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Chicken karyotype analysis/ SCEs

Chicken karyotype analysis/ SCEs. Ciaran Morrison. Diagrammatic representation of the chicken karyotype. The DT40 karyotype. Modal karyotype is: 2 Gga-1, 3 Gga-2, 2 Gga-3, 2 Gga-4, 2 Gga-5, 1 Gga-Z. From: Sonoda et al. (1998) EMBO J. 17 : 598-608.

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Chicken karyotype analysis/ SCEs

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  1. Chicken karyotype analysis/ SCEs Ciaran Morrison

  2. Diagrammatic representation of the chicken karyotype

  3. The DT40 karyotype Modal karyotype is: 2 Gga-1, 3 Gga-2, 2 Gga-3, 2 Gga-4, 2 Gga-5, 1 Gga-Z. From: Sonoda et al. (1998) EMBO J. 17: 598-608

  4. Variation/ mosaicism in the DT40 karyotype 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: 299-307 From: Sonoda et al. (1998) EMBO J. 17: 598-608

  5. Count the macrochromosomes

  6. 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)

  7. 5 2 3 1 5 2 3 4 Z 2 1 4 Anna Stephan

  8. Anna Stephan

  9. Anna Stephan

  10. Chromosome aberrations From: Sonoda et al. (1998) EMBO J. 17: 598-608

  11. 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)

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

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

  14. Telomere analysis in DT40s Chickens have high levels of telomere repeat sequence, notably in the microchromosomes. DT40s have shorter-than-normal telomeres, although the interstitial sequences are retained. From: O’Hare & Delany (2009) Chromosome Res. 17: 947-64.

  15. Recent information on centromeres in DT40s • Shang WH, Hori T, Toyoda A, Kato J, Popendorf K, Sakakibara Y, Fujiyama A, Fukagawa T. • Genome Res. 2010 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.

  16. 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).

  17. Sister chromatid exchanges Sonoda, E. et al. 1999. Mol. Cell. Biol. 19(7):5166-5169

  18. SCE visualisation + BrdU 1 cell cycle Equally-labelled sisters

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

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

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

  22. 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) Fluorescence plus Giemsa (‘FPG’) Simpson, LJ and Sale, JE (2006) ‘Sister chromatid exchange assay’, in Buerstedde and Takeda (eds.) Reviews and Protocols in DT40 Research 399-403.

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