Chapter 8
Cloning

The moral issues posed by human cloning are profound and have implications for today and for future generations. Today's overwhelming and bipartisan House action to prohibit human cloning is a strong ethical statement, which I commend. We must advance the promise and cause of science, but must do so in a way that honors and respects life.

—President George W. Bush, July 2001

We must not say to millions of sick or injured human beings "go ahead and die and stay paralyzed because we believe … a clump of cells is more important than you are."

—Representative Jerrold Nadler (D-NY), July 2001

The Human Genome Project defines three distinct types of cloning. The first is the use of highly specialized deoxyribonucleic acid (DNA) technology to produce multiple, exact copies of a single gene or other segment of DNA to obtain sufficient material to examine for research purposes. This process produces cloned collections of DNA known as clone libraries. The second kind of cloning involves the natural process of cell division to create identical copies of the entire cell. These copies are called a cell line. The third type of cloning, reproductive cloning, is the one that has received the most attention in the mass media. This is the process that generates complete, genetically identical organisms such as Dolly, the famous Scottish sheep cloned in 1996 and named after the entertainer Dolly Parton.

Cloning may also be described by the technology used to perform it. For example, the term recombinant DNA technology describes the technology and mechanism of DNA cloning. Also known as molecular cloning, or gene cloning, it involves the transfer of a specific DNA fragment of interest to researchers from one organism to a self-replicating genetic element of another species such as a bacterial plasmid. (See Figure 8.1.) The DNA under study may then be reproduced in a host cell. This technology has been in use since the 1970s and is a standard practice in molecular biology laboratories.

Just as GenBank is an online public repository of the human genome sequence, the Clone Registry database is a sort of "public library." Used by genome sequencing centers to record which clones have been selected for sequencing, which sequencing efforts are currently under way, and which are finished and represented by sequence entries in GenBank, the Clone Registry may be freely accessed by scientists worldwide. To effectively coordinate all of this information, a standardized system of naming clones is essential. The nomenclature used is shown in Figure 8.2.

CLONING GENES

Molecular cloning is performed to enable researchers to have many copies of genetic material available in the laboratory for the purpose of experimentation. Cloned genes allow researchers to examine encoded proteins and are used to sequence DNA. Gene cloning also allows researchers to isolate and experiment on the genes of an organism. This is particularly important in terms of human research; in instances where direct experimentation on humans might be dangerous or unethical, experimentation on cloned genes is often practical and feasible.

Cloned genes are also used to produce pharmaceutical drugs, insulin, clotting factors, human growth hormone, and industrial enzymes. Before the widespread use of molecular cloning, these proteins were difficult and expensive to manufacture. For example, before recombinant DNA technology, insulin (a pancreatic hormone that regulates blood glucose levels) used by people with diabetes was extracted and purified from cow and pig pancreases. Because the amino acid sequences of insulin from cows and pigs are slightly different from those in human insulin, some patients experienced adverse immune reactions to the nonhuman "foreign insulin." The recombinant human version of insulin is identical to human insulin so it does not produce an immune reaction.

Figure 8.3 shows how a gene is cloned. First, a DNA fragment containing the gene being studied is isolated from chromosomal DNA using restriction enzymes. It is joined with a plasmid (a small ring of DNA found in many bacteria that can carry foreign DNA) that has been cut with the same restriction enzymes. When the fragment of chromosomal DNA is joined with its cloning vector (cloning vectors, such as plasmids and yeast artificial chromosomes, introduce foreign DNA into host cells), it is called a recombinant DNA molecule. Once it has entered into the host cells, the recombinant DNA can be reproduced along with the host cell DNA.

Another molecular cloning technique that is simpler and less expensive than the recombinant cloning method is the polymerase chain reaction (PCR). PCR has also been dubbed "molecular photocopying" because it amplifies DNA without the use of a plasmid. Figure 6.5 in chapter 6 shows how PCR is used to generate a virtually unlimited number of copies of a piece of DNA.

REPRODUCTIVE CLONING

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 with 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 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 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 8.4 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 mtDNA. 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 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, and rabbits.

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.

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 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 that was different from the process used to clone Dolly the sheep. The most immediate benefit of this new technology was to improve livestock—cloning enables breeders to take some 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 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 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, Cesare Galli et al., describe their cloning technique in "Pregnancy: A Cloned Horse Born to Its Dam Twin" (Nature, August 7, 2003).

