Research in the News: Creating a Cloned Sheep Named Dolly
Deborah Barnes, Ph.D.
Who would have thought that a Scottish sheep named Dolly could cause an international uproar? But she has, and it's all because of her unorthodox beginnings. Dolly is not an ordinary sheep, produced through the mating of a ewe and a ram. She is a clone, an exact genetic replica of her donor "mother," a six-year-old female sheep. And that fact, together with another recent announcement that scientists in Oregon have cloned monkeys from embryonic cells, has triggered debates among researchers, ethicists, and politicians all over the world.
Cover of the Nature journal issue announcing the birth of Dolly
The ethical controversy over any future experiments that involve the cloning of human beings -- which has NOT occurred -- is likely to dominate news coverage of the subject for months to come. Before engaging in such a debate, it is important to understand what the process of cloning involves, and why Dolly is so special. It is also important to understand some of the key questions that Dolly raises.
What is a Clone
The noun "clone" and the verb "to clone" are not used consistently. In biology, a clone is a cell or an organism that is genetically identical to another cell or organism. Many simple organisms such as bacteria reproduce themselves by copying their DNA and splitting in half. The two bacteria that result from this form of asexual reproduction are genetically identical; they are clones of each other. In contrast, during the process of sexual reproduction, the nucleus of a sperm cell, which carries the father's DNA, fuses with the nucleus of the egg cell, which contains the mother's DNA. The resulting offspring carry genetic material from both parents and are not identical to either parent.
The verb "to clone" refers to the process of creating cloned cells or organisms. The process differs, depending on the kinds of cells used in the cloning procedure and the desired result. Usually, when scientists clone an animal, they take the nucleus of a cell -- which contains chromosomes made of deoxyribonucleic acid (DNA) and proteins -- and place it into an egg cell (also called an oocyte) from which the nucleus has been removed. The egg cell then divides to produce an embryo that develops into an animal, if the procedures work as planned.
Diagram of the laboratory procedures used to create cloned Rhesus monkeys. Early embryos called blastocysts were created using standard in vitro fertilization techniques (collecting eggs and sperm from adult monkeys [top right] and allowing fertilization to occur in plastic laboratory dishes). After the fertilized eggs divided several times to form blastocysts, they were separated into individual embryonic cells. During a process called nuclear transfer, the nucleus of each embryonic cell was fused (using an electrical field) with an oocyte from a different monkey after the nucleus of the egg cell had been removed (left). The fused cell developed in a culture dish to form a new blastocyst and was transferred to a surrogate mother monkey, which gave birth. The two Rhesus monkeys produced by this method were clones of the embryonic cells (produced from fertilized eggs); they were not clones of either parent monkey.
In previous cloning experiments with cattle and mice, the "donor" chromosomes came from very early embryos or, in the case of cloned frogs, from young tadpoles. Very early embryos from mammals are called blastocysts, balls of immature cells that are produced after a fertilized egg divides several times, but before any specialized tissues begin to develop. Recently, two Rhesus monkeys were produced from embryonic cells in blastocysts by a method called nuclear transfer.
How was Dolly Created?
Dolly is different. She was generated from a specialized adult cell, not from an unspecialized embryonic cell.
To create Dolly, Ian Wilmut of the Roslin Institute in Edinburgh, Scotland, and his colleagues used a cell derived from the udder of a six-year-old sheep in the final stage of pregnancy. The researchers fused the adult udder cell with an oocyte that was ready to be fertilized, but taken from a different sheep. The scientists had previously removed the nucleus from the oocyte, and they used an electrical current to fuse it with the udder cell. The key to Dolly's success was to make the nucleus of the donor cell "quiet" so that it stopped behaving like an udder cell and could be reprogrammed to become an embryo.
Diagram of the laboratory procedures used to create Dolly. Donor udder cells were removed from an adult sheep (top right), grown in laboratory culture dishes, and arrested in the G0 phase of the cell cycle. A single udder cell (arrested in G0) was fused with an oocyte from a different sheep after the nucleus of the egg cell had been removed (left). The fused cell developed in a culture dish to form a blastocyst and was then transferred to a surrogate mother sheep, which gave birth. Of 277 fused cells that were prepared in this way, only one survived to become the cloned sheep named Dolly.
