Chicken karyotype analysis/ SCEs

Ciaran Morrison

Diagrammatic representation of the chicken karyotype

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.

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

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)

1

12

2

2

3

3

4

4

5

5

Z

Anna Stephan

Anna Stephan

Anna Stephan

Chromosome aberrations

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

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 gapCtb: Chromatid-type breakCsg: Chromosome gapCsb: Chromosome breakCte: 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: 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.

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.

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. 1999. Mol. Cell. Biol. 19(7):5166-5169

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 revealsdifferential labelling

SCE visualisation

2nd cell cycle

+ BrdU

Differentially-labelled sisters

Giemsa staining revealsdifferential 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 399-403.

Fluorescence plus Giemsa (‘FPG’)