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CLONING - Failure exceeds success
Posted 1 January 2002
This article by Gina Kolata appeared in the New York Times, 11 December 2001 (also see our overview).
Four years ago, Dr. Tanja Dominko went to a laboratory in Oregon with high hopes that she would soon be cloning monkeys. The lab was generously financed with federal grants, there were plenty of monkeys to work with and most experts thought that since Dolly the sheep had just been created by cloning, monkeys would not be far behind.
She left a year ago, with a cloning portfolio that she calls her gallery of horrors. After three years, and about 300 attempts, the best she got was a placenta with no fetus. Most of the time, she saw grotesquely abnormal embryos containing cells without chromosomes, where the cell's DNA resides; or cells with three or four nuclei and one time even nine; or cells that looked more like cancer cells than the cells of a healthy animal.
So Dr. Dominko joined a long line of cloning researchers unable to create clones. And the story she tells, she says, is a seldom-heard cautionary tale.
Researchers who have occasional success cloning one species, like cows, are meeting failure with others, like dogs. For them - even for the scientists who made Dolly - cloning success is the exception, not the rule. A vast majority of efforts fail, even in species that have at one time or another been cloned.
Still, cloning technology is potentially highly useful, so researchers keep trying. Companies are cloning to make copies of valuable animals. They also are cloning to genetically modify animals so that humans can use their organs or so that the animals will secrete useful medicines in their milk. But, cloning experts say, the day when scientists can easily, reproducibly and routinely produce a bountiful supply of cloned animals - or people - seems far in the distant future.
Now Dr. Dominko works for Advanced Cell Technology, the Worcester, Mass., company that is a vigorous advocate of commercial cloning technology, and that made the startling assertion last month that it was trying to clone humans.
So far, though, the researchers at Advanced cell Technology have not succeeded - most of the eggs died without dividing even once. Their best attempt was an embryo that grew to six cells before dying.
They had hoped to use cloned human embryos to extract stem cells, which are embryo cells that can in theory develop into any cell or body tissue and that the company hopes to use to treat diseases.
With cloning, the embryos and so the stem cells could be made genetically identical to a patient who needs the treatment. But stem cells appear only after about five days of growth and the company's embryos were not even close.
This lack of success was not a surprise to leading stem cell researchers and cloning experts who attended a meeting last week on regenerative medicine, which is based on the idea of using new cells and organs to regenerate failing human bodies. Most of these researchers have concluded that routinely cloning animals or growing human embryo clones long enough to extract stem cells could be years or even decades away, and Dr. Dominko said her experience with monkeys might tell why.
She said her work suggested that even if researchers could grow human embryo clones to the blastocyst stage, the stage of development occurring when stem cells arise, most of the blastocysts might be unusable. The embryos may have few stem cells, and the few that are present may have abnormal chromosomes.
"My results do not mean that it can't be done," Dr. Dominko said. But, she and others said, the monkey story, which she told in a lecture and in interviews at the meeting in Washington on Dec. 3, is something of a reality check in a context of cloning hype and, at times, near-hysteria.
"I think this is really a profound story," she said. "We can make all sorts of things that look like embryos, but what are we really making?"
It also raises questions about the success of cloning in general. Dr. Ian Wilmut, the scientist who created Dolly, a sheep that was the first animal cloned from cells of an adult, said at the meeting that there were as many species in which cloning failed, as those in which it succeeded. While scientists have cloned sheep, cattle, mice, goats and pigs, no one has been able to clone rabbits, rats, cats, dogs or monkeys.
"Even the same teams that succeeded with other species failed with these." Dr. Wilmut said.
In general, he said, just 1 to 4 percent of cloning efforts in the species where it has worked results in the birth of a live animal. That, he said, indicates that cloning appears to create serious abnormalities in almost all embryos.
The monkey work began because scientists and the National Institutes of Health urgently wanted genetically identical monkeys for research. The hope was to use them in testing drugs and vaccines, for example, or understanding the likelihood of getting diseases. Cloning seemed a natural way to proceed.
In 1997, Dr. Don P. Wolf, a senior scientist at the Oregon Regional Primate Research Center, announced an important advance. He had taken a cell from a monkey embryo and used it to clone a monkey that would have been the identical twin of that embryo if it had been allowed to develop.
