Appendix N. 
395 
A principal biological barrier that prevents clones from being 
normal is the “epigenetic" difference (such as distinct patterns of 
DNA methylation'^) between the chromosomes inherited from mother 
and from father, i.e. the difference between the “maternal" and the 
“paternal” genome of an individual. Such methylation of specific 
DNA sequences is known to be responsible for shutting down the 
expression of nearby genes. Parent-specific methylation meirks are 
responsible for the expression of imprinted genes and cause only one 
copy of an imprinted gene, derived either from sperm or egg, to be 
active while the other allele is inactive (Ferguson-Smith and Surani, 
2001). When sperm and oocyte genomes are combined at 
fertilization, the parent-specific marks established during oogenesis 
and spermatogenesis persist in the genome of the zygote (Fig 3A). Of 
interest for this discussion is that within hours after fertilization, 
most of the global methylation marks (with the exception of those on 
imprinted genes) are stripped from the sperm genome whereas the 
genome of the oocyte is resistant to this active demethylation 
process (Mayer et al., 2000). This is because the oocyte genome is in 
a different “oocyte-appropriate" epigenetic state than the sperm 
genome. The oocyte genome becomes only partially demethylated 
within the next few days by a passive demethylation process. The 
result of these post-fertilization changes is that the two parental 
genomes are epigenetically different (as defined by the patterns of 
DNA methylation) in the later stage embryo and remain so in the 
adult in imprinted as well as non-imprinted sequences. 
In cloning, the epigenetic differences that are established during 
gametogenesis may be erased because both parental genomes of the 
somatic donor cell are introduced into the egg from the outside and 
are thus exposed equally to the demethylation activity present in the 
egg cytoplasm (Fig 3B). This predicts that imprinted genes should be 
particularly vulnerable to inappropriate methylation and associated 
dysregulation in cloned animals. The results summarized eairlier are 
consistent with this prediction. For cloning to be made safe, the two 
parental genomes of a somatic donor cell would need to be 
physically separated and separately treated in an “oocyte- 
appropriate" and a “sperm-appropriate” way, respectively. At 
present, it seems that this is the only rational approach to guarantee 
the creation of the epigenetic differences that are normally 
established during gametogenesis. Such an approach is beyond our 
present abilities. These considerations imply that serious biologicel 
berriers exist that interfere with faithful reprogramming after nuclear 
•transfer. It is a safe conclusion that these biological barriers 
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