Chicken karyotype analysis/ SCEs Ciaran Morrison

Diagrammatic representation of the chicken karyotype

The DT40 karyotype From: Sonoda et al. (1998) EMBO J. 17: Modal karyotype is: 2 Gga-1, 3 Gga-2, 2 Gga-3, 2 Gga-4, 2 Gga-5, 1 Gga-Z.

Variation/ mosaicism in the DT40 karyotype From: Sonoda et al. (1998) EMBO J. 17: Variations observed in karyotypes during extended culture periods, within the same cultures. Particular variations included: 2 Gga-2, 1 Gga-4; 1 Gga-3; 1 Gga-1 Chang & Delany (2004) Chromosome Res. 12:

Count the macrochromosomes

Metaphase spreads-basics Block cells in metaphase (colcemid for up to 4h; longer results in v. high condensation) Hypotonically swell cells. Fix cells and drop on to slides. Dry and stain with Giemsa (or DAPI)

Z Anna Stephan

Chromosome aberrations From: Sonoda et al. (1998) EMBO J. 17:

Chromosome aberrations Analysis allows the observation of DNA damage in chromosomes. Analysis may indicate specific repair deficiencies or abnormalities (e.g., abnormalities in both sisters is often from post-replicative repair, i.e. HR)

‘A Spotter’s Guide’ Ctg: Chromatid-type gap Ctb: Chromatid-type break Csg: Chromosome gap Csb: Chromosome break Cte: Chromatid exchange

Scoring suggestion Record data for each macrochromosome in a spread. Sum for population (100 metaphases)

Telomere analysis in DT40s From: O’Hare & Delany (2009) Chromosome Res. 17: Chickens have high levels of telomere repeat sequence, notably in the microchromosomes. DT40s have shorter-than- normal telomeres, although the interstitial sequences are retained.

Recent information on centromeres in DT40s Shang WH, Hori T, Toyoda A, Kato J, Popendorf K, Sakakibara Y, Fujiyama A, Fukagawa T. Genome Res Jun 9. [Epub ahead of print] The chicken genome has a hybrid centromere model, involving either long arrays of tandem repeats on some chromosomes or relative short spans of non-tandem-repeat sequences on other chromosomes.

Sister chromatid exchanges Equal exchanges between sister chromatids (post-replicative). Reflect homologous recombinational repair activities in cells. Frequently used in mutagenesis/ toxicity assays as readouts for environmental stressors. Greatly elevated in certain human diseases (Bloom’s syndrome).

Sonoda, E. et al Mol. Cell. Biol. 19(7): Sister chromatid exchanges

SCE visualisation 1 cell cycle + BrdU Equally-labelled sisters

SCE visualisation 2nd cell cycle + BrdU Differentially-labelled sisters

SCE visualisation 2nd cell cycle + BrdU Differentially-labelled sisters Giemsa staining reveals differential labelling

SCE visualisation 2nd cell cycle + BrdU Differentially-labelled sisters Giemsa staining reveals differential labelling and exchanges

SCEs: observation Requires labelling for 2 cell cycles; optimise! Visualisation of differential labelling may include Hoechst staining (to magnify differences between the labelled/ unlabelled sisters) Simpson, LJ and Sale, JE (2006) ‘Sister chromatid exchange assay’, in Buerstedde and Takeda (eds.) Reviews and Protocols in DT40 Research Fluorescence plus Giemsa (‘FPG’)