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

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.

Sex reversal triggers the rapid transition from genetic to temperature-dependent sex

“…Metaphase chromosome spreads were prepared from fibroblast cultures of tail tissue following ref. 33. Metaphases for all individuals were stained with 49,6-diamidino-2-phenylindole (DAPI), the chromosome number identified and compared with the normal P. vitticeps karyotype 34 , to eliminate the possibility of chromosome abnormality.…”

The ZW sex microchromosomes of an Australian dragon lizard share no homology with those of other reptiles or birds

“…The relevant protocols for sexing P. vitticeps, cell culture, and chromosome preparation have been described elsewhere (Ezaz et al 2008(Ezaz et al , 2005. P. vitticeps BAC clones containing homologs of snake and chicken sex chromosome genes were isolated using overlapping oligonucleotides (overgo)-based genomic library screening (Ross et al 1999).…”

ANOLISSEX CHROMOSOMES ARE DERIVED FROM A SINGLE ANCESTRAL PAIR

“…We prepared chromosome spreads from both males and females of five Anolis species (A. carolinensis, A. sagrei, A. grahami, A. lineatopus, and A. distichus) from fibroblast cultures established from tail clips (Ezaz et al 2008;Main et al 2012). Cells were arrested in metaphase using vinblastine sulfate (1 mg/ml) for 2-3 h. We collected cells after trypsin digestion and incubated them in a hypotonic solution (0.07 M KCl) for 20 min at 37 • C. After hypotonic treatment, cells were centrifuged and fixed through a series of five washes in methanol:acetic acid (3:1).…”