A simple non-invasive protocol to establish primary cell lines from tail and toe explants for cytogenetic studies in Australian dragon lizards (Squamata: Agamidae)
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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.
KeywordsReptiles Lizards Cell lines Tail Toe Non-invasive Chromosomes
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.
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