Nuclear transfer has been used in mammals as both a valuable tool
forembryonic studies and as a method for the multiplication of
'elite' embryos.
Offspring have only been reported when early embryos,
or embryo-derived cells primary culture were used as nuclear donors.
Here we provide the first report, to our knowledge,
of live mammalian offspring following nuclear transfer
from an established cell line. Lambs were born after cells
derived from sheep embryos, which had been cultured for 6 to 13
passages, were induced to quiescence by serum starvationbefore
transfer of their nuclei into enucleated oocytes.
Induction ofquiescence in the donor cells may modify the donor
chromatin structure tohelp nuclear reprogramming
and allow development.
This approach will provide the same powerfull opportunities for
analysisand modification of gene function in livestock species
that are availablein the mouse through the use of embryonic stem cells.
Briefing notes in relation to Nature paper on nuclear transfer
#Can you describe simply what has been done in these experiments?
This is the first time that offspring have been produced by nuclear
transfer from an established cell line.
#What is involved in nuclear transfer?
You have two different cells in nuclear transfer:
an unfertilised egg and a donor cell.
The donor cells were obtained by culture of cells fromsheep embryos
over a period of several months.
In this way it was possibleto obtain many thousand genetically
identical cells.
The donor embryo was from an all white breed:
the Welsh Mountain breed.
The recipients eggs were recovered from Scottish Blackface ewes.
By micromanipulation the chromosomes were removed from the eggs
beforethe nucleus of the donor cell was introduced by cell fusion.
The electric current which is used to fuse the cells also triggers
theegg to begin development.
These new embryos were then transferred torecipient sheep to discover
if they were able to develop to lambs. When the lambs were born they
were all genetically identical females.They were all white,
Welsh Mountain lambs, as it is the transferred nucleuswhich determines
the characteristics of the offspring. #What is the key
to this success? We cannot be sure as the experiment has only
been conducted once withone cell type and it seems likely that
several factors are involved. However, we suggest that the
induction of a quiescent state in the donorcells may be one important
factor. Early development is controlled by RNAand proteins that are
produced in the unfertilised egg while it is stillin the ovary.
At a species specific stage of development these compoundsare
destroyed and products of the embryos own genes assume control of
development. In a normal cell the chromosomes are very active and
there isan open structure to some of the DNA to provide access
to the cell machinery.When a nucleus from such a cell is
transferred into an oocyte it is assumedthat development depends upon
changes to chromosomal structure to first ofall inhibit activity
from the chromosome and then to re-initiate activityat the appropriate
stage of development. The nucleus in quiescent cells iscomparatively
inactive.
We hypothesise that in this case the recipient eggis more
readily able to "reprogramme" gene expression in the new embryos.
#Why did so few of the embryos develop to lambs?
We do not know because the experiment has only been carried out once
with one cell type. However,it should be appreciated that these
results representa very significant advance and this is a new
approach to nuclear transfer.Improvements to the efficiency are to
be expected as other experimentsare carried out. #What is the
biological significance of this research? The fact that cells
which had been in culture for a prolonged period andhad begun to
differentiate were ab
