Analyse der Chromosomenverteilung in menschlichen Fibroblasten mittels 3D-Vielfarben Fluoreszenz in situ Hybridisierung

In human diploid fibroblasts (HDFs) the cell nucleus is oval in
shape, quite large in xy-diameter (10-20µm), but flat in
z-direction (5µm). In these nuclei chromosome territories typically
lie side by side or slightly above each other. The question whether
these arrangements are ordered or variable has yielded conflicting
answers. We hybridised an improved 7-fluorochrome MFISH probe set
on 3D-preserved cell nuclei, fixed with buffered 4%
paraformaldehyde. A LEICA wide field microscope with an
8-filter-wheel and an automated z-step motor was used for imaging
of the 7 fluorochromes plus DAPI. Multicolor images from nuclei
were taken as serial sections in z-direction. After deconvolution,
a specifically developed Program goldFISH Saracoglu K. et al. 2001
was used to classify the images according to the labelling scheme.
The classification algorithm corresponds to the procedure
previously used for metaphase spreads, now adopted to 3-D studies
of chromosome territory arrangements in the cell nucleus. The
analysis of 30 G0-fibroblast nuclei and 30 prometaphase rosettes
revealed a pronounced variability of chromosome territory
neighbourhoods, as described by Allison D. C. et al. 1999, but in
contrast to Nagele R. et al. 1995. However we noted a distinct
radial order: small chromosomes were located close to the centre
while large chromosomes were positioned towards the nuclear rim.
This non-random radial positioning could also be observed in
prometaphase rosettes