The resulting embryo -- which became Dolly -- carried all of the chromosomes from the donor udder cell and none of the nuclear chromosomes from the host egg cell. Therefore, Dolly is an exact genetic copy -- a clone -- of her donor-cell "mother." Wilmut and his colleagues published their findings in a scientific paper in the 27 February, 1997, issue of Nature.
Why did researchers create Dolly?
Wilmut and his collaborators created Dolly because they are trying to find ways to produce livestock that carry specific genetic traits. Two biotechnology companies funded the research. One, called PPL Therapeutics, is trying to find ways to produce animals that carry certain proteins in their milk. The theory is that if researchers can develop animals with desirable characteristics, they can then clone those animals to produce entire herds that carry the same traits.
Why is Dolly so special?
While some people worry about cloning humans, most scientists celebrate Dolly. Her very existence contradicts a long-standing idea about embryonic development, and scientists enjoy research results that turn an old theory upside down. However, it is important to remember that the researchers in Scotland tried 277 times to create cloned sheep, and they succeeded only once. They are still trying to improve the techniques used to create Dolly.
Dolly is special because she disproves the notion that cells from an adult animal are too specialized to generate a new organism. Researchers have known for a long time that during embryonic development, cells become specialized because some of their genes are turned off (inactivation) and others are turned on (activation). Before Dolly, scientists thought that many genes in adult cells are permanently turned off. They believed that only the genes in the fertilized egg, embryo, or very young animal are totipotent -- all-powerful, fully activated, and capable of generating a new organism. But Dolly, it seems, proves the old theory wrong.
"Dolly is remarkable because she was cloned from an adult, somatic cell," says Alan Wolffe of the laboratory of molecular embryology at the National Institute of Child Health and Human Development in Bethesda. A somatic cell is a cell of the body that carries the full number of chromosomes for that species -- 54 chromosomes for sheep and 46 for humans. Somatic cells such as muscle cells, nerve cells, and skin cells are usually specialized to perform certain functions.
Despite the enthusiasm about Dolly, not all scientists are convinced that she was generated from an adult cell that was fully specialized or differentiated. Some researchers think that Dolly may have resulted from an udder cell that retained some of the characteristics of embryonic cells. The skeptics point out that some of the cells in the udder of a pregnant sheep (such as Dolly's genetic "mother") are not fully differentiated. This is because the udder tissue in a sheep or cow is mammary tissue -- similar to that in the breast of a human. During pregnancy, cells in mammary tissue make the glands and ducts that produce and carry milk. So, some scientists reason, certain cells in the mammary tissue of a pregnant female sheep such as Dolly's donor "mother" are capable of developing into different kinds of cells -- possibly making it easier to reprogram them to an embryo-like state.
Despite some lingering questions about the properties of the cell used to generate Dolly, she remains an international phenomenon. Dolly proves that it is possible to clone an apparently healthy animal from the nucleus of an adult cell.
Why was Dolly a success?
The key to Dolly's success was to make the nucleus of the donor udder cell behave like the nucleus of a normal, fertilized egg that could develop into a baby sheep. The process involved several key steps that involved the preparation of the donor udder cell and the preparation of the host oocyte.
To prepare the donor udder cell, Wilmut and his colleagues removed cells from the udder of a pregnant sheep, and grew them in plastic culture dishes in an incubator. Then, after the cells had divided several times, the researchers changed the growth medium -- the nutrient-rich fluid used to grow cells in culture. Instead of containing 10 percent blood serum, the new growth medium for the udder cells contained only 0.5 percent serum, which literally "starved" the cells of nutrients. And, in the case of generating Dolly, serum-starvation blocked the udder cells from dividing further, a phenomenon called cell-cycle arrest.
To understand how Dolly was created, it is necessary to understand the normal cell cycle. The cell cycle is a series of molecular events that allows a cell to reproduce itself. In normally dividing cells, the cell cycle progresses through four phases -- mitosis or cell division (M), a cell-growth phase called "gap" 1 (G1), a period of DNA synthesis in which the DNA replicates itself (S), and a second "gap" phase (G2) in which the cell, which now carries twice its normal amount of genetic material, prepares to divide.
