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Why is cloning so hard?

Scientists have identified how a single gene may help explain why mammalian cloning is such an inefficient process

Cold Spring Harbor Laboratory

Although in recent years our papers and magazines have been rife with images of Dolly, Copy Cat, and the like, the reality is that cloning mammals is still an extremely inefficient endeavor. By tracking the expression of a gene called Oct4, a joint effort from the laboratories of Dr John McLaughlin and Dr. Hans Schöler and at the University of Pennsylvania School of Veterinary Medicine has developed a way to assay the progress of newly cloned mammalian embryos, and evaluate the possibility for successful development. Their results, published in the May 15 issue of Genes & Development, cast a long shadow of doubt over current prospects for reproductive human cloning.

Cloning [the transfer of a somatic (body) cell nucleus into an enucleated egg] depends upon the faithful reprogramming of the donor nucleus. In order to properly direct embryonic development, the donor nucleus must abort its former genetic program and adopt the gene expression profile of an embryonic nucleus. It is thought that a failure in donor nucleus reprogramming may underlie the doomed fate of most cloned embryos. To test this hypothesis, the researchers analyzed cloned mouse embryos for the expression of Oct4 -- a gene that is not expressed in somatic cells, but is expressed in the embryo -- as a means to evaluate the fidelity of cloned embryo's genetic reprogramming.

Oct4 encodes a transcription factor (a protein that regulates the expression of other genes) that is crucial for normal embryonic development and viability. Oct4 expression is strictly regulated during development. In the blastocyst stage embryo, Oct4 is only expressed in the inner cell mass (ICM; the portion of the embryo that eventually gives rise to all fetal tissues). In the adult, Oct4 is only expressed in germ cells.

Because Oct4 is not expressed in adult somatic cells, but is required in the embryo, the Penn team used Oct4 to test whether or not the donor nucleus has been successfully reprogrammed to turn on Oct4 in the ICM.

They used donor nuclei from mouse cumulus cells, the adult somatic cells that surround ovulated eggs. The researchers found that the majority of cumulus-cloned embryos did not successfully reprogram their Oct4 gene expression pattern. Only 34% of blastocyst-stage, cumulus-cloned embryos expressed Oct4 in the ICM. The remainder of the clones either did not express Oct4 at all (11.3%), or aberrantly expressed Oct4 in other regions of the embryo as well as the ICM (54.7%). Furthermore, the researchers found that many of the clones that did correctly express Oct4 in the ICM did not do so at normal levels, thus further reducing the number of viable clones.

To further observe the developmental potential of cloned blastocyst-stage embryos, the research team used specially designed cell culture conditions to mimic the implantation of the blastocyst into the uterus of a surrogate mother. The ability of the clones to divide and form outgrowths in vitro serves as an experimental model of their ability to develop in utero.

Compared to embryos formed through in vitro fertilization (IVF) methods, the cumulus-cloned embryos exhibited a marked decrease in their ability to form outgrowths. Of the outgrowths formed, less than half expressed Oct4, and those that did generally displayed abnormally low levels (compared to IVF controls). Overall, the researchers were able to correlate aberrant Oct4 expression with dampened developmental potential of cloned embryos.

An important observation, though, was that the small number of clone outgrowths that did adequately express Oct4 were capable of forming embryonic stem cell lines. As Dr. Schöler explains, although "this small number of cells in the embryo probably would not have been sufficient to allow development...it certainly was sufficient to allow the derivation of an embryonic stem cell line." As embryonic stem cells are the fundamental building blocks of therapeutic cloning, this demonstration supports existing evidence as to the scientific feasibility of therapeutic cloning. The work simultaneously shows the infeasibility of reproductive human cloning.

Dr. Schöler is quick to point out that "embryonic stem cells can be carefully tested prior to being used in the patient. In reproductive cloning, only the organism provides proof of whether the procedure worked or not."

As the results demonstrate, cloned mouse embryos seldom display normal Oct4 expression, indicating a failure of most clones to adequately reset their genetic program. While misexpression of Oct4 is, itself, sufficient to disrupt embryonic development, it is likely that Oct4 is not the only gene aberrantly expressed in cloned embryos. Further research will determine what other genes are misregulated during the development of cloned embryos, and how these may negatively impact the fate of clones - and reproductive cloning - in general.

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