Cloning - Reproductive Cloning

cloned clone cell dolly

Another way to describe and classify cloning is by its purpose. Organismal or reproductive cloning is a technology used to produce a genetically identical organism—an animal that has the same nuclear DNA as an existing, or even an extinct, animal.

The reproductive cloning technology used to create animals is called somatic cell nuclear transfer (SCNT). In SCNT scientists transfer genetic material from the nucleus of a donor adult cell to an enucleated egg (an egg from which the nucleus has been removed). This FIGURE 8.5
Process used to clone Dolly
SOURCE: "The Process Used to Clone Dolly," in Genetics, vol. 1, A–D, Macmillan Reference USA, Gale Group, 2002
eliminates the need for fertilization of an egg by a sperm. The reconstructed egg containing the DNA from a donor cell is treated with chemicals or electric current in order to stimulate cell division. Once the cloned embryo reaches a suitable stage, it is transferred to the uterus of a surrogate (female host), where it continues to grow and develop until birth. Figure 8.5 shows the entire SCNT process that culminates in the transfer of the embryo into the surrogate mother.

Organisms or animals generated using SCNT are not perfect or identical clones of the donor organism or "parent" animal. Although the clone's nuclear DNA is identical to the donor's, some of the clone's genetic materials come from the mitochondria in the cytoplasm of the enucleated egg. Mitochondria, the organelles that serve as energy sources for the cell, contain their own short segments of DNA called mDNA. Acquired mutations in the mDNA contribute to differences between clones and their donors and are believed to influence the aging process.

Dolly the Sheep Paves the Way for Other
Cloned Animals

In 1952 scientists transferred a cell from a frog embryo into an unfertilized egg, which then developed into a tadpole. This process became the prototype for cloning. Ever since, scientists have been cloning animals. The first mammals were also cloned from embryonic cells in the 1980s. In 1997 cloning became headline news when, following more than 250 failed attempts, Ian Wilmut (1944–) and his colleagues at the Roslin Institute in Edinburgh, Scotland, successfully cloned a sheep, which they named Dolly. Dolly was the first mammal cloned from the cell of an adult animal, and since then researchers have used cells from adult animals and various modifications of nuclear transfer technology to clone a range of animals including sheep, goats, cows, mice, pigs, cats, rabbits, and the gaur named Noah.

To create Dolly, the Roslin Institute researchers transplanted a nucleus from a mammary gland cell of a Finn Dorsett sheep into the enucleated egg of a Scottish blackface ewe and used electricity to stimulate cell division. The newly formed cell divided and was placed in the uterus of a blackface ewe to gestate. Born several months later, Dolly was a true clone—genetically identical to the Finn Dorsett mammary cells and not to the blackface ewe, which served as her surrogate mother. Her birth revolutionized the world's understanding of molecular biology, ignited worldwide discussion about the morality of generating new life through cloning, prompted legislation in dozens of countries, and launched an ongoing political debate in the U.S. Congress. In fact, when Dolly was cloned, the event touched off widespread fears that the technology would soon be used to create cloned humans. A 1997 CNN/USA Today/Gallup Poll found that 87% of Americans polled believed human cloning would be a bad development for humanity, and 88% believed it would be morally wrong.

Dolly was the object of intense media and public fascination. She proved to be a basically healthy clone and produced six lambs of her own through normal sexual means. Before her death by lethal injection on February 14, 2003, Dolly had been suffering from lung cancer and arthritis. An autopsy (postmortem examination) of Dolly revealed that, other than her cancer and arthritis, she was anatomically like other sheep.

In February 1997 Don Wolf (1939–) and his colleagues at the Oregon Regional Primate Center in Beaverton successfully cloned two rhesus monkeys using laboratory techniques that had previously produced frogs, cows, and mice. It was the first time that researchers used a nuclear transplant to generate monkeys. The monkeys were created using different donor blastocysts (early-stage embryos), so they were not clones of one another—each monkey was a clone of the original blastocyst that had developed from a fertilized egg. Unfortunately, neither of the cloned monkeys survived past the embryonic stage.