But serum-starved cells depart from the cell cycle and arrest in a phase called G0. The nucleus of the udder cell used to generate Dolly was arrested in G0, which allowed it to be reprogrammed to form an embryo.
Meanwhile, the Scottish researchers also had to prepare the host oocyte, which they obtained from a different sheep. They collected oocytes that were ready to be fertilized, meaning that the nuclei of the oocytes carried only half the normal number of chromosomes. The researchers removed the nucleus from each egg cell and used a mild electrical current to fuse it with a nucleus from an udder cell. At that point, a series of events occurred that is still not well understood.
Researchers know that the cytoplasm of an oocyte contains proteins that encourage embryonic development. They do not know what all of those proteins are. In the case of Dolly, some of the proteins in the egg cell cytoplasm reprogrammed the nucleus of the donor udder cell so that it could divide to form an embryo. This series of events involved stripping away proteins that are normally bound to the DNA of adult cells and replacing them with proteins that are normally bound to the DNA of embryonic cells. (Different kinds of proteins are bound to DNA; a major group is called histones.)
After the DNA-linked proteins were removed and replaced, other proteins in the oocyte cytoplasm began to turn on genes in the udder cell nucleus. These events normally occur after an egg cell is fertilized by a sperm cell. At this point in the case of Dolly, the nucleus of the udder cell began to behave like the nucleus of a fertilized egg. The gene-activating proteins are called transcription factors, because they allow the DNA that makes up the genes to be transcribed into a related molecule called messenger RNA (ribonucleic acid). The conversion -- or transcription -- of DNA into messenger RNA is the first step toward making the new proteins that a growing embryo (or any other kind of cell) requires.
Once the donor, udder-cell nucleus and the host, egg-cell cytoplasm interacted successfully, the embryo that became Dolly began to develop normally. After it divided several times and became a tiny ball of cells called a blastocyst, the researchers placed it into the uterus of an adult female sheep -- Dolly's surrogate mother. The sheep carried Dolly through a normal pregnancy and gave birth to the lamb that has caused so much controversy. When Wilmut and his collaborators analyzed the DNA of Dolly's cells, they found that it was identical to the DNA of the donor udder cell. That is why Dolly is a clone.
Making the Monkeys
In February 1997, researchers in Oregon produced two Rhesus monkeys using laboratory techniques that had previously worked with frogs, cattle, and mice. They collected eggs from and sperm from adult monkeys and allowed fertilization to occur in plastic laboratory dishes, a standard in vitro fertilization (IVF) technique. After the fertilized eggs had divided several times to produce very early embryos called blastocysts -- tiny balls of only 6 to 12 cells -- the researchers transferred one of these cells into an "enucleated" oocyte. They allowed the oocyte to divide several times and then placed it into the uterus of a surrogate mother, where embryonic development occurred.
This was the first time that researchers had been able to use a nuclear transfer procedure -- taking the nucleus from an embryonic cell and fusing it with an enucleated oocyte -- to generate monkeys. Everyone paid attention to the research because monkeys are so closely related to humans, at least in evolutionary terms. People reasoned that if scientists could clone monkeys, they might also be able to clone humans.
Don Wolf, of the Oregon Regional Primate Center in Beaverton, headed the research team that generated the monkeys. The most important difference between the monkeys and Dolly is that unspecialized embryonic cells were used to create the monkeys, whereas a specialized adult cell was used to create Dolly.
To generate the monkeys, the Oregon researchers used standard in vitro fertilization techniques. They collected sperm and eggs from adult monkeys and allowed fertilization to take place in plastic laboratory dishes, called an "in vitro" environment. The eggs that became fertilized began to divide until a blastocyst or ball of 6 to 12 cells resulted. The researchers removed the coating (zona pellucida) that surrounds the blastocyst and separated the cells. Each cell carries the same genetic material -- because each developed from the same fertilized oocyte. These embryonic cells became the potential "donor" cells for the cloning procedure.
