the procedure and an example bench setup are included (Figure 1).
<ol>
	<li>Connect the tattoo gun and foot pedal to the power supply. Position the foot pedal underneath the working surface.</li>
	<li>Assembling the tattoo gun
	<ol>
		<li>Using an Allen wrench or...

Optimizing Amphibian Marking: A Guide to Tattooing Xenopus

<ol>
	<li>Hubrecht, R. C., Carter, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+3Rs+and+humane+experimental+technique:+Implementing+change.">The 3Rs and humane experimental technique: Implementing change.</a> Animals (Basel). 9 (10), 754 (2019).</li><li>Elkan, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Xenopus+pregnancy+test.">The Xenopus pregnancy test.</a> British Med J. 2, 1253-1256 (1938).</li><li>Vize, P. D., Zorn, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+genomic+data+and+browser+resources.">Xenopus genomic data and browser resources.</a> Dev Biol. 426 (2), 194-199 (2017).</li><li>Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Xenopus: A Laboratory Manual. , (2023).</li><li>Borodinsky, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+laevis+as+a+model+organism+for+the+study+of+spinal+cord+formation,+development,+function+and+regeneration.">Xenopus laevis as a model organism for the study of spinal cord formation, development, function and regeneration.</a> Front Neural Circuits. 11, 90 (2017).</li><li>Hoppler, S., Conlon, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus:+Experimental+access+to+cardiovascular+development,+regeneration+discovery,+and+cardiovascular+heart-defect+modeling.">Xenopus: Experimental access to cardiovascular development, regeneration discovery, and cardiovascular heart-defect modeling.</a> Cold Spring Harb Perspect Biol. 12 (6), a037200 (2020).</li><li>Fisher, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenbase:+key+features+and+resources+of+the+Xenopus+model+organism+knowledgebase.">Xenbase: key features and resources of the Xenopus model organism knowledgebase.</a> Genetics. 224 (1), iyad018 (2023).</li><li>Slater, P. G., Hayrapetian, L., Lowery, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+laevis+as+a+model+system+to+study+cytoskeletal+dynamics+during+axon+pathfinding.">Xenopus laevis as a model system to study cytoskeletal dynamics during axon pathfinding.</a> Genesis. 55 (1-2), (2017).</li><li>Bermudez, J. G., Chen, H., Einstein, L. C., Good, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+biology+of+cell+boundary+conditions+through+confinement+of+Xenopus+cell-free+cytoplasmic+extracts.">Probing the biology of cell boundary conditions through confinement of Xenopus cell-free cytoplasmic extracts.</a> Genesis. 55 (1-2), (2017).</li><li>Stooke-Vaughan, G. A., Davidson, L. A., Woolner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+as+a+model+for+studies+in+mechanical+stress+and+cell+division.">Xenopus as a model for studies in mechanical stress and cell division.</a> Genesis. 55 (1-2), (2017).</li><li>Blackburn, A. T. M., Miller, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+congenital+kidney+diseases+in+Xenopus+laevis.">Modeling congenital kidney diseases in Xenopus laevis.</a> Dis Model Mech. 12 (4), dmm038604 (2019).</li><li>Tandon, P., Conlon, F., Furlow, J. D., Horb, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+genetic+toolkit+in+Xenopus:+Approaches+and+opportunities+for+human+disease+modeling.">Expanding the genetic toolkit in Xenopus: Approaches and opportunities for human disease modeling.</a> Dev Biol. 426 (2), 325-335 (2017).</li><li>Wheeler, G. N., Br&#228;ndli, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+vertebrate+models+for+chemical+genetics+and+drug+discovery+screens:+Lessons+from+zebrafish+and+Xenopus.">Simple vertebrate models for chemical genetics and drug discovery screens: Lessons from zebrafish and Xenopus.</a> Dev Dyn. 236 (6), 1287-1308 (2009).</li><li>Carron, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+origin+of+Hox13-dependent+skin+appendages+in+amphibians.">Evolutionary origin of Hox13-dependent skin appendages in amphibians.</a> Nat Commun. 15 (1), 2328 (2024).</li><li>Schultz, T. W., Dawson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Housing+and+husbandry+of+Xenopus+for+oocyte+production.">Housing and husbandry of Xenopus for oocyte production.</a> Lab Animal. 32 (2), 34-39 (2003).</li><li>Cadillac, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Animal identification systems used for mice. , (2006).</li><li>Donnelly, M. A., Guyer, C., Juterbock, J. E., Alford, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+marking+amphibians.">Techniques for marking amphibians.</a> Meas Monitor Biol Div: Std Meth Amphibians. , 277-284 (1994).</li><li>Hutchens, S. J., Deperno, C. S., Matthews, C. E., Pollock, K. H., Woodward, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visible+implant+fluorescent+marker:+A+reliable+marking+alternative+for+snakes.">Visible implant fluorescent marker: A reliable marking alternative for snakes.</a> Herpetol Rev. 39 (3), 301-303 (2008).</li><li>Hoogstraten-Miller, S., Dunham, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+identification+methods+for+African+clawed+frogs+(Xenopus+laevis).">Practical identification methods for African clawed frogs (Xenopus laevis).</a> Lab Animal. 26 (7), 36-38 (1997).</li><li>Loopstra, J. A., Zwart, P., Verhoeff-de Fremery, R., Vervoordeldonk, F. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marking+of+African+clawed+toads+(Xenopus+laevis).+Improvement+of+a+skin+autograft+technique.">Marking of African clawed toads (Xenopus laevis). Improvement of a skin autograft technique.</a> New Dev Biosci: Their Implicat Lab Animal Sci. , 311-316 (1988).</li><li>R&#246;del, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+tropicalis.">Xenopus tropicalis.</a> AmphibiaWeb. , (2001).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Worldwide+Tattoo+Supply.">Worldwide Tattoo Supply.</a> Stealth 2 Rotary Tattoo Machine Intro &amp; Maintenance. , (2015).</li><li>Khan, S. U., Mufti, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hot+history+and+cold+future+of+brands.">The hot history and cold future of brands.</a> J Manager Sci. 1 (1), 75-84 (2007).</li><li>Catlett, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> All About Lip Tattoo: what to Expect, Cost, Risks, Touch-ups &amp; More. , (2019).</li></ol>