An important distinction between the process that created Dolly and the one that produced the monkeys was that unspecialized embryonic cells were used to create the monkeys, whereas a specialized adult cell was used to create Dolly. The Oregon experiment was followed closely in the scientific and lay communities because, in terms of evolutionary biology and genetics, primates are closely related to humans. Researchers and the public speculated that if monkeys could be cloned, it might become feasible to clone humans.

In May 2001 BresaGen Limited, an Australian biotechnology firm, announced the birth of that country's first cloned pig. The pig was cloned from cells that had been frozen in liquid nitrogen for more than two years, and the company used technology it described as new and quite different from the process used to clone Dolly the sheep. The most immediate benefit of this technology is to improve livestock—cloning enables breeders to take a small number of animals with superior genetics and rapidly produce more. Biomedical scientists were especially attentive to this research because of its potential for xenotransplantation—the use of animal organs for transplantation into humans. Pig organs genetically modified so that they are not rejected by the human immune system could prove to be a boon to medical transplantation.

During the same year the first cat was cloned, and the following year rabbits were successfully cloned. In January 2003 researchers at Texas A & M University reported that cloned pigs behaved normally—as expected for a litter of pigs—but were not identical to the animals from which they were cloned in terms of food preferences, temperament, and how they spent their time. The investigators found that the cloned pigs' behavior was as variable as a control group (normally bred) of pigs in nearly every way. They played, ate, slept, fought, and responded to outside stimuli with the same range of behavior as the others. Even their physical characteristics were comparable to the control group in variation, and there was variation between the cloned pigs. The researchers explained the variation as arising from the environment and epigenetic (not involving DNA sequence change) factors, causing the DNA to line up differently in the clones. Epigenetic activity is defined as any gene-regulating action that does not involve changes to the DNA code and that persists through one or more generations, and it may explain why abnormalities such as fetal death occur more frequently in cloned species.

On May 4, 2003, a cloned mule—the first successful clone of any member of the horse family—was born in Hayden, Idaho. The clone was not just any mule, but the brother of the world's second-fastest racing mule. Named "Idaho Gem," the cloned mule was created by researchers at the University of Idaho and Utah State University. The researchers attributed their success to changes in the culture medium they used to nurture the eggs and embryos.

In August 2003 scientists at the Laboratory of Reproductive Technology in Cremona, Italy, were the first to clone a horse. The Italian scientists described the cloning techniques in the August 7, 2003, issue of the journal Nature (Cesare Galli et al., "Pregnancy: A Cloned Horse Born to Its Dam Twin," vol. 424, no. 6949, August 7, 2003).

While the mule was cloned from cells extracted from a mule fetus, the cloned horse's DNA came from her adult mother's skin cells. There were other differences as well. The University of Idaho researchers harvested fertile eggs from mares, removed the nucleus of each egg, and inserted DNA from cells of a mule fetus. The reconstructed eggs were then surgically implanted into the wombs of female horses. In contrast, the Italian scientists harvested hundreds of eggs from mare carcasses, cultured the eggs, removed their DNA, and replaced it with DNA taken from either adult male or female horse skin cells.

In May 2004 the first bull was cloned from a previously cloned bull in a process known as "serial somatic cell cloning" or "recloning." Prior to the bull, the only other successful recloning efforts involved mice. The successful research team, led by Dr. Xiangzhong (Jerry) Yang, director of the University of Connecticut's Center for Regenerative Biology, described their techniques in the May 23, 2004, issue of Nature Biotechnology. Their effort was also cited in the Guinness Book of World Records as the "largest clone in the world."

At the close of 2004 a Korean research team reported cloning macaque monkey embryos, which would be used as a source of stem cells. In early 2005 conservationists focused research efforts on cloning rare and endangered species. The Audubon Center for Research of Endangered Species in New Orleans, Louisiana, embarked on efforts to clone an African wild cat, Felis lybica.

