Cytotechnology

, Volume 58, Issue 3, pp 135–139 | Cite as

A simple non-invasive protocol to establish primary cell lines from tail and toe explants for cytogenetic studies in Australian dragon lizards (Squamata: Agamidae)

  • Tariq Ezaz
  • Denis O’Meally
  • Alexander E. Quinn
  • Stephen D. Sarre
  • Arthur Georges
  • Jennifer A. Marshall Graves
Brief Report

Abstract

Primary cell lines were established from cultures of tail and toe clips of five species of Australian dragon lizards: Tympanocryptis pinguicolla, Tympanocryptis sp., Ctenophorus fordi, Amphibolurus norrisi and Pogona vitticeps. The start of exponential cell growth ranged from 1 to 5 weeks. Cultures from all specimens had fibroblastic morphology. Cell lines were propagated continuously up to ten passages, cryopreserved and recovered successfully. We found no reduction in cell viability after short term (<6 months) storage at −80 °C. Mitotic metaphase chromosomes were harvested from these cell lines and used in differential staining, banding and fluorescent in situ hybridisation. Cell lines maintained normal diploidy in all species. This study reports a simple non-invasive method for establishing primary cell lines from Australian dragon lizards without sacrifice. The method is likely to be applicable to a range of species. Such cell lines provide a virtually unlimited source of material for cytogenetic, evolutionary and genomic studies.

Keywords

Reptiles Lizards Cell lines Tail Toe Non-invasive Chromosomes 

References

  1. Cogger HG (2000) Reptiles and amphibians of Australia, 6th edn. Reed New Holland, SydneyGoogle Scholar
  2. Ezaz T, Quinn AE, Miura I, Sarre SD, Georges A, Graves JAM (2005) The dragon lizard Pogona vitticeps has ZZ/ZW micro-sex chromosomes. Chromosome Res 13:763–776. doi:10.1007/s10577-005-1010-9 CrossRefGoogle Scholar
  3. Ezaz T, Quinn AE, Georges A, Sarre SD, O’Meally D, Graves JAM (2009) Molecular marker suggests rapid changes of sex-determining mechanisms in Australian dragon lizards. Chromosome Res. doi:10.1007/s10577-008-9019-5 Google Scholar
  4. Freshney IR (2006) Culture of animal cells: manual of basic technique, 4th edn. Wiley-Liss, Inc., New YorkGoogle Scholar
  5. Harlow PS (2004) Temperature-dependent sex determination in lizards. In: Valenzuela N, Lance VA (eds) Temperature dependent sex determination in vertebrates. Smithsonian Institution, Washington DC, pp 11–20Google Scholar
  6. Hugall AF, Foster R, Hutchinson M, Lee MSY (2008) Phylogeny of Australasian agamid lizards based on nuclear and mitochondrial genes: implications for morphological evolution and biogeography. Biol J Linn Soc 93:343–358CrossRefGoogle Scholar
  7. Mansell JM, Elliott RJ, Gaskin JM (1989) Initiation and ultrastructure of a reptilian fibroblast cell line obtained from cutaneous fibropapillomas of the green turtle, Chelonia mydas. In Vitro Cell Dev Biol 25:1062–1064. doi:10.1007/BF02624142 CrossRefGoogle Scholar
  8. Masters JR, Stacey GN (2007) Changing medium and passaging cell lines. Nat Protocols 9:2276–2284. doi:10.1038/nprot.2007.319 CrossRefGoogle Scholar
  9. Olmo E (2005) Chromorep: a reptile chromosomes database. http://193.206.118.100/professori/chromorep.pdf. Accessed 24 January 2005
  10. Sarre S, Georges A, Quinn A (2004) The ends of a continuum: genetic and temperature-dependent sex determination in reptiles. Bioessays 26: 639–645 CrossRefGoogle Scholar
  11. Simpson SB, Cox PG (1967) Vertebrate regeneration system: culture in vitro. Science 157:1330–1332. doi:10.1126/science.157.3794.1330 CrossRefGoogle Scholar
  12. Witten GJ (1983) Some karyotypes of Australian agamids (Reptilia: Lacertilia). Aust J Zool 31:533–540. doi:10.1071/ZO9830533 CrossRefGoogle Scholar
  13. Zhang Q, Cooper RK, Wolters WR, Tiersch TR (1998) Isolation, culture, and characterization of a primary fibroblast cell line from channel catfish. Cytotechnology 26:83–90. doi:10.1023/A:1007911619537 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Tariq Ezaz
    • 1
    • 2
  • Denis O’Meally
    • 1
  • Alexander E. Quinn
    • 2
  • Stephen D. Sarre
    • 2
  • Arthur Georges
    • 2
  • Jennifer A. Marshall Graves
    • 1
  1. 1.Comparative Genomics Group, Research School of Biological SciencesThe Australian National UniversityCanberraAustralia
  2. 2.Institute for Applied EcologyUniversity of CanberraCanberraAustralia

Personalised recommendations