Roslin Institute and Dolly the Sheep

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Dolly the sheep, who became famous as the first mammal to be cloned from an adult cell, has died.
The news was confirmed on Friday by the Roslin Institute, the Scottish research centre which created her.

A decision was taken to "euthanize" six-year-old Dolly after a veterinary examination showed that she had a progressive lung disease, the institute said in a statement.  Dolly became the first mammal clone when she was born on 5 July 1996.  She was revealed to the public the following year.

Post-mortem
Dr Harry Griffin, from the institute, said: "Sheep can live to 11 or 12 years of age and lung infections are common in older sheep, particularly those housed inside.  "A full post-mortem is being conducted and we will report any significant findings"

Dolly was a sheep created totally by design - even her name was picked specifically to be appealing.  It came about during the latter stages of labour when Dolly was born.  Stockmen involved in the delivery thought of the fact that the cell used came from a mammary gland and arrived at Dolly Parton, the country and western singer.
Cloning row 

Her birth was only announced seven months later and was heralded as one of the most significant scientific breakthroughs of the decade.  But it also prompted a long-running argument over the ethics of cloning, reaching further levels with the latest allegations of human cloning.


Dolly, a Finn Dorset, bred normally on two occasions with a Welsh mountain ram called David.  She first gave birth to Bonnie in April 1998 and then to three more lambs in 1999.  But in January last year her condition caused concern when she was diagnosed with a form of arthritis.




Museum piece
The condition would usually be expected in older animals and another debate erupted over what could properly be judged as Dolly's true age, and the risks of premature ageing in clones.

Professor Ian Wilmut, who led the team that created her, said at the time that the arthritis showed their cloning techniques were "inefficient" and needed more work.  Dr Patrick Dixon, a writer on the ethics of human cloning, said the nature of Dolly's death would have a huge impact on possibility of producing a cloned human baby.

He said: "The real issue is what Dolly died from, and whether it was linked to premature ageing, she was not old - by sheep standards - to have been put down."

'Profound effects'

Speaking to BBC News 24 on Friday, Prof Wilmut said Dolly's birth should be the important issue.
"The fact that we were able to produce an animal from the cell of another adult - it had profound effects on biological research and in medicine." 

Professor Richard Gardner, chair of the Royal Society working group on stem cell research and therapeutic cloning, said: "We must await the results of the post-mortem on Dolly in order to assess whether her relatively premature death was in any way connected with the fact that she was a clone.  "If there is a link, it will provide further evidence of the dangers inherent in reproductive cloning and the irresponsibility of anybody who is trying to extend such work to humans."

Dolly has been promised to the National Museum of Scotland and will be put on display in Edinburgh in due course.

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I've decided to make cloning one of the first topics in this first issue of Science Explained because the folks who created Dolly are acquaintances of mine. (Yes, I am a name dropper, aren't I?) A few kilometres from my home lives Dolly, the world's first mammalian clone; not counting identical twins. (They're clones too.)

What makes Dolly different from identical twins is that she was grown from a cell taken from an ADULT animal! Many bright, well-respected scientists said it couldn't be done. Dr Ian Wilmut, who is in charge of the lab that created Dolly, admits that he had his doubts. However the hard work and imaginative thinking of his staff made it all possible.

How did they do it and what did they do?
First some background to teach you the basics of developmental biology.

An oocyte (pronounced "oh-oh-sight") is an unfertilized egg and it has no chance of developing into an animal unless it's fertilized. A recently fertilized egg is called a zygote (pronounced "zye-goat"). Funny how the last two letters in the alphabet describe the first stage of an individual animal. For example, a frog zygote normally divides and grows into a complete animal, a tadpole. Later that tadpole will develop into an adult frog.

A cell from a frog's gut should always remain a frog-gut-cell because it has "differentiated". Differentiation is the natural process whereby cells specialize into a certain kind of cell. As a frog embryo grows and develops its cells differentiate into nerve cells, blood cells, fat cells and many other different kinds of cells. That's what differentiation is all about. Differentiation is important because without it an animal could never be anything but a blob of unspecialized cells. As a mass of embryo cells divide and differentiate they "create" the animal. This incredible process of differentiation turns zygotes into animals and it's all controlled by the genes. Although the exact process is still poorly understood, scientists agree that differentiation must have something to do with changes in the nucleus of cells. You may recall that the nucleus is the part of the cell containing the genetic material (the DNA all coiled up in organized structures called chromosomes).

What do frog cells have to do with Dolly?

Like most scientific "breakthroughs" the earlier work done by others provided the foundation on which to try something new.

