Skip to main content
SpringerLink
Log in

COMET Assay for Detection of DNA Damage During Axolotl Tail Regeneration

  • Protocol
  • First Online:
Salamanders

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2562))

  • 936 Accesses

Abstract

The purpose of this chapter is to evaluate DNA damage during axolotl tail regeneration using an alkaline comet assay. Our method details the isolation of cells from regenerating and non-regenerating tissues and the isolation of peripheral blood for single-cell gel electrophoresis. Also, we detail each of the steps for the development of the comet assay technique which includes mounting the isolated cells on an agarose matrix, alkaline electrophoresis, and DNA damage detection.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Franzke B, Neubauer O, Wagner KH (2015) Super DNAging-new insights into DNA integrity, genome stability and telomeres in the oldest old. Mutat Res Rev Mutat Res 766:48–57

    Article  CAS  Google Scholar 

  2. Turgeon MO, Perry NJS, Poulogiannis G (2018) DNA damage, repair, and cancer metabolism. Front Oncol 8:15

    Article  Google Scholar 

  3. Chatterjee N, Walker GC (2017) Mechanisms of DNA damage, repair, and mutagenesis. Environ Mol Mutagen 58:235–263

    Article  CAS  Google Scholar 

  4. Friedberg EC (2003) DNA damage and repair. Nature 421:436–440

    Article  Google Scholar 

  5. Lin X, Kapoor A, Gu Y et al (2020) Contributions of DNA damage to Alzheimer’s disease. Int J Mol Sci 21

    Google Scholar 

  6. Kermi C, Aze A, Maiorano D (2019) Preserving genome integrity during the early embryonic DNA replication cycles. Genes (Basel) 10

    Google Scholar 

  7. Ostling O, Johanson KJ (1984) Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 123:291–298. https://doi.org/10.1016/0006-291X(84)90411-X

    Article  CAS  PubMed  Google Scholar 

  8. Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191. https://doi.org/10.1016/0014-4827(88)90265-0

    Article  CAS  PubMed  Google Scholar 

  9. Collins AR (2004) The comet assay for DNA damage and repair: principles, applications, and limitations. Appl Biochem Biotechnol Part B Mol Biotechnol 26:249–261

    CAS  Google Scholar 

  10. Langie SAS, Azqueta A, Collins AR (2015) The comet assay: past, present, and future. Front Genet 6:266

    Article  Google Scholar 

  11. Firsanov D V., Solovjeva L V., Mikhailov VM, Svetlova MP (2016) Methods for the detection of DNA damage. In: genome stability: from virus to human application. Elsevier Inc., pp 635–649

    Google Scholar 

  12. Figueroa-González G, Pérez-Plasencia C (2017) Strategies for the evaluation of DNA damage and repair mechanisms in cancer. Oncol Lett 13:3982–3988

    Article  Google Scholar 

  13. Santa-Gonzalez GA, Gomez-Molina A, Arcos-Burgos M et al (2016) Distinctive adaptive response to repeated exposure to hydrogen peroxide associated with upregulation of DNA repair genes and cell cycle arrest. Redox Biol 9:124–133. https://doi.org/10.1016/j.redox.2016.07.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. de Lapuente J, Lourenço J, Mendo SA et al (2015) The comet assay and its applications in the field of ecotoxicology: a mature tool that continues to expand its perspectives. Front Genet 6:180

    Article  Google Scholar 

  15. Burlibaşa L, Gavrilacaron L (2011) Amphibians as model organisms for study environmental genotoxicity. Appl Ecol Environ Res 9:1–15. https://doi.org/10.15666/aeer/0901_001015

    Article  Google Scholar 

  16. Wouters A, Ploem JP, Langie SAS et al (2020) Regenerative responses following DNA damage - β-catenin mediates head regrowth in the planarian Schmidtea mediterranea. J Cell Sci 133. https://doi.org/10.1242/jcs.237545

  17. Barghouth PG, Thiruvalluvan M, LeGro M, Oviedo NJ (2019) DNA damage and tissue repair: what we can learn from planaria. Semin Cell Dev Biol 87:145–159