Meanwhile, the researchers injected female monkeys with hormones to make them ovulate. They recovered several mature oocytes from the monkeys and removed the nucleus from each egg cell using a tiny glass needle. The process is called enucleation. The enucleated oocytes served as the recipient or "host" cells for the cloning procedure.
To create the monkey clones, the Oregon researchers used a mild electric current that caused a single donor nucleus (from an embryonic cell) to fuse with the host oocyte (an enucleated egg). This process is called nuclear transfer, and it generates an egg cell that behaves as if it has been fertilized by a sperm cell. The artificially "fertilized" eggs divided by mitosis and became blastocysts in plastic culture dishes that contain a nutrient-rich liquid. The dishes and blastocysts were kept warm in an incubator.
In the final step, the researchers transferred two or three blastocysts into the uterus of a surrogate mother monkey. If the procedure works as planned, the surrogate mother carries at least one of the dividing embryos through a normal pregnancy and gives birth to a healthy infant Rhesus monkey.
Most embryos produced by nuclear transfer do not survive these procedures. In the Oregon experiment, only two monkeys survived. They were generated from different donor blastocysts and so are not clones of each other. Instead each monkey is a clone of the original blastocyst that had developed from a fertilized egg.
More Ideas About Clones...And Differentiation
In many ways, the cells of multicellular organisms -- daffodils or maple trees, frogs, sheep, or humans -- are genetic clones because they all come from the same fertilized egg cell. During embryonic development, all of the cells of these organisms are produced by mitosis (cell division) from the fertilized oocyte. Each time mitosis occurs, a cell copies its DNA and then splits in half. The two daughter cells that result carry the same number and kind of chromosomes and genes, which are made of the same DNA as the oocyte. So in terms of their genetic content, all the cells of a multicellular organism -- except the egg cells and sperm cells -- are clones. But even though the cells of a multicellular organism contain identical genetic material, they do not look or act the same. An adult multicellular plant or animal is not simply a massive ball of identical cells.
This is because a complex process occurs during development that causes the cells of an embryo to assume different shapes and functions. During the process called differentiation, cells begin to acquire unique molecular and structural characteristics that allow them to perform specific functions. In mammals, for instance, red blood cells carry oxygen; skeletal muscle cells contract and move the body; and nerve cells conduct electrical impulses that signal other cells to respond. One of the greatest challenges in developmental biology is to understand how a single fertilized egg becomes a complex, differentiated multicellular organism.
What accounts for differentiation during embryonic development? How can cells that carry the same DNA, genes, and chromosomes look and function so differently? The simplest explanation is that, as an embryo grows, the genes in different cells become turned on or off in different patterns or combinations. One combination of active and inactive genes results in a skeletal muscle cell. A different combination of active and inactive genes results in a nerve cell. Researchers have learned much about the molecular processes that turn genes on and off during development and adulthood, although many questions remain unanswered.
Dolly, the cloned sheep, has changed the way that scientists think about a related question: Are the genes in an adult, differentiated cell permanently turned "on" or "off?" Put another way, is it possible to reprogram a differentiated, adult cell so that as it divides, it is capable of doing what a fertilized egg cell can do -- producing an entirely new organism? Dolly's existence suggests that differentiated cells from adult mammals can be reprogrammed to behave as if they were embryonic cells.
Researchers in Scotland created Dolly by fusing an adult, differentiated cell obtained from a sheep's udder with a fertilized egg cell from which they had removed the nucleus. The scientists, led by Wilmut of the Roslin Institute in Edinburgh, based their techniques on methods developed in the 1960s by Gurdon, Briggs, and King. Those researchers experimented with the African clawed frog, Xenopus laevis, and won a Nobel prize for their research. They used the nucleus from a tadpole's intestinal cell to generate an adult frog, but they were never able to use the nucleus from an adult cell to create an adult frog.
Wilmut and his colleagues have spent many years developing their experimental techniques, which are still far from perfect. But they have succeeded in doing what has never before been accomplished -- cloning an apparently healthy adult mammal from an adult differentiated cell.