Cloning Endangered Species

Reproductive cloning technology may be used to repopulate endangered species such as the African bongo antelope, Sumatran tiger, and the giant panda, or animals that reproduce poorly in zoos or are difficult to breed. On January 8, 2001, scientists at Advanced Cell Technology (ACT), a biotechnology company in Massachusetts, announced the birth of the first clone of an endangered animal, a baby bull gaur—a large wild ox from India and Southeast Asia—named Noah. Noah was cloned using the nuclei of frozen skin cells taken from an adult male gaur that had died eight years earlier. The skin cell nuclei were joined with enucleated cow eggs, one of which was implanted into a surrogate cow mother. Unfortunately, the cloned gaur died from an infection within days of its birth. The same year scientists in Italy successfully cloned an endangered wild sheep. Cloning an endangered animal is different from cloning a more common animal because cloned animals need surrogate mothers to be carried to term. The transfer of embryos is risky, and researchers are reluctant to put an endangered animal through the rigors of surrogate motherhood, opting to use nonendangered domesticated animals whenever possible.

Cloning extinct animals is even more challenging than cloning living animals because the egg and the surrogate mother used to create and harbor the cloned embryo are not the same species as the clone. Furthermore, for most already extinct animal species like the woolly mammoth or dinosaur there is insufficient intact cellular and genetic material from which to generate clones. In the future, carefully preserving intact cellular material of imperiled species may allow for their preservation and propagation.

In "In Cloning Noah's Ark" (Scientific American, November 2000), ACT cloning researchers Robert Lanza, Betsy Dresser, and Philip Damiani reported that they achieved their highest success rates—10% of attempts yielding live births—when cloning domestic cattle implanted into cows of the same species. Lanza, Dresser, and Damiani noted that the process was as much an art as a science, particularly when cloning involved transplanting an embryo into another species.

Although Lanza, Dresser, and Damiani conceded that cloning endangered species is controversial, they asserted that it is a viable way to manage species that are in danger of extinction. They called for the establishment of a genetic trust—a worldwide network of storehouses—to hold frozen tissue from all the endangered species from which it would be possible to collect DNA samples.

On April 1, 2003, ACT announced the birth of a healthy clone of a Javan banteng, an endangered cattle-like animal native to Asian jungles. The clone was created from a single skin cell, taken from another banteng before it died in 1980, which had remained frozen until it was used to create the clone. The banteng embryo gestated in a standard beef cow in Iowa.

Born April 1, 2003, the cloned banteng is expected to develop normally, growing its characteristic horns and reaching an adult weight of as much as 1,800 pounds. He was nicknamed "Stockings" and, as of 2005, lived at the San Diego Zoo. Hunting and habitat destruction have reduced the number of banteng, which once lived in large numbers in the bamboo forests of Asia, by more than 75% in the past two decades. By 2005 just 3,000–5,000 banteng remained worldwide.

Reproductive Human Cloning

In December 2002 a religious sect known as the Raelians made news when their private biotechnology firm, Clonaid, announced that after creating several hundred cloned human embryos and performing ten implantation experiments on human subjects they had successfully delivered "the world's first cloned baby." The announcement, which could not be independently verified or substantiated, generated unprecedented media coverage and was condemned in the scientific and lay communities. At least some of the media frenzy resulted from the unique beliefs of the Raelians—namely, the sect contends that humans were created by extraterrestrial beings. According to sect founder and former journalist Claude Vorilhon, who is now known as Rael, he was contacted in 1973 by an extraterrestrial being who emerged from a flying saucer and told him that people from another planet created humans in laboratories. Since then the Raelians have grown into an international movement with more than 40,000 members. Their interest in cloning arises from their belief that the human soul departs when the body dies. In the Raelian worldview the key to eternal life is not the soul but the re-creation of individuals from their DNA. As of May 2005, Clonaid claimed to have produced at least thirteen cloned children, but had yet to offer any proof of their existence.

Clonaid's announcement brought attention on the fact that several laboratories around the world had embarked on clandestine efforts to deliver a cloned human embryo. For example, in 2002 a U.S. fertility specialist, Panayiotis Zavos, claimed to be collaborating with about two dozen researchers internationally to produce human clones. Another doctor focusing on fertility issues, Severino Antinori, attracted media attention when he maintained that hundreds of infertile couples in Italy and thousands in the United States had already enrolled in his human cloning initiative. Neither these researchers nor anyone else had offered proof of successful reproductive human cloning as of June 1, 2005.

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