chicken karyotype analysis/ sces ciaran morrison

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Chicken karyotype analysis/ SCEs Ciaran Morrison

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Page 1: Chicken karyotype analysis/ SCEs Ciaran Morrison

Chicken karyotype analysis/ SCEs

Ciaran Morrison

Page 2: Chicken karyotype analysis/ SCEs Ciaran Morrison

Diagrammatic representation of the chicken karyotype

Page 3: Chicken karyotype analysis/ SCEs Ciaran Morrison

The DT40 karyotype

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

Modal karyotype is: 2 Gga-1, 3 Gga-2, 2 Gga-3,2 Gga-4, 2 Gga-5, 1 Gga-Z.

Page 4: Chicken karyotype analysis/ SCEs Ciaran Morrison

Variation/ mosaicism in the DT40 karyotype

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

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

Page 5: Chicken karyotype analysis/ SCEs Ciaran Morrison

Count the macrochromosomes

Page 6: Chicken karyotype analysis/ SCEs Ciaran Morrison

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)

Page 7: Chicken karyotype analysis/ SCEs Ciaran Morrison

1

12

2

2

3

3

4

4

5

5

Z

Anna Stephan

Page 8: Chicken karyotype analysis/ SCEs Ciaran Morrison

Anna Stephan

Page 9: Chicken karyotype analysis/ SCEs Ciaran Morrison

Anna Stephan

Page 10: Chicken karyotype analysis/ SCEs Ciaran Morrison

Chromosome aberrations

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

Page 11: Chicken karyotype analysis/ SCEs Ciaran Morrison

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)

Page 12: Chicken karyotype analysis/ SCEs Ciaran Morrison

‘A Spotter’s Guide’

Ctg: Chromatid-type gapCtb: Chromatid-type breakCsg: Chromosome gapCsb: Chromosome breakCte: Chromatid exchange

Page 13: Chicken karyotype analysis/ SCEs Ciaran Morrison

Scoring suggestion

Record data for each macrochromosome in a spread.

Sum for population (100 metaphases)

Page 14: Chicken karyotype analysis/ SCEs Ciaran Morrison

Telomere analysis in

DT40s

From: O’Hare & Delany (2009) Chromosome Res. 17: 947-64.

Chickens have high levels of telomere repeat sequence, notably in the microchromosomes.

DT40s have shorter-than-normal telomeres, although the interstitial sequences are retained.

Page 15: Chicken karyotype analysis/ SCEs Ciaran Morrison

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.

Page 16: Chicken karyotype analysis/ SCEs Ciaran Morrison

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).

Page 17: Chicken karyotype analysis/ SCEs Ciaran Morrison

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

Sister chromatid exchanges

Page 18: Chicken karyotype analysis/ SCEs Ciaran Morrison

SCE visualisation

1 cell cycle

+ BrdU

Equally-labelled sisters

Page 19: Chicken karyotype analysis/ SCEs Ciaran Morrison

SCE visualisation

2nd cell cycle

+ BrdU

Differentially-labelled sisters

Page 20: Chicken karyotype analysis/ SCEs Ciaran Morrison

SCE visualisation

2nd cell cycle

+ BrdU

Differentially-labelled sisters

Giemsa staining revealsdifferential labelling

Page 21: Chicken karyotype analysis/ SCEs Ciaran Morrison

SCE visualisation

2nd cell cycle

+ BrdU

Differentially-labelled sisters

Giemsa staining revealsdifferential labelling

and exchanges

Page 22: Chicken karyotype analysis/ SCEs Ciaran Morrison

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 399-403.

Fluorescence plus Giemsa (‘FPG’)

Page 23: Chicken karyotype analysis/ SCEs Ciaran Morrison