Decades ago a fellow named Gurdon developed the method of "nuclear transfer". (To learn more about the history of nuclear transfer read what Gurdon himself has to say.) This is a two step process.

First he used delicate needles and a good microscope to suck out the nucleus from a frog oocyte, producing an "enucleated oocyte". (That's an oocyte without a nucleus.) With the genetic material removed the enucleated oocyte would not divide or differentiate even when fertilized. That was no surprise. (A cell is nothing without its nucleus.)
But the results from Gurdon's second step shocked a lot of people! He used the same equipment and skill to transfer the nucleus from a frog's gut cell into an enucleated oocyte. That's nuclear transfer, the transfer of a nucleus from one cell to another, creating a "new cell" with a different nucleus. Many of these new cells which Gurdon created behaved like a zygote. They divided and divided and divided just like a normal developing embryo, producing a ball of cells. And this ball of cells differentiated! Nerve cells, skin cells, blood cells appeared just as they would in a normal embryo. After the normal length of time Gurdon had tadpoles! Because the tadpoles had all come from the gut cells of the same adult, they all had the same genetic material. So they were all clones, identical twins of each other. But unlike normal identical twins they were made from differentiated cells.
Gurdon had proven something that many scientists had argued about. He proved that differentiation was REVERSIBLE. Gurdon's method of nuclear transfer turned back the hands of time, in a developmental sense. Gurdon's method of nuclear transfer made clones from adult cells!

Naturally this got a lot of scientists thinking about cloning. But there were two problems.

First, Gurdon's nuclear transferred tadpoles never grew into frogs! Other folks repeated his experiments and got similar results. Nuclear transfer couldn't clone frogs from frog cells; all you got were tadpoles. No one knew why. Even today, no one knows why the tadpoles made by nuclear transfer die instead of growing into frogs. Weird.

The second problem was that Gurdon's method seemed to work only with frogs (or perhaps I should say "tadpoles"). When scientists tried nuclear transfer with mice, cattle or indeed any mammal, they got nowhere. The "new cells" sometimes divided a few times, but not for long and none of them differentiated properly. You just couldn't clone mammals. By the early 1980's most scientists accepted the idea that something very special allows frogs to be (partially) cloned (into tadpoles). Whatever that process was, it was not found in mammals. The textbooks made it very clear. Differentiation was (sort of) reversible in frogs but not in mammals.
Bummer.

So what exactly did the scientists at the Roslin Institute do?
Well, Keith Campbell, a fellow working for Dr Wilmut, thought that maybe the cell cycle had something to do with this cloning trouble.

The cell cycle is often described as a circle of cell life and division, but I think that can be a bit confusing for some people, so let's try to remember that by "cycle" we mean it happens again and again.

A cell divides into two "daughter cells" and both of these cells live, "eat", grow, copy their genetic material and divide again producing two more daughter cells. Because each daughter cell has a copy of the same genes in its nucleus, daughter cells are "clones" of each other just like identical twins. This "twining" goes on and on with each cell cycle. This is a natural process.

The cell cycle fascinates biologists. Very fast cell cycles occur during development causing a single cell to make many copies of itself as it grows and differentiates into an embryo. Some very fast cell cycles also occur in adult animals. Hair, skin and gut cells have very fast cell cycles to replace cells that naturally die. And cancer is a disease caused by cells cycling out of control. It's no wonder that biologists think the cell cycle is so important.



But there is a kind of "parking spot" in the cell cycle called "quiescence" (pronounced "kwee-S-ence"). A quiescent ("kwee-S-cent") cell has left the cell cycle, it has stopped dividing. Quiescent cells might reenter the cell cycle at some later time, or they might not. It depends on the type of cell. Most nerve cells stay quiescent forever. On the other hand, some quiescent cells may later reenter the cell cycle in order to make more cells. (For example, when a young girl starts to develop breasts.)

Many biologists (including myself) thought that to make a clone you should transfer the nucleus from a fast dividing cell. It made sense because fast cycling cells are exactly what makes an embryo grow. Besides, the gut cells used to make the tadpole clones were fast cycling cells. Many biologists tried to make clones by transferring the nucleus from fast dividing cells but all of those experiments were unsuccessful. (I tried injecting the fast growing cells from chicken feathers into hen eggs in the hope of cloning birds, but it didn't work.)

Keith (Dr Campbell) thought about it in a different way. He wondered if a quiescent nucleus would be a better donor. True, it was not cycling (that's what makes it quiescent, by definition) but Keith thought maybe that's what the nucleus needs for it to be successfully transferred. Maybe the cell needs time to "rest" before starting to make a whole new animal. Maybe the nucleus needs time, lots of time, to get its DNA in order. Maybe...?
Maybe quiescent cells would work!

So they tried it with cells from sheep.

The folks at the Roslin Institute do a lot of work with sheep as part of their partnership with a company called Pharmaceutical Proteins Limited Therapeutics (PPL Therapeutics). Earlier they had made transgenic sheep (sheep with human genes transferred into them, but that's another story).

They used cells from an adult sheep's mammary (breast) glands for the "donor" nucleus. They grew the cells in tissue culture, an artificial situation that is commonly used in laboratories to grow large numbers of cells in bottles. Tissue culture allows scientists to fiddle with the cells and alter their characteristics. That is exactly what Dr Campbell did. He "starved" the cells of important nutrients and the cells stopped growing and dividing. They became quiescent. (Keith knew they would become quiescent when starved of nutrients because other researchers had proven that years ago; but few folks really cared because who needs quiescent cells?)

And then he made Dolly?
Yes, but creating Dolly was not easy.  Using techniques similar to those used 20 years ago by Gurdon, Bill Ritchie (a technician working with Dr Campbell) removed the nucleus from an oocyte that was collected from a Scottish Blackface ewe.  (Ewes are female sheep. The Scottish Blackface breed is a common breed of sheep in Scotland easily identified by its black face.)


Oocytes have a "shell" of proteins and fibers (called the zona pellucida) and it is through this protective coat that Bill injected the nucleus from a quiescent mammary cell into the enucleated oocyte. That cell nucleus was from a different breed of sheep called a Finn Dorset, which happens to be a pure white breed of sheep. He then used a tiny pulse of electricity to cause the new nucleus to fuse with the enucleated oocyte's cytoplasm. (Cytoplasm is the solution inside the cell.) This electricity also helps "kick start" cells into "activity" so they are more likely to divide. This new, fused cell (containing the Finn Dorset mammary cell nucleus in the cytoplasm of a previously enucleated Blackface oocyte) was transferred into the reproductive "chamber" of a Blackface ewe (the same breed that provided the oocyte).

Bill and his fellow researchers than repeated this process 276 times! That's right, 276 times.  I told you this wasn't easy.

After 148 days, a normal length of time for the Finn Dorset breed of sheep, Dolly was born.



As you can see she is a healthy, normal looking Finn Dorset. (Dolly's the wee one on the left) born to a Blackface ewe (her mom's on the right). This proves that Dolly wasn't the product of a sneaky mating; Dolly's Blackface mom could not produce a white faced sheep no matter who was the father. (It has to do with the genetics of sheep breeds.) But just to be sure, the scientists DNA "fingerprinted" Dolly and her "mom" and proved that Dolly's DNA matched the cells from the tissue culture, not the cells from the ewe that gave birth to her.

Dolly is a normal (Finn Dorset) sheep. Contrary to the reports in some of the trash newspapers, she has not eaten her keeper or her fellow sheep. She does not shoot laser beams out of her eyes or talk. Dolly is a friendly, normal, healthy sheep who enjoys being petted, especially if you have some food in your hand!

This amazing research was published in Nature and you can read all the technical details there. You may be surprised to learn that clones had been made at the Roslin Institute before, but those clones were made from the nucleus of embryo cells not adult cells.

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• Which type?

• Technology

• Progress AD (After Dolly)

• Limitations of nuclear transfer

• Method of Nuclear Transfer in Livestock

• Applications

• Ethics

Much confusion happens when people see the word "clone" used. Depending on the age of the dictionary, the definition of biological cloning can be:

• A group of genetically identical individuals descended from the same parent by asexual reproduction. Many plants show this by producing suckers, tubers or bulbs to colonise the area around the parent.

• A group of genetically identical cells produced by mitotic division from an original cell. This is where the cell creates anew set of chromosomes and splits into two daughter cells. This is how replacement cells are produced in your body when the old ones wear out.

• A group of DNA molecules produced from an original length of DNA sequences produced by a bacterium or a virus using molecular biology techniques. This is what is often called molecular cloning or DNA cloning

• The production of genetically identical animals by 'embryo splitting'. This can occur naturally at the two cell stage to give identical twins. In cattle, when individual cells from 4- and 8-cell embryos and implanted in different foster mothers, they can develop normally into calves and this technique has been used routinely within cattle breeding schemes for over 10 years.

• The creation of one or more genetically identical animals by transferring the nucleus of a body cell into an egg from which the nucleus has been removed. This is also known as Nuclear Transfer (NT) or cell nuclear replacement (CNR) and is how Dolly was produced.