    Article  CAS  Google Scholar 

  18. Sousounis K, Bryant DM, Fernandez JM et al (2020) Eya2 promotes cell cycle progression by regulating DNA damage response during vertebrate limb regeneration. elife 9. https://doi.org/10.7554/eLife.51217

  19. Ponomareva LV, Athippozhy A, Thorson JS, Voss SR (2015) Using Ambystoma mexicanum (Mexican axolotl) embryos, chemical genetics, and microarray analysis to identify signaling pathways associated with tissue regeneration. Comp Biochem Physiol Part C Toxicol Pharmacol 178:128–135. https://doi.org/10.1016/j.cbpc.2015.06.004

    Article  CAS  Google Scholar 

  20. Echeverri K, Tanaka EM (2002) Ectoderm to mesoderm lineage switching during axolotl tail regeneration. Science (80) 298:1993–1996. https://doi.org/10.1126/science.1077804

    Article  CAS  Google Scholar 

  21. Gauron C, Rampon C, Bouzaffour M et al (2013) Sustained production of ROS triggers compensatory proliferation and is required for regeneration to proceed. Sci Rep 3. https://doi.org/10.1038/srep02084

  22. McCusker CD, Athippozhy A, Diaz-Castillo C et al (2015) Positional plasticity in regenerating Amybstoma mexicanum limbs is associated with cell proliferation and pathways of cellular differentiation regeneration and repair. BMC Dev Biol 15:1–17. https://doi.org/10.1186/s12861-015-0095-4

    Article  Google Scholar 

  23. Al Haj Baddar NW, Chithrala A, Voss SR (2019) Amputation-induced reactive oxygen species signaling is required for axolotl tail regeneration. Dev Dyn 248:189–196. https://doi.org/10.1002/dvdy.5

    Article  CAS  PubMed  Google Scholar 

  24. Tice RR, Agurell E, Anderson D et al (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environmental and Molecular Mutagenesis. Environ Mol Mutagen, In, pp 206–221

    Google Scholar 

  25. Denis JF, Sader F, Ferretti P, Roy S (2015) Culture and transfection of axolotl cells. Methods Mol Biol 1290:187–196. https://doi.org/10.1007/978-1-4939-2495-0_15

    Article  PubMed  Google Scholar 

  26. Końca K, Lankoff A, Banasik A et al (2003) A cross-platform public domain PC image-analysis program for the comet assay. Mutat Res Genet Toxicol Environ Mutagen 534:15–20. https://doi.org/10.1016/S1383-5718(02)00251-6

    Article  Google Scholar 

  27. Gyori BM, Venkatachalam G, Thiagarajan PS et al (2014) OpenComet: an automated tool for comet assay image analysis. Redox Biol 2:457–465. https://doi.org/10.1016/j.redox.2013.12.020

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

Belfran Carbonell was funded by Minciencias grant 936-2019. This work was funded by the University of Antioquia funding.

Author information

Authors and Affiliations

  1. Grupo Genética Regeneración y Cáncer, Institute of Biology, University of Antioquia, Medellin, Colombia

    Belfran Carbonell, Jennifer Álvarez, Gloria A. Santa-González & Jean Paul Delgado

  2. Biomedical Innovation and Research Group, Faculty of Applied and Exact Sciences, Instituto Tecnologico Metropolitano, Medellin, Colombia

    Gloria A. Santa-González

  3. Department of Integrated Basic Studies, Faculty of Dentistry, University of Antioquia, Medellin, Colombia

    Belfran Carbonell

Authors
  1. Belfran Carbonell
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jennifer Álvarez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gloria A. Santa-González
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jean Paul Delgado
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jean Paul Delgado .

Editor information

Editors and Affiliations

  1. Department of Biology, University of Kentucky, Lexington, KY, USA

    Ashley W. Seifert

  2. Department of Biology, Wake Forest University, Winston-Salem, NC, USA

    Joshua D. Currie

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Carbonell, B., Álvarez, J., Santa-González, G.A., Delgado, J.P. (2023). COMET Assay for Detection of DNA Damage During Axolotl Tail Regeneration. In: Seifert, A.W., Currie, J.D. (eds) Salamanders. Methods in Molecular Biology, vol 2562. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2659-7_12

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2659-7_12

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2658-0

  • Online ISBN: 978-1-0716-2659-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation