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Abnormalities in clones
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Study shows normal-looking clones may be abnormal

By Nadia Halim
Whitehead Institute

Scientists have found the first evidence to show that even seemingly normal-looking clones may harbor serious abnormalities affecting gene expression that may not manifest themselves as outward characteristics.

The findings, reported in the July 6 issue of Science by researchers at the Whitehead Institute for Biomedical Research and University of Hawaii, confirm the previous suspicion that reproductive cloning is not only inefficient, but may actually be unsafe.

In the study, scientists from the laboratories of MIT Professor of Biology Rudolf Jaenisch and Professor Ryuzo Yanagimachi of the University of Hawaii sought to examine the mechanisms underlying poor survival and gross overgrowth in cloned animals.

They made mouse clones from embryonic stem cells and monitored the activity of imprinted genes -- developmental genes that are controlled by special tags that do not affect the base sequence itself. They looked to see if these tags were faithfully reproduced in cloned mice and in the donor cells used to make the clones. This would tell them if the problems with cloning resulted from aberrations in donor cells or as a result of the cloning procedure itself.

Scientists found to their surprise that much of the problem lay in the makeup of the donor embryonic stem cells, which they found to be extremely unstable in culture. As they divide in culture, these cells lose the tags that tell an imprinted gene to be either turned on or off during development. The researchers found that even clones made from sister stem cells had differences in their gene expression.

Despite this instability, many embryos survived to adulthood, suggesting that mammalian development is surprisingly tolerant of aberrant gene regulation.

"This suggests that even apparently normal clones may have subtle aberrations of gene expression that are not easily detected in the cloned animal," Jaenisch said.

HOW IT'S DONE

The cloning procedure involves removing the nucleus, or the genetic command center, of an egg and replacing it with the nucleus from an adult cell or an embryonic stem cell. Ideally, the egg resets the developmental clock of the nucleus back to a state compatible with early embryonic growth and gives rise to a new organism that is genetically identical to the donor cell. Researchers had thought that using the nucleus from an embryonic stem cell might require less reprogramming than using the nucleus from an adult cell, such as a mammary cell, which has already committed to a certain function.

Embryonic stem cells are unique in that they can give rise to an entire organism without using the nuclear transfer procedure. The researchers found that animals developed from mouse embryonic stem cells by this technique exhibited irregular gene expression just as animals derived from nuclear transfer using embryonic stem cells, confirming that these abnormalities likely existed in the cells before the cloning procedure was performed.

"While embryonic stem cells may be easier to developmentally reprogram than adult cells, they may have a different set of problems arising from errors occurring during culturing that cannot be fixed by cloning," said David Humpherys, a graduate student in the Jaenisch lab.

This may mean researchers will need to find ways to better preserve gene regulatory tags during cell culture techniques. Researchers also still need to test whether animals cloned from adult cells will show similar defects.

"However, despite their instability, it is important to remember that embryonic stem cells when combined with normal cells -- as in making chimeras or when used in transplantation -- may function fine. In other words, embryonic stem cells might work fine when used as cell therapy, but when they are used to make whole animals would likely produce organisms that are abnormal," Jaenisch said.

"While these new results are a concern for reproductive cloning, misexpression of imprinted genes likely has little significance for therapeutic cloning. Imprinted genes have defined roles in fetal development, but may serve no crucial role in the differentiated cells of the adult," said Humpherys.

Widespread dysregulation in clones
Nuclear transfer technique is a culprit in causing abnormalities. | By Nicole Johnston



The incidence of abnormal gene expression in cloned mammals is more extensive than widely believed, Rudolph Jaenisch and colleagues reported this week in the Proceedings of the National Academy of Sciences. Furthermore, many abnormalities are a consequence of the cloning procedure, whereas others derive from the donor nucleus, said the authors from the Whitehead Institute for Biomedical Research and Massachusetts Institute of Technology.

Cloning mammals involves taking a mature donor nucleus and reprogramming it to a state that supports embryonic development, while suppressing previously transcribed genes involved in cell differentiation in the donor. The majority of cloned mammals die before or soon after birth. The few that do survive, however, are affected by developmental abnormalities to varying degrees.

The extent of these abnormalities was revealed by Jaenisch and colleagues using oligonucleotide microarray analysis, which enabled them to evaluate the expression of more than 10,000 genes in the livers and placentas of cloned mice. Donor nuclei were obtained from embryonic stem cells cultured in the lab and cumulus cells (cells in the ovary that nourish the developing egg and are easiest to clone) obtained directly from the mice. Until now, gene expression analysis in cloned animals has only looked at a small number of genes from preimplantation embryos.

What they found was worse than anyone anticipated. Hundreds of genes of the 10,000 studied were dysregulated, comprising roughly 4% of the mouse genome. Significantly, abnormal expression of many of the same genes was found whether the nuclei were obtained from cultured cells or taken directly from the mice, fingering the nuclear transfer technique and not cell culture as a significant cause of many abnormalities. "It means that clones that develop despite enormous dysregulation are not normal," said Jaenisch.

This news won't bode well for biotech companies intent on making hay from cloning mammals, or rogue groups claiming to be able to clone human beings. "What is true with other mammals will be true with humans," Jaenisch explained. "It's totally irresponsible to think you can use the nuclear transfer procedure to produce any normal individuals. Those clones will suffer the same fate and be as abnormal as [cloned] mice."

Nor does he think that tide will turn anytime in the foreseeable future. "Reprogramming the donor nucleus during egg maturation is the principle barrier to cloning," said Jaenisch. "There is no good [alternative] way to accomplish this and no progress to improve the procedure of cloning."