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Cloned Mice Have Genomic Flaws
New findings have profound implications for reproductive cloning in humans

By Nancy Touchette


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Cloning

Ever since Dolly the sheep was cloned in Scotland five years ago, researchers, entrepreneurs, politicians, and ethicists have grappled with the possibility of someday cloning humans. Sheep, cows, mice, goats, kittens, and pigs have all been cloned. But for many, the idea of cloning humans has seemed like science fiction.

Then last year, Advanced Cell Technology in Worcester, Massachusetts, announced it had cloned a human embryo that progressed to the six-cell stage. This year, an Italian doctor claims to have cloned and implanted a human embryo into a woman's uterus. And for $200,000 the company Clonaid promises to clone a human baby.

Among the issues raised by the idea of cloning humans, safety is one of the more tangible. Prominent researchers have been arguing that cloning animals—and humans in particular—is risky business. Only a small percentage of cloned animals survive to birth, and those that do are usually subject to a host of health problems including obesity, frequent bouts of pneumonia, and liver failure.

Now, a new study by Rudolf Jaenisch and his colleagues at the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, shows that the genomes of cloned mice are severely compromised. Using DNA microarray technology, the team analyzed more than 10,000 genes from liver and placenta cells in cloned mice and found that up to four percent of the genes do not function normally.


Genetic material being removed from an egg cell.

The results strongly argue against the safety of attempting human cloning for reproductive purposes and raise questions about therapeutic cloning that should be answered before clinical research goes forward.

"There have been two camps regarding cloning in mammals," says Jaenisch. "In one camp, people have looked at the clones and thought 'They can't be normal.' Others said, 'They must be normal, because they survive.'"

Typically, less than 10 percent of cloned embryos survive. Those that do survive are often oversized at birth, have abnormally large placentas, and die at a younger age than their naturally bred counterparts.

Jaenisch says previous studies on cloned mammals have pointed to problems with the genome integrity, but it wasn't clear just how big these problems are. Researchers have already identified a handful of genes whose activity was not normal in the cloned embryos, affecting their development.

Indeed, it appears that developmental problems in clones arise not from genetic mutations, but from changes in the normal program of gene activation, or expression.

In the present study, Jaenisch's team found that aberrant gene expression is much more widespread than originally suspected. Using a special technique called DNA microarray analysis, in which large numbers of genes are examined on a gene chip, the researchers were able to measure the functioning of more than 10,000 genes in cells from cloned mice.

The Whitehead researchers created cloned mice from either adult or embryonic stem cells—cells derived from the early embryo that have the potential to develop into any cell in the body. They then looked at gene expression in placenta and liver cells.


Diagram representing changes in gene activity.

The result was clear differences in how genes functioned in the cloned cells. In placental cells, more than 200 genes were more or less active compared to normal embryos. The same was true for dozens of genes in liver cells.

Given all the errors in gene expression, Jaenisch says it is amazing the embryos even survive to birth. "There is a fair amount of tolerance to incorrect gene expression. But development is like a symphony," he says. "Everyone has to play correctly for the symphony to sound perfect. You can make a lot of mistakes and the symphony will go on, but it will sound awful."

Just what is causing the aberrant gene expression remains a mystery. When an animal is cloned, the nucleus of a mature adult cell is removed and inserted into an egg whose nucleus has been removed. In the new environment the genetic material must somehow be reprogrammed to turn off the genes specific to the adult cell and turn on the genes needed for embryonic development.

"You could imagine a liver cell happily minding its own business being a liver cell and expressing genes that make it a liver cell," says Colin Stewart of the National Cancer Institute in Frederick, Maryland. But if the liver cell is put into an egg cell, he adds, then it is exposed to factors researchers don't know anything about.


Cloned female mouflon with her foster mother.

"Suddenly," Stewart continues, "that happy nucleus humming away as a liver nucleus has to switch off all the genes that say 'Make me a liver cell' and turn on the genes that say 'Now make me an embryo.' The whole slate has to be wiped clean."

Jaenisch observed the greatest aberration in gene activity in the placenta of the developing mice. He suspects that problems with genes in the placenta are greater than those in the liver, because the developing embryo doesn't have as much time to undergo the reprogramming process.

Many researchers, including Jaenisch, nevertheless support the continued cloning of animals for commercial use. Progeny of cloned animals are completely normal because their egg and sperm undergo the normal process of reprogramming and the cloning process does not affect the primary structure of DNA.

However, the rush to move therapeutic cloning from the lab to the clinic should proceed cautiously, according to Richard Chaillet of the University of Pittsburgh. Therapeutic cloning involves transferring a nucleus from an adult cell to an egg cell and then removing and culturing embryonic stem cells from the clone.

In principal, the cultured cells could then be induced to differentiate into specific types of cells. These could be transplanted into patients to treat a host of diseases and conditions such as Parkinson's, diabetes, heart disease, and even spinal cord injury.

"This study sends the message that we need to better understand what is going on," says Chaillet. "If your goal is to get normal clones, we will have to understand this nebulous reprogramming process better."

Adds Jaenisch, "There is a message here for people who want to clone humans for reproductive purposes: It is totally irresponsible."

See related GNN articles
»Modified cloning strategy succeeds with rabbits
»Endangered wild sheep clone reported to be healthy

Kierszenbaum, A.L. Genomic imprinting and epigenetic reprogramming: Unearthing the garden of forking paths. Mol Reprod Dev. Published online September 17, 2002.
Humpherys, D. et al. Abnormal gene expression in cloned mice derived from ES cell and cumulus cell nuclei. Proc Natl Acad Sci USA. Published online September 16, 2002.

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