chicken karyotype analysis/ sces ciaran morrison
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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’)