The mule was cloned from cells extracted from a mule fetus, whereas the cloned horse's DNA came from her adult mother's skin cells. There were other differences as well. The University of Idaho and Utah State University 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. Before the bull, the only other successful recloning efforts involved mice. Chikara Kubota, X. Cindy Tian, and Xiangzhong Yang, the successful research team, describe their techniques in "Serial Bull Cloning by Somatic Cell Nuclear Transfer" (Nature Biotechnology, May 23, 2004). 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 South Korean research team reported cloning macaque monkey embryos, which would be used as a source of stem cells. Conservationists then focused research efforts on cloning rare and endangered species. In April 2005 Texas A&M University announced the first successfully cloned foal in the United States. That same month, Korean scientists at Seoul National University (SNU) cloned a dog they dubbed "Snuppy." In May 2005 the Brazilian Agricultural Research Corporation, Embrapa, reported the creation of two cloned calves from a Junquiera cow, which is an endangered species.

Cloning Endangered Species

Reproductive cloning technology may be used to repopulate endangered species such as the African bongo antelope, the 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 such as 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 April 2003 ACT announced the birth of a healthy clone of a Javan banteng, an endangered cattlelike 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 developed normally, growing its characteristic horns and reaching an adult weight of about 1,800 pounds. He was nicknamed Stockings and, as of 2007, 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% from 1983 to 2003.

In August 2005 the Audubon Nature Institute in New Orleans, Louisiana, reported that two unrelated endangered African wildcat clones had given birth to eight babies. Their births confirmed that clones of wild animals can breed naturally, which is vitally important for protecting endangered animals on the brink of extinction.

Reproductive Human Cloning

In December 2002 a religious sect known as the Raelians made news when their private biotechnology firm, Clonaid, announced that 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 beliefs of the Raelians—namely, the sect contends that humans were created by extraterrestrial beings. In 2005 Clonaid claimed to have produced at least thirteen cloned children, but as of 2007 had not yet offered any proof of their existence.

Clonaid's announcement brought attention to the fact that several laboratories around the world had embarked on clandestine efforts to clone a human embryo. For example, in 2002 a U.S. fertility specialist, Panayiotis Zavos, claimed to be collaborating with about two dozen international researchers 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 early 2007.

THERAPEUTIC CLONING

Therapeutic cloning (also called embryo cloning) is the creation of embryos for use in biomedical research. The objective of therapeutic cloning is not to create clones but to obtain stem cells. Stem cells are "master cells" capable of differentiating into multiple other cell types. This potential is important to biomedical researchers because stem cells may be used to generate any type of specialized cell, such as nerve, muscle, blood, or brain cells. Many scientists believe that stem cells can not only provide a ready supply of replacement tissue but also may hold the key to developing more effective treatments for common disorders such as heart disease and cancer as well as degenerative diseases such as Alzheimer's and Parkinson's. Researchers believe that in the future it may be possible to induce stem cells to grow into complete organs.

Advocates of therapeutic cloning point to other treatment benefits such as using stem cells to generate bone marrow for transplants. They contend that scientists could use therapeutic cloning to manufacture perfectly matched bone marrow using the patient's own skin or other cells. This would eliminate the problem of rejection of foreign tissue associated with bone marrow transplant and other organ transplantation. Stem cells also have the potential to repair and restore damaged heart and nerve tissue. Furthermore, there is mounting evidence to suggest that stem cells from cloned embryos have greater potential as medical treatments than stem cells harvested from unused embryos at fertility clinics, which are created by in vitro fertilization and are now the major source of stem cells for research. These prospective benefits are among the most compelling arguments in favor of cloning to obtain embryonic stem cells.

Stem cells used in research are harvested from the blastocyst after it has divided for five days, during the earliest stage of embryonic development. Many people regard human embryos as human beings or at least potential human beings and consider their destruction, or even using techniques to obtain stem cells that might imperil their future viability, as immoral or unethical.

In November 2001 the ACT researchers Jose B. Cibelli et al. reported in "Somatic Cell Nuclear Transfer in Humans: Pronuclear and Early Embryonic Development" (e-biomed: The Journal of Regenerative Medicine, November 26, 2001) that they had created a cloned human embryo, and, unlike groups that had claimed to have done this before, they published their results. The ACT press release "Advanced Cell Technology, Inc. (ACT) Today Announced Publication of Its Research on Human Somatic Cell Nuclear Transfer and Parthenogenesis" (November 25, 2001, http://www.advancedcell.com) boasted that this achievement offered "the first proof that reprogrammed human cells can supply tissue" and asserted that this accomplishment was a vital first step toward the objective of therapeutic cloning—using cloned embryos to harvest embryonic stem cells able to grow into replacement tissue perfectly matched to individual patients. To clone the human embryos, Cibelli et al. collected women's eggs and painstakingly removed the genetic material from the eggs with a thin needle. A skin cell was inserted inside each of eight enucleated eggs, which were then chemically stimulated to divide. Just three of the eight eggs began dividing, and only one reached six cells before cell division ceased.