Technology
Nuclear transfer involves transferring the nucleus from a diploid cell ( containing 30-40,000 genes and a full set of paired chromosomes) to an unfertilised egg cell from which the maternal nucleus has been removed. The technique involves several steps (see diagram below). The nucleus itself can be transferred or the intact cell can be injected into the oocyte. In the latter case, the oocyte and donor cell are normally fused and the 'reconstructed embryo' activated by a short electrical pulse. In sheep, the embryos are then cultured for 5-6 days and those that appear to be developing normally ( usually about 10%) are implanted into foster mothers.

Nuclear transfer is not a new technique. It was first used in 1952 to study early development in frogs and in the 1980's the technique was used to clone cattle and sheep using cells taken directly from early embryos. In 1995, Ian Wilmut, Keith Campbell and colleagues created live lambs- Megan and Morag - from embryo derived cells that had been cultured in the laboratory for several weeks. This was the first time live animals had been derived from cultured cells and their success opened up the possibility of introducing much more precise genetic modifications into farm animals.

In 1996, Roslin Institute and collaborators PPL Therapeutics created Dolly, the first animal cloned from a cell taken from an adult animal. The announcement of her birth in February 1997 started the current fascination in all things cloned. Until then, almost all biologists thought that the cells in our bodies were fixed in their roles: the creation of Dolly from a mammary gland cell of a six year old sheep showed this was not the case and the achievement was voted Science Breakthrough of the Year at the end of 1997.

progress AD (After Dolly)
At first Dolly was a 'clone alone' but in August 1998, a group in Hawaii published a report of the cloning of over 50 mice by nuclear transfer. Since then, research groups around the world have reported the cloning of cattle, sheep, mice, goats and pigs. Equally competent groups have had no success in cloning rabbits, rats, monkeys, cats or dogs.

There are differences in early development between species that might influence success rate. In sheep and humans, the embryo divides to between the 8- and 16- cell stage before nuclear genes take control of development, but in mice this transition occurs at the 2 cell stage. In 1998, a Korean group claimed that they had cloned a human embryo by nuclear transfer but their experiment was terminated at the 4-cell stage and so they had no evidence of successful reprogramming.

Success rates remain low in all species, with published data showing that on average only about 1% of 'reconstructed embryos' leading to live births. With unsuccessful attempts at cloning unlikely to be published, the actual success rate will be substantially lower. Many cloned offspring die late in pregnancy or soon after birth, often through respiratory or cardiovascular dysfunction. Abnormal development of the placenta is common and this is probably the major cause of foetal loss earlier in pregnancy. Many of the cloned cattle and sheep that are born are much larger than normal and apparently normal clones may have some unrecognised abnormalities.
The high incidence of abnormalities is not surprising. Normal development of an embryo is dependent on the methylation state of the DNA contributed by the sperm and egg. and on the appropriate reconfiguration of the chromatin structure after fertilisation. Somatic cells have very different chromatin structure to sperm and 'reprogramming' of the transferred nuclei must occur within a few hours of activation of reconstructed embryos. Incomplete or inappropriate reprogramming will lead to dysregulation of gene expression and failure of the embryo or foetus to develop normally or to non-fatal developmental abnormalities in those that survive.
Improving success rates is not going to be easy. At present, the only way to assess the 'quality' of embryos is to look at them under the microscope and it is clear that the large majority of embryos that are classified as 'normal' do not develop properly after they have been implanted. A substantial effort is now being made to identify systematic ways of improving reprogramming. One focus is on known mechanisms involved in early development, and in particular on the 'imprinting' of genes. Another is to use technological advances in genomics to screen the expression patterns of tens of thousands of genes to identify differences between the development of 'reconstructed embryos' and those produced by in vivo or in vitro fertilisation.


Limitations of nuclear transfer
It is important to recognise the limitations of nuclear transfer. Plans to clone extinct species have attracted a lot of publicity. One Australian project aims to resurrect the 'Tasmanian tiger' by cloning from a specimen that had been preserved in a bottle of alcohol for 153 years and another research group announced plans to clone a mammoth from 20,000 year old tissue found in the Siberian permafrost. However, the DNA in such samples is hopelessly fragmented and there is no chance of reconstructing a complete genome. In any case, nuclear transfer requires an intact nucleus, with functioning chromosomes. DNA on its own is not enough: many forget that Jurrasic Park was a work of fiction.