Tattooing humans is an art form dating back thousands of years, and for as long as humans have tattooed themselves, they have also tattooed or branded animals23. The equipment and techniques for marking animals, particularly mammals, are well-established, well-documented, and widely accessible. While marking animals was originally for distinguishing livestock and theft deterrents23, its role in biomedical research has become just as important.
A ...

Animal models are useful tools for investigating questions pertaining to human health. In practice, biomedical research using animal models requires careful organization and maintenance of a healthy animal colony. Best practices for ethical animal handling and husbandry aim to reduce the number of animals needed for experimentation and refine practices to ensure animal welfare1. The clawed frog genus, including Xenopus laevis (X. laevis; African clawed frog) and Xenopus tropicalis (X. tropicalis; Western clawed frog), have been used in biomedical research since the 1930s when X. laevis were used by South African doctors to conduct the first pregnancy tests2. While modern pregnancy tests no longer require frogs, the role of Xenopus research persists. Advantages of using Xenopus for biomedical research include their well-annotated genomes3, year-round inducible ovulation of large clutches of eggs4, and externally laid eggs amenable to in vitro fertilization. These features make them a useful asset for vertebrate embryology and development5,6,7, basic molecular and cellular biology7,8,9,10, and for modeling human disease7,11,12,13.
Reliable methods for tracking individual Xenopus animals are essential for recording biological replicates and improving rigor and reproducibility in research. As Xenopus are frequently housed in groups, animal marking allows researchers to easily track individual animals4. Maintaining an accurate record of animals can save time and resources and improve the ability to track animals&#39; health. For example, individual identification of animals can improve organization workflows for generating transgenic Xenopus lines, as this requires multiple generations of frogs with specific genotypes verified by sequencing14, which requires organization and individual identification of animals. This is particularly true when these mutations lack easily discernible adult phenotypes. Similarly, the use of Xenopus oocytes and embryos to study basic cellular and developmental biology benefits from tracking individual animals. After inducing ovulation, animals need to rest for a minimum of 3 months to prevent health complications such as hyper-ovulation syndrome15. Individual identification methods ensure that animals are not induced to ovulate too frequently.
Marking and tracking animals also enables lab personnel to track animals&#39; health concerns. Animals of the same genotype falling ill can indicate excessive inbreeding or unanticipated health concerns associated with the transgene. Similarly, animals falling ill after recent ovulation can indicate issues with reagents, materials, or techniques. Tracking animals and their health enables lab personnel and veterinarians to follow up when concerns resurface and take preventative measures to prevent future illness. In mammals, there are numerous identification methods. Permanent methods for mice include ear punching, ear tags, tattooing, and subcutaneous microchips16. These can clearly and reliably differentiate animals within a colony or cage and can be easily administered by laboratory personnel. Methods such as ear punching are minimally invasive, require only one piece of specialized equipment, and work for animals of most ages. While these systems are straightforward and useful for mice, their use in frogs presents a unique set of challenges. Frogs and other amphibians lack a pinna (external ear structure). Some researchers have attached tags to the animal&#39;s jaw, toe, or hind limb17,18. This approach resulted in various problems: jaw tags caused irritation, and agitated frogs attempted to pull off tags with their forelimbs17. Toe tags pierced the webbing between toes, impairing movement and carrying the risk of becoming lost. As such, amphibians require their own methods for identification. Historically, toe-clipping has also been used to mark amphibians17,19. The toe is clipped with a sharp pair of scissors, and the animal can be identified by the length of toes on the forefeet and hind feet or by the angle at which the toe has grown back (in salamanders). However, this method poses the ethical concern that toe clipping may impair the animal&#39;s movement17. In addition, this can cause bleeding and introduce a risk of infection. Another established marking system is skin autografts, in which skin is taken from one part of the frog and surgically attached to another part. For example, a method is described for marking a frog&#39;s back or shoulder using a light-colored skin graft from its chest20. Skin grafts also come with limitations and risks: the procedure is invasive and introduces the risk of Aeromonas hydrophila infection, or red leg, a potentially fatal affliction; complete healing of the autograft takes up to 6 weeks; and with the methods described, only 6 frogs can be housed together because of limited places to put an autograft20.