That same year investigators at the South Australian Research and Development Institute used lambs to experiment with therapeutic cloning. The goal was to replace cells stricken with Parkinson's disease with healthy ones derived from a cloned embryo. In 2003 researchers in Italy reported successfully using adult stem cells to cure mice that had a form of multiple sclerosis. The scientists injected the diseased mice with stem cells that had been extracted from the brains of adult mice reproduced in the laboratory. Postmortem examination of the mice showed that the stem cells had migrated to and then repaired damaged areas of the nerves and brain.

In August 2003 a Chinese research team led by Huizhen Sheng, an American-trained scientist working at the Shanghai Second Medical University, reported that it had made human embryonic stem cells by combining human skin cells with rabbit eggs. Their accomplishment was published in the Chinese scientific journal Cell Research, a peer-reviewed publication of the Shanghai Institute of Cell Biology and the Chinese Academy of Sciences. The researchers removed the rabbit eggs' DNA and injected human skin cells inside them. The eggs then grew to form embryos containing human genetic material. After several days the embryos were dissected to extract their stem cells.

In February 2004 scientists at Seoul National University in South Korea reported in the journal Science that they had successfully cloned healthy human embryos, removed embryonic stem cells, and grown them in mice. In January 2006, following a lengthy investigation, Seoul National University concluded that the research reported in Science had been fabricated. As a result, the journal retracted the article along with another study by the same author. In May 2006, the investigator, Hwang Woo-suk, was charged with fraud, embezzlement, and violating South Korea's bioethics statutes.

In 2005 Wilmut was granted a license by the British government to clone human embryos to generate stem cell lines to study motor neuron disease (MND). Wilmut and his colleagues are working to clone embryos to generate stem cells that would in turn become motor neurons with MND-causing gene defects. By observing the stem cells grow into neurons, the researchers hope to discover what causes the cells to degenerate. Their research involves comparing the stem cells with healthy and diseased cells from MND patients to gain a better understanding of the illness and to test potential drug treatments.

Human reproductive cloning remains illegal in Britain but therapeutic cloning—creating embryos as a source of stem cells to cure diseases—is allowed on an approved basis. The license granted to Wilmut and his colleagues is the second one granted by Britain's Human Fertilisation and Embryology Authority.

In July 2006 the researchers Deepa Deshpande et al. restored movement to paralyzed rats using a new method that demonstrates the potential of embryonic stem cells to restore function to humans suffering from neurological disorders. They published their results in "Recovery from Paralysis in Adult Rats Using Embryonic Stem Cells" (Annals of Neurology, July 2006). Although clinical trials in humans are still years away, the results of this research represent an important advance in the quest for a cure for paralysis and other neurological disorders.

In October 2006 Kevin A. D'Amour et al., in "Production of Pancreatic Hormone-Expressing Endocrine Cells from Human Embryonic Stem Cells" (Nature Biotechnology, October 19, 2006), reported developing a process to turn human embryonic stem cells into pancreatic cells that can produce insulin and other hormones. The researchers anticipate testing these cells in animals in 2008 and if the animal studies are successful, then clinical trials in human patients may begin as soon as 2009.

Three studies—Volker Schächinger et al. in "Intracoronary Bone Marrow-Derived Progenitor Cells in Acute Myocardial Infarction," Ketil Lunde et al. in "Intracoronary Injection of Mononuclear Bone Marrow Cells in Acute Myocardial Infarction," and Birgit Assmus et al. in "Transcoronary Transplantation of Progenitor Cells after Myocardial Infarction"—describing the use of stem cells in the treatment of heart disease were published in the September 21, 2006, issue of the New England Journal of Medicine. The studies produced conflicting results: Schächinger and his colleagues reported benefits for patients who had suffered myocardial infarction (heart attack). Lunde and his contributors found no benefit from stem cell treatment of such patients. Assmus and her collaborators studied patients with chronic heart failure, who did show improvement after treatment. In the editorial "Cardiac Cell Therapy—Mixed Results from Mixed Cells" in the same issue of the journal, Antony Rosenzweig writes that the three studies "provide a realistic perspective on this approach while leaving room for cautious optimism and underscoring the need for further study."

