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.
References
Cogger HG (2000) Reptiles and amphibians of Australia, 6th edn. Reed New Holland, Sydney
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
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
Freshney IR (2006) Culture of animal cells: manual of basic technique, 4th edn. Wiley-Liss, Inc., New York
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 11–20
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 93:343–358
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
Masters JR, Stacey GN (2007) Changing medium and passaging cell lines. Nat Protocols 9:2276–2284. doi:10.1038/nprot.2007.319
Olmo E (2005) Chromorep: a reptile chromosomes database. http://193.206.118.100/professori/chromorep.pdf. Accessed 24 January 2005
Sarre S, Georges A, Quinn A (2004) The ends of a continuum: genetic and temperature-dependent sex determination in reptiles. Bioessays 26: 639–645
Simpson SB, Cox PG (1967) Vertebrate regeneration system: culture in vitro. Science 157:1330–1332. doi:10.1126/science.157.3794.1330
Witten GJ (1983) Some karyotypes of Australian agamids (Reptilia: Lacertilia). Aust J Zool 31:533–540. doi:10.1071/ZO9830533
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
Acknowledgments
We thank Wendy Dimond and Tobias Uller for tissue samples of T. pinguicolla and C. fordii, respectively. Riccardo Natoli for cell photography. Animal collection, handling, and sampling were performed according to the guidelines of the Australian Capital Territory Animal Welfare Act 1992 (Section 40) and licences issued by the Queensland, South Australia, Victoria, Australian Capital Territory, and New South Wales state governments. All experiments were performed with the approval of the University of Canberra Animal Experimentation Ethics Committee (Proposal CEAE 04/04) and the Australian National University Animal Experimentation Ethics Committee (Proposals R.CG.02.00 and R.CG.08.03). This work is supported by Australian Research Council Discovery Grants (DP0449935 and DP0881196) awarded to Jenny Graves, S·S. and A.G., and S·S. and A.G, respectively.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Ezaz, T., O’Meally, D., Quinn, A.E. et al. A simple non-invasive protocol to establish primary cell lines from tail and toe explants for cytogenetic studies in Australian dragon lizards (Squamata: Agamidae). Cytotechnology 58, 135–139 (2008). https://doi.org/10.1007/s10616-009-9182-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10616-009-9182-3