Less invasive marking approaches include glass beads and transponder chips17,19. In the glass bead method, glass beads are threaded onto a small suture and sewn into the frog&#39;s skin. This provides greater variability than skin autografts, with at least 60 distinctive color combinations. There is, however, a risk that the suture can come out and result in the beads being lost. Alternatively, a microchip transponder can be implanted under the skin in the frog&#39;s dorsal lymph sac. This is considered the most permanent marking method and enables a potentially infinite number of animals to be individually identified and cataloged. However, this is also the most expensive method, as individual microchips are expensive, and a large colony would be costly to mark. Microchips also require a special scanner to read19. One common approach for Xenopus identification is referring to animals&#39; natural coloring and patterning. This works especially well for frogs such as X. laevis, which have distinct patterns that remain throughout adulthood. However, these patterns can change over time with stress, and coloring can appear different when frogs are moved between transparent and tinted containers15. Additionally, this identification method is less useful for X. tropicalis, which has less distinct marking patterns compared to X. laevis, or for albino animals, which have no color markings21. Even for species with distinct markings, lab personnel can interpret the placement and size of markings differently, which can cause errors in identification. Because of this, photographing animals is most reliable in conjunction with an additional identification method. Therefore, we seek to mark and identify Xenopus animals using a technique that is easily discernable, permanent, and minimally invasive.
There are limited published resources describing methods for tattooing amphibians. Tattooing has been described alongside other branding techniques, including heat brands, silver nitrate brands, and freeze brands17. In the same resource, tattooing was done by drawing numerals with a 27G hypodermic needle, and the process was noted not to cause infection, in contrast to the other branding techniques, which used a wire shaped into a numeral or other mark. In another source, an electric tattooing machine (described as a vibrating needle) was used to mark frogs, but little detail was provided on the technique17,19. The authors warn that by disturbing the frog&#39;s protective slime layer, this procedure increases the risk of red leg. While there is no marking or identification method that is both completely noninvasive (such as photographs) and permanent (such as microchips), tattooing provides an effective compromise. Tattooing is relatively straightforward compared to other techniques, such as skin autografts. Additional benefits include a smaller learning curve and relatively inexpensive equipment. Tattooing aquatic amphibians comes with certain challenges, which can intimidate researchers and impair successful animal marking. This paper aims to provide researchers with well-documented methods for tattooing adult Xenopus with a rotary tattoo machine.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3 needle round liners</td>
			<td>Worldwide Tattoo Supply</td>
			<td>1203RLB</td>
			<td>Packaged sterile</td>
		</tr>
		<tr>
			<td>5 Needle Round Disposable ULTRA</td>
			<td>Worldwide Tattoo Supply</td>
			<td>HTIPRS5-U</td>
			<td>Packaged sterile</td>
		</tr>
		<tr>
			<td>5 needle round liners</td>
			<td>Worldwide Tattoo Supply</td>
			<td>1205RLB</td>
			<td>Packaged sterile</td>
		</tr>
		<tr>
			<td>7 needle round liners</td>
			<td>Worldwide Tattoo Supply</td>
			<td>1207RLB</td>
			<td>Packaged sterile</td>
		</tr>
		<tr>
			<td>Clip Cord</td>
			<td>Worldwide Tattoo Supply</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Foot pedal</td>
			<td>Worldwide Tattoo Supply</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Inkpots</td>
			<td>Worldwide Tattoo Supply</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes, delicate task wipes</td>
			<td>Fisher Scientific</td>
			<td>06-666A</td>
			<td></td>
		</tr>
		<tr>
			<td>RCA Connection</td>
			<td>Worldwide Tattoo Supply</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Scream Ink Pitch Black, 1oz</td>
			<td>Worldwide Tattoo Supply</td>
			<td>SI101</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel grips</td>
			<td>Worldwide Tattoo Supply</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Stealth 2.0 Rotary Tattoo Machine</td>
			<td>Worldwide Tattoo Supply</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Stealth 2.0 Rotary Tattoo Machine Box Set</td>
			<td>Worldwide Tattoo Supply</td>
			<td>STEALTH2-SET</td>
			<td></td>
		</tr>
		<tr>
			<td>Styrofoam island</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>This is the lid of a styrofoam cold shipping container</td>
		</tr>
		<tr>
			<td>Tricaine (ethyl 3-aminobenzonate methanesulfate)</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td>CAUTION: IRRITANT</td>
		</tr>
		<tr>
			<td>Unbleached paper towels</td>
			<td>Grainger</td>
			<td>2U229</td>
			<td>Paper towels MUST be unbleached, bleach is toxic to amphibians</td>
		</tr>
		<tr>
			<td>Voltage Supply</td>
			<td>Worldwide Tattoo Supply</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Wash bottle (with frog-safe water)</td>
			<td>Fisher Scientific</td>
			<td>FB0340923T</td>
			<td>Frog safe water is dechlorinated, pH 7.0-8.5, conductivity 1200-1800 uS</td>
		</tr>
		<tr>
			<td>X. laevis adult female</td>
			<td>Xenopus1</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Zip-top plastic bag</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>This bag should be large enough to hold the styrofoam island</td>
		</tr>
	</tbody>
</table>