Rick Weiss, in "Stem Cell Work Shows Promise and Risks" (Washington Post, October 23, 2006), reports that research conducted at the University of Rochester Medical Center using nerve cells grown from human embryonic stem cells to treat rats afflicted with Parkinson's disease produced mixed results. The treatment reduced the animals' symptoms, but caused tumors in the rodents' brains. The researchers acknowledged that their work showed both the promise and risks associated with stem cell treatments.

Research Promises Therapeutic Benefits without Cloning

In "Homologous Recombination in Human Embryonic Stem Cells" (Nature Biotechnology, March 2003), Thomas P. Zwaka and James A. Thomson report that they used human embryonic stem cells to splice out individual genes and substituted different genes in their place. Their accomplishment was heralded as a first step toward the goal of regenerating parts of the human body by transplanting either stem cells or tissues grown from stem cells into patients. Zwaka and Thomson used electrical charges and chemicals to make the cells' membranes permeable; the cells allowed the customized genes to enter, and they then found and replaced their counterparts in the cells' DNA.

The ability to make precise genetic changes in human stem cells could be used to boost their therapeutic potential or make them more compatible with patients' immune systems. Some researchers assert that the success of this bioengineering feat might eliminate the need to pursue the hotly debated practice of therapeutic cloning, but others caution that such research could heighten concerns among those who fear that stem cell technology will lead to the creation of "designer babies," which are bred for specific characteristics such as appearance, intelligence, or athletic prowess.

In May 2003 the University of Pennsylvania researcher Hans R. Schöler and his colleagues announced another historic first: The researchers transformed ordinary mouse embryo cells into egg cells in laboratory dishes ("Scientists Produce Mouse Eggs from Embryonic Stem Cells, Demonstrating Totipotency Even In Vitro," ScienceDaily, May 2, 2003). Schöler selected from a population of stem cells the ones that bore certain genetic traits suggesting the potential to become eggs. They then isolated those in laboratory dishes. Eventually, the cells morphed into two kinds of cells, including young egg cells. The eggs matured normally and appeared to be healthy in terms of their appearance, size, and gene expression. When cultured for a few days, the eggs also underwent spontaneous division and formed structures resembling embryos, a process called parthenogenesis. This finding implies that the eggs were fully functional and likely could be fertilized with sperm.

Once refined, this technology could be applied to produce egg cells in the laboratory that would enable scientists to engineer traits into animals and help conservationists rebuild populations of endangered species. It offers researchers the chance to observe mammalian egg cells as they mature, a process that occurs unseen within the ovary. The technology also offers an unparalleled opportunity to learn about meiosis (reduction division), the process of cell division during which an egg or sperm disgorges half of its genes so it can join with a gamete of the opposite sex. There are many potential medical benefits as well. For example, women who cannot make healthy eggs could use this technology to ensure healthy offspring.

Like many new technologies, transforming cells into eggs simultaneously resolves existing ethical issues and creates new ones. For example, because the embryonic stem cells spontaneously transformed themselves into eggs, this procedure overcomes many of the ethical objections to cloning, which involves creating offspring from a single parent. However, it also paves the way for the creation of "designer eggs" from scratch and, if performed with human cells, could redefine the biological definitions of mothers and fathers.

In September 2003 efforts to transform stem cells into sperm were successful. Toshiaki Noce and his colleagues in Tokyo, Japan, observed male mouse embryonic stem cells that developed spontaneously, with some cells actually becoming germ cells. When the researchers transplanted the germ cells into testicular tissue, the cells underwent meiosis and formed sperm cells. One possible medical application of this technology would be to assist couples who are infertile because the male cannot produce healthy sperm. One of the ethical issues that might arise would be the potential for two men to both be biological fathers of a child. Another is the potential to generate a human being who never had any parents using two laboratory-grown stem cells, one transformed into a sperm and the other into an egg. Many ethicists advise consideration of such issues before permitting human experimentation.

In 2004 the National Institutes of Health (NIH) reported that researchers from the University of Pennsylvania School of Veterinary Medicine used cells from mice to grow sperm progenitor cells in a laboratory culture (November 3, 2004, http://www.nih.gov/news/pr/nov2004/nichd-03.htm). Known as spermatogonial stem cells, the progenitor cells are incapable of fertilizing egg cells but give rise to cells that develop into sperm. The researchers transplanted the cells into infertile mice, which were then able to produce sperm and father offspring that were genetically related to the donor mice.