In similar research in other species, such cloning from embryo cells has been much easier than making a clone of an adult animal, or even of a fetus. But it was a first step for researchers, and it was the first time it had been done in monkeys. Success in cloning monkey fetuses or even adults seemed within reach.
Since Dr. Wolf was planning to retire, the center brought in Dr. Gerald P. Schatten, an expert on fertilization, to replace him. Dr. Schatten hired Dr. Dominko and several other experts in embryo development to do the work.
But after Dr. Wolf's success with cloning the monkey embryo cell - which neither he nor anyone else was eve able to repeat - Dr. Wolf decided not to retire after all. And he and Dr. Schatten ended up with competing laboratories, on adjacent floors of the research building, supported by the National Institutes of Health and focusing on cloning monkeys.
"We were all very, very hopeful," Dr. Dominko said. The investigators were trying variations on the same method that worked for Dr. Wilmut when he made Dolly. They would take a monkey egg, remove its genetic material and slip in the cell of another monkey. If cloning worked, the egg would use the genetic material from the new cell to direct the development of a monkey that was genetically identical to the one that provided the cell.
The researchers began by trying to repeat Dr. Wolf's work. They got embryos that were clones, but most were horribly deformed and died. The few that lived long enough to be transferred to a monkey's uterus died soon afterward. Dr. Dominko said she transferred 50 embryos. But, she said, "we never obtained a single pregnancy."
She decided to go back and take apart the cloning process, trying to figure out what was going wrong. To clone, scientists subject eggs and embryos to some pretty harsh treatment, including adding chemicals and dyes; poking, prodding and pulling on the eggs; and subjecting them to electric shocks. One of those treatments, she thought, might be doing the damage.
Or perhaps, Dr. Dominko thought, the problem was simply in the very first step in cloning, the mechanical injury of removing the DNA from an egg. She designed an experiment. She would take the DNA out of an egg as if she were to do cloning. But then she would put it back and fertilize the egg and see if it developed normally.
The results, she said, were the same sorts of grossly abnormal embryos that she saw with cloning.
But when she tested other steps in cloning they, too, created deformed embryos.
"The process is so complex that there is not one variable that makes or breaks it, " Dr. Dominko said. "That is what is so frustrating about dissecting this thing in primates - it will never be feasible. You would need a whole primate colony with thousands of animals. How many questions can you ask on a single egg? One. If you are lucky, you ask the right one."
It may be that a law of large numbers is what made cloning work in other species, she and other experts said. After all, Dr. Dominko used 300 monkey eggs in three years. That, said Dr. Ray Page, a colleague at Advanced Cell Technology, "is a bad experiment on a bad day in a cow lab." Researchers get thousands of cow eggs from slaughterhouses, Dr. Page said. That makes it much easier to try cloning often enough, with enough slight variations in technique, that it eventually works.
Dr. Page said he learned this from bitter experience at a Blacksburg. Va., laboratory where he worked for a biotechnology company, PPL Therapeutics trying to clone cattle and pigs. The group finally succeeded, Dr. Page said, but only after thousands of frustrating attempts. "None of us expected it to take so long."
"That's the history of cloning," Dr. Page said. When it finally works, no one really knows why. "For some reason things just seem to click."
Another Advanced Cell researcher, Dr. Teruhiko Wakayama, who had cloned mice at the University of Hawaii, tried thousands of times before it worked.
Others were luckier - or very skilled. Dr. Steven Willadsen, a cloning pioneer in Windermere, Fla., said he cloned the first sheep from an embryo cell in the 1980s. It worked the first time he tried it - he got a healthy lamb.
But one problem in the field, said Dr. Ryuzo Yanagimachi, a mouse cloning expert at the University of Hawaii, is that scientists talk only about their successes, not their many failures.
"The ones that survive are the exceptions, not the rule," Dr. Yanagimachi said. "People do not report negative results."
Maybe they should, Dr. Dominko said, especially if cloning is to be used to make commercial products, like stem cells.
"If you want to make it into something that will have commercial value, not only do you have to pull a volume of material out of it, but the process has to be repeatable," she said. "Your success cannot be 1 or 2 percent. A 2 percent success rate is not a success, it's a biological accident. Where's the other 98 percent? Show me them."
"Unfortunately", she said, "in monkeys, that's all we have to show."
Also see our overview, Cloning Animals, by Kathy Archibald - Animal Aid's Scientific Researcher.