The full term development of sheep, cows, goats, pigs,
and mice has been achieved through the transfer of somatic
cell nuclei into enucleated oocytes. However, only a small
percentage of conceptuses survive to term and are characterized
by high mortality rate and widespread epigenetic abnormalities
due to misexpression of many genes. We are using nuclear
transplantation procedures to compare the potency of stem
cells, differentiated cells and of transformed cells to
direct embryonic development.
Nuclear cloning represents a general and unbiased approach
to probe whether cellular differentiation involves epigenetic
as opposed to genetic alterations. The utility of the
approach was demonstrated by the generation of monoclonal
mice from mature B and T cells demonstrating that the
genetic alterations that occurred in a single donor cell,
i.e., the somatic rearrangements of the IgG and TCR loci,
could be amplified and were present in all tissues of
the monoclonal mice. Following a similar logic we used
the nuclear transfer procedure to assess whether irreversible
alterations occur during the maturation of cortical or
olfactory neurons and could be visualized in a cloned
animal. Only one allele of about 1,500 receptor genes
is expressed in a given olfactory neuron but the mechanism
of receptor choice and monoallelic expression is obscure.
We have derived cloned mice from mature olfactory neurons
that expressed the P2 olfactory receptor. The analysis
of the cloned mice suggest that neuronal maturation and
olfactory receptor choice does not involve genetic alterations
that would interfere with nuclear potency to generate
mice. Our results also indicate that receptor choice in
olfactory neurons is fully reversible.
The malignant state of tumor cells is known to be caused
by genetic as well as epigenetic changes of the genome,
prompting us to use nuclear transfer as an approach to
distinguish between these changes. As nuclear donors we
are using embryonic carcinoma cells and somatic cancer
cells, including leukemia cells and solid tumors such
as melanoma cells. Our results suggest that the genome
of some somatic cancer cells can be reprogrammed to direct
at least some embryonic development after transfer of
the nucleus into the oocyte indicating that the tumor
phenotype is largely determined by epigenetic alterations.
In contrast, when we derived ES cells by nuclear cloning
from embryonic carcinoma (EC) donor cells, we did not
observe a gain in developmental potency suggesting that
genetic rather than epigenetic changes are responsible
for the phenotype of EC cells.
