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BRAVE NEW WORLD

TIMES MEDICAL WRITER

Last weekend, Scottish researchers shocked the biological world by announcing that they had successfully cloned an adult mammal. Dr. Ian Wilmut and his colleagues at the Roslin Institute near Edinburgh took DNA from an udder cell of an adult ewe, inserted it into a sheep egg and grew it to birth in a surrogate mother.

They now have a healthy 7-month-old ewe named Dolly that has captured the attention of the world.

Here are answers to some of the most commonly asked questions about this groundbreaking feat and cloning in general.

Q: What is cloning?

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A: Cloning is the production of an exact genetic duplicate of a living organism. In normal sexual reproduction, an egg and a sperm--each containing half the normal genetic complement of an adult--fuse, combining their DNA to produce the complete genetic blueprint of a third adult.

In cloning, all of the genetic material comes from one parent, and the offspring is genetically identical to that parent.

Q: Why was Wilmut’s success such a surprise?

A: Because virtually all previous attempts at cloning mammals had “failed miserably,” according to reproductive scientist Lee Silver of Princeton University. Scientists could readily clone plants, but they made little progress in animals.

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The best results previously were in amphibians such as frogs, where researchers could successfully implant adult DNA in eggs and achieve a viable embryo. But the cloned frogs always died in the tadpole stage, never making it to adulthood.

Q: Why did they fail?

A: The problem is that something happens to DNA as the embryo grows to adulthood and individual cells become specialized, turning into skin or brain or kidney cells, for instance.

Each cell contains all the genetic information necessary to make a complete adult organism. But as an embryonic cell slowly turns into a skin cell, for example, those genes that are not needed in a skin cell are shut off. In a human adult skin cell, 85% to 90% of the 100,000 or so genes in the human blueprint are inactivated.

If cloning is to be successful, those inactivated genes must be turned back on. Scientists had tried various methods of doing this but found that large segments of the DNA remained inactivated after it had been inserted into an egg. In effect, the egg was trying to build an adult using only 20% of a blueprint and, of course, it didn’t work.

Researchers didn’t know how genes were shut off during embryonic development, but they envisioned two mechanisms. During normal cellular activity, unneeded genes are often inactivated by proteins that bind to the DNA double helix structure. That kind of inactivation is reversible.

Alternatively, the DNA could be irreversibly inactivated during growth and maturation by chemically modifying it so that it could no longer be used as a blueprint for the whole organism. If this were the case, cloning would be impossible. The consistent failures at cloning had led researchers to think that DNA was being irreversibly altered.

“That was what we believed until last Saturday,” Silver said. “They proved the dogma wrong,” added reproductive biologist James Murray of UC Davis.

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One of the most important findings in Wilmut’s report is that the DNA inactivation is, in fact, apparently reversible, according to biologist Colin Stewart of the National Cancer Institute.

Q: What exactly did Wilmut do to overcome the problem?

A: Through trial and error, he discovered that stopping the growth of adult cells at a particular time allowed genes to become unblocked.

Wilmut’s team removed a few cells from the udder of a 6-year-old ewe and began growing them in a laboratory dish. After the cells had been growing for a few days, the team stopped supplying them with crucial nutrients, which halted the cells’ growth.

They then took each cell and, using an electric current, fused it with a sheep egg cell from which the DNA had been removed so as not to interfere with the cloning process. (Researchers already knew that it is better to use an unfertilized egg cell than one that had been fertilized.) The electric current also triggers the cloned egg to begin dividing and maturing.

The embryo was then implanted in the uterus of a surrogate mother, where it could grow to term in the same manner as a fertilized egg produced by in vitro fertilization. The team implanted a total of 273 fused eggs and got one lamb.

Wilmut was not following a grand scientific plan in his experiments, but merely varying conditions for the attempted cloning in a systematic manner. “He was just playing games, tinkering with things,” Silver said. “Fortunately, he tinkered with the right thing.”

Wilmut has performed the same feat using cells taken from early embryos and from late-stage embryos, rather than adult sheep, producing a total of seven sheep clones to date.

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Q: Why sheep?

A: Sheep have long been used for in vitro fertilization research because, for reasons that still elude biologists, the technique works best in them. “Sheep are easy,” is a common maxim among reproductive biologists. They are also cheap and are not as big and difficult to handle as many other mammals.

But there is nothing unique about sheep, Murray said. Many reproductive advances were achieved in mice and cattle, as well. Experts agree that Wilmut’s breakthrough could just as well have been achieved in another species. But the Roslin Institute is a center for studying sheep, sheep are extremely important to the Scottish economy and Wilmut’s team has been studying genetic engineering in sheep for 15 years.

Q: Why did so few embryos grow into lambs?

A: The team doesn’t know yet, but the low success rate is not surprising. In the early days of in vitro fertilization research, one success in several hundred attempts was considered acceptable. Even today, after years of study, the success rate in women over 40 is only about seven in 400. Reproduction remains much harder in the lab than in the bedroom, but researchers are confident the success rate will improve as their techniques are refined.

Q: Can other mammals, including humans, be cloned?

A: Almost certainly, experts agree. Such advances in reproductive technology are often readily transferred from one species to another, and there is nothing about the current research to suggest that it is any different. The technique is also relatively simple, and a large number of laboratories should be capable of doing it. In fact, scientists say it would be surprising if some researcher is not already trying it in cattle or primates.

Biologically, a human is just another mammal and there is nothing unique about humans that would make it more difficult to clone them.

Q: Why did they do it?

A: Although there are a broad variety of benefits from cloning, Wilmut and others want to use it for genetic engineering of animals to improve their productivity or yield drugs, among other things. That technique--which focuses on adding a small number of genes to an animal rather than reproducing an adult and has vast commercial implications--is still a scattershot process that is hard to control. DNA for one or more genes is injected into a fertilized egg, which is then implanted in a host. Only about 1% to 2% of the time does the researcher obtain the desired animal.

It is much easier to genetically engineer individual cells than whole animals, and researchers can make a broader variety of changes in cells. Theoretically, a genetically engineered cell could then be cloned to produce a desired animal.

Researchers are already working, for example, to produce human drugs in cows’ and goats’ milk. Others are attempting to make cows and goats that produce more milk. Some researchers are adding human genes to pigs so that the animals can be used as donors for human organ transplants. Genetic engineering might also be used to make leaner, more healthy animals.

That is why the research is sponsored primarily by industry, and not by government.

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Ewe 2

Years of failed experiments preceeded the feat of genetic engineering that created Dolly, the cloned sheep.

How mammals reproduce

Normal: An egg and a sperm each have only half a set of DNA. They must fuse and join their DNA to get the whole blueprint for an organism.

Cloning: A full set of DNA is obtained from one parent. That DNA is inserted into a sea of embryonic proteins in an egg from which the nucleus has been removed, setting the stage for growth.

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Why cloning hasn’t worked?

As a fertilized egg grows into an adult, unneeded DNA is systematically shut off. In an adult skin or brain cell, for example, 85% to 90% of DNA is inactivated. If that DNA is placed in an egg cell in an attempt to clone it, the proteins that inactivate the DNA remain in place, thus an embryo cannot grow.

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What the scientists did

1) Wilmut’s team removed cells from a ewe’s udder and starved them so they would stop growing--a process that unblocked the DNA.

2) They also removed the nucleus from an unfertilized egg from another sheep.

3) The udder cell and the egg were fused with an electric current.

4) The fused egg was placed in a third ewe, the surrogate mother, where it grew into an embryo just as if an egg had been fertilized in the normal manner.

Source: AP, Los Angeles Times


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