methods for tattooing xenopus laevis with a rotary tattoo machine

Animal models expand the scope of biomedical research, furthering our understanding of developmental, molecular, and cellular biology and enabling researchers to model human disease. Recording and tracking individual animals allows researchers to reduce the number of animals required for study and refine practices to improve animal wellbeing. Several well-documented methods exist for marking and tracking mammals, including ear punching and ear tags. However, methods for marking aquatic amphibian species are limited, with the existing resources being outdated, ineffective, or prohibitively costly. In this manuscript, we outline methods and best practices for marking Xenopus laevis with a rotary tattoo machine. Proper tattooing results in high-quality tattoos, making individuals easily distinguishable for researchers and posing minimal risk to animals' health. We also highlight the causes of poor-quality tattoos, which can result in tattoos that fade quickly and cause unnecessary harm to animals. This approach allows researchers and veterinarians to mark amphibians, enabling them to track biological replicates and transgenic lines and to keep accurate records of animal health.

Author Spotlight: Refining Xenopus laevis Marking Techniques for Biomedical Studies

Methods for Tattooing Xenopus laevis with a Rotary Tattoo Machine

Our research is focused on investigating the organization of the cell cortex using Xenopus egg extracts and supported lipid bilayers. We are trying to understand how dynamic cytoskeletal patterns are regulated within the cortex and how these patterns support basic cell functions like cell division. This protocol outlines an effective and safe method for marking Xenopus laevis.This approach enables researchers to track genetic lines and experimental replicates and to monitor animal health. Tattooing is a cost-effective permanent marking technique for amphibians. It's much less invasive than other permanent methods, such as skin graft or tags, it's also more reliable than qualitative methods, like photos or pattern recognition.

High-quality tattoos will have dark, legible strokes on the frog&#39;s chest and can be clearly differentiated from several feet away (Figure 5A). In general, larger numbers and markings are better for readability, but longer names and numbers can be made smaller to fit comfortably on the frog&#39;s chest. Tattoo longevity is more difficult to judge, but high-quality frog tattoos should remain dark and legible for at least 3-6 months (Figure 5D-D&#39;</s...

Here, we describe methods for tattooing adult Xenopus laevis (African clawed frog) with a rotary tattoo machine. Proper tattooing results in dark, easily legible numerals that last for several months and make animals easily distinguishable for research and recordkeeping purposes.