Other obvious requirements for cloning are an appropriate supply of oocytes and surrogate mothers to carry the cloned embryos to term. Cloning of endangered breeds will be possible by using eggs and surrogates from more common breeds of the same species. It may be possible to clone using a closely related species but the chance of successfully carrying a pregnancy to term would be increasingly unlikely if eggs and surrogate mothers are from more distantly related species. Proposals to 'save' the Panda by cloning, for example, would seem to have little or no chance of success because it has no close relatives to supply eggs or carry the cloned embryos.
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Method of Nuclear Transfer in Livestock

Applications
Nuclear transfer can viewed in two ways: as a means to create identical copies of animals or as a means of converting cells in culture to live animals. the former has applications in livestock production, the latter provides for the first time an ability to introduce precise genetic modifications into farm animal species.

Cloning in Farm Animal production
Nuclear transfer can in principle be used to create an infinite number of clones of the very best farm animals. In practice, cloning would be limited to cattle and pigs because it is only in these species that the benefits might justify the costs. Cloned elite cows have already been sold at auction for over $40,000 each in the US but these prices reflect their novelty value rather than their economic worth. To be effective, cloning would have to be integrated systematically into breeding programmes and care would be needed to preserve genetic diversity. It would also remains to be shown that clones do consistently deliver the expected commercial performance and are healthy and that the technology can be applied without compromising animal welfare.

Production of Human therapeutic proteins
Human proteins are in great demand for the treatment of a variety of diseases. Whereas some can be purified from blood, this is expensive and runs the risk of contamination by AIDS or hepatitis C. Proteins can be produced in human cell culture but costs are very high and output small. Much larger quantities can be produced in bacteria or yeast but the proteins produced can be difficult to purify and they lack the appropriate post-translational modifications that are needed for efficacy in vivo.

By contrast, human proteins that have appropriate post-translational modifications can be produced in the milk of transgenic sheep, goats and cattle. Output can be as high as 40 g per litre of milk and costs are relatively low. PPL Therapeutics, one of the leaders in this field and their lead product, alpha-1-antitrypsin, is due to enter phase 3 clinical trials for treatment of cystic fibrosis and emphysema in 2001.. Nuclear transfer allows human genes to be inserted at specific points in the genome, improving the reliability of their expression and allows genes to be deleted or substitutes as well as added.

Xenotransplantation
The chronic shortage of organs means that only a fraction of patients who could benefit actually receive transplants. Genetically modified pigs are being develop as an alternative source of organs by a number of companies, though so far the modifications have been limited to adding genes. Nuclear transfer will allow genes to be deleted from pigs and much attention is being directed to eliminating the alpha-galactosyl transferase gene. This codes for an enzyme that creates carbohydrate groups which are attached to pig tissues and which would be largely responsible for the immediate rejection of an organ from a normal pig by a human patient.

Cell Based Therapies
Cell transplants are being developed for a wide variety of common diseases, including Parkinson's Diseases, heart attack, stroke and diabetes. Transplanted cells are as likely to be rejected as organs but this problem could be avoided if the type of cells needed could be derived from the patients themselves. The cloning of adult animals from a variety of cell types shows that the egg and early embryo have the capability of 'reprogramming' even fully differentiated cells. Understanding more about the mechanisms involved may allow us to find alternative approaches to 'reprogramming' a patient's own cells without creating ( and destroying ) human embryos.


Ethics
Many ethical and moral concerns have arisen over the potential applications of the cloning technology. The technology is still in its infancy and in the meantime, society as a whole has time to contemplate which uses of the technology might be acceptable and which would not. The suddenness of the news of the cloning of the first adult animal caught almost all commentators by surprise and some suggested that we should have fully discussed the implications of our work before we started. The public may see science as a series of 'breakthroughs' but in reality progress is much more continuous. Where in the sequence of events that led to Dolly should we have consulted and with whom? It is also impossible to predict all potential applications of a new technology. Most will be beneficial but all technology can be misused in one way or another. The solution is not to regulate the technology itself but how it is applied.

Those concerned that scientists were "playing at God" seemed to ignore how much mankind has altered the cards that we were originally dealt. Animals were first domesticated about 5000 years ago and selective breeding since has produced modern strains of livestock, plants and pets which are very different from their original progenitors. In medicine, our current life expectancy of well over 70 years is a result of direct intervention in nature, from improved prenatal care, vaccination and use of antibiotics. The human condition is still far from perfect and there is no particular reason now to call a general halt to what most people view as progress.

Roslin believes it has a clear social responsibility to keep the public informed of the results of its research and is a very active participant in the ongoing public debates about cloning, animal experimentation, genetic modification and human stem cell research.