Cloning in Cattle

In mammalian cell nuclei chromosome territories (CTs) occupy
positions correlating with their gene-density and chromosome size.
While this global radial order has been well documented, the
question of whether a global neighborhood order is also maintained
has remained a controversial matter. To answer this question I grew
clones (of HeLa, HMEC and human diploid fibroblast cells) for up to
5 divisions (32 cells) and performed 3D FISH experiments to
visualize the nuclear positions of 3 different CT pairs. Using
different landmark-based registration approaches I assessed the
similarity of CT arrangements in daughter cells and cousins. As
expected from a symmetrical chromatid movement during mitotic
anaphase and telophase, I was able to confirm previous findings of
a pronounced similarity of CT arrangements between daughter cells.
However, already after two cell cycles the neighborhood order in
cousins was nearly completely lost. This loss indicates that a
global neighborhood order is not maintained. Further, I could show
in the present thesis that a gene density correlated distribution
of CTs, which has already been shown in different cell types of
various species appears to be independent of the cell cycle.
Moreover I could provide evidence that the nuclear shape plays a
major role in defining the extent of this gene-density correlated
distribution, as nuclei of human, old world monkey and bovine
fibroblasts showed an increased difference in the radial
distribution of gene poor/dense CTs when their nuclei were
artificially reshaped from a flat ellipsoid to a nearly spherical
nucleus. The observation that a gene-density correlated
distribution of CTs has been found in nuclei from birds to humans
argues for a significant, yet undiscovered functional impact. So
far CTs have been investigated mainly in cultured cells and to some
extent in tissues, yet little is known about the origin and fate of
CTs during early development. To gain insights into the very early
organization of CTs in preimplantation embryos I have developed a
fluorescence in situ hybridization (FISH) protocol, which enables
the visualization of CTs in three dimensionally preserved embryos.
Using this protocol I have investigated CTs of bovine chromosomes
19 and 20, representing the most gene-rich and gene-poor
chromosomes, respectively. Equivalent to the distributions
described in other species I could confirm a gene density related
spatial CT arrangement in bovine fibroblasts and lymphocytes with
CT 19 being localized more internally and CT 20 more peripherally.
Importantly, I did not find a gene density related distribution of
CTs 19 and 20 in early embryos up to the 8-cell stage. Only in
embryos with more than 8 cells a significant difference in the
distribution of both chromosomes became apparent that increased
upon progression to the blastocyst stage. Since major genome
activation in bovine embryos occurs during the 8- to 16-cell stage,
my findings suggest an interrelation between higher order chromatin
arrangements and transcriptional activation of the embryonic
genome. Using another experimental set up I analyzed the topology
of a developmentally regulated transgene utilizing bovine nuclear
transfer (NT) embryos derived from fetal fibroblasts, which
harbored a mouse Oct4/GFP reporter construct integrated at a single
insertion site on bovine chromosome 13. I analyzed the intranuclear
distribution of the transgene as well as its position in relation
to its harboring chromosome in donor cell nuclei and day 2 NT
embryos, where the transgene is still inactive as well as in day 4
NT embryos, where transgene expression starts, and day 7 NT
embryos, where expression is highly increased. Compared to donor
cell nuclei I found a more peripheral location of both BTA 13 CTs
and the Oct4/GFP transgene in day 2, day 4 and day 7 NT embryos,
although there was a trend of the transgene and both BTA 13 CTs to
re-localize towards the nuclear interior from d2 to d7 embryos.
Moreover, I found the transgene located at the surface of its
harboring CT 13 in donor fibroblasts, whereas during
preimplantation development of NT embryos it became increasingly
internalized into the chromosome 13 territory, reaching a maximum
in d7 NT embryos, i.e. at the developmental stage when its
transcription levels are highest. These latter experiments show
that the transfer of a somatic nucleus into a chromosome depleted
oocyte triggers a large scale positional change of CTs 13 and of an
Oct4/GFP transgene and indicate a redistribution of this
developmentally regulated Oct4/GFP transgene during activation and
upregulation in developing NT embryos.