This breakthrough has many potential applications, including developing new treatments for male infertility and extending the reproductive lives of endangered species. Researchers will also attempt to genetically manipulate the sperm cells grown in a culture medium and then implant the cells into animals. In this way they could introduce new traits into laboratory animals and livestock, such as disease resistance. The culture technique offers researchers additional opportunities to investigate the potential of spermatogonial stem cells as a source for adult stem cells to replace diseased or injured tissue.

New Methods of Obtaining Stem Cells without Destroying Embryos

In "Embryonic and Extraembryonic Stem Cell Lines Derived from Single Mouse Blastomeres" (Nature, January 12, 2006), Young Chung et al. report that embryonic stem cell cultures could be derived from single cells of mouse embryos. Irina Klimanskaya et al., in "Human Embryonic Stem Cell Lines Derived from Single Blastomeres" (Nature, August 23, 2006), describe a technique for removing a single cell—called a blastomere—from a three-day-old embryo with eight to ten cells and using a biochemical process to create embryonic stem cells from the blastomere. The method of removing a cell from the embryo is much like the technique used for preimplantation genetic diagnosis, which is performed to screen the cell for genetic defects. The researchers note that human embryonic stem cell lines derived from a single blastomere were comparable to lines derived with conventional techniques. Although Klimanskaya and her colleagues assert that the new method "will make it far more difficult to oppose this research," opponents of stem cell research contend that the new technique is morally unacceptable because even a single cell removed from an early embryo may have the potential to produce a life.

Another technique reported in 2006 can obviate the need for embryonic stem cells. Erika Check notes in "Simple Recipe Gives Adult Cells Embryonic Powers" (Nature, July 6, 2006) that researchers in the United Kingdom discovered the gene, called nanog, that is the key to "reprogramming" adult cells back to an embryonic state. The reprogramming of adult cells using nanog may make it possible for scientists to generate cells that specialize and develop into every type of cell in the body without the controversial use of human embryonic stem cells.

OPINIONS SHAPE PUBLIC POLICY

The difficulty and low success rate of much animal reproductive cloning (an average of just one or two viable offspring result from every one hundred attempts) and the as-yet-inadequate understanding about reproductive cloning have prompted many scientists to deem it unethical to attempt to clone humans. Many attempts to clone mammals have failed, and about one-third of clones born alive suffer from anatomical, physiological, or developmental abnormalities that are often debilitating. Some cloned animals have died prematurely from infections and other complications at rates higher than conventionally bred animals, and some researchers anticipate comparable outcomes from human cloning. Furthermore, scientists cannot yet describe or characterize how cloning influences intellectual and emotional development. Even though the attributes of intelligence, temperament, and personality may not be as important for cattle or other primates, they are vital for humans. Without considering the myriad religious, social, and other ethical concerns, the presence of so many unanswered questions about the science of reproductive cloning has prompted many investigators to consider any attempts to clone humans as scientifically irresponsible, unacceptably risky, and morally unallowable.

On August 9, 2001, President George W. Bush (http://www.whitehouse.gov/news/releases/2001/08/20010809-2.html) announced his decision to allow federal funds to be used for research on existing human embryonic stem cell lines as long as the derivation process (which begins with the removal of the inner cell mass from the blastocyst) had already been initiated and the embryo from which the stem cell line was derived no longer had the possibility of development as a human being. The president established the following criteria that research studies must meet to qualify for federal funding:

• The stem cells must have been drawn from an embryo created for reproductive purposes that was no longer needed for these purposes.

• Informed consent must have been obtained for the donation of the embryo and no financial inducements provided for donation of the embryo.

In January 2002 the Panel on Scientific and Medical Aspects of Human Cloning was convened by the National Academy of Sciences; the National Academy of Engineering; the Institute of Medicine Committee on Science, Engineering, and Public Policy; and the National Research Council, Division on Earth and Life Studies Board on Life Sciences. Following the panel, the report Scientific and Medical Aspects of Human Cloning (January 2002, http://www7.nationalacademies.org/cosepup/Human_Cloning.html) was issued that called for a ban on human reproductive cloning.

The panel recommended a legally enforceable ban with substantial penalties as the best way to discourage human reproductive cloning experiments in both the public and private sectors. It cautioned that a voluntary measure might be ineffective because many of the technologies needed to accomplish human reproductive cloning are widely accessible in private clinics and other organizations that are not subject to federal regulations.

The panel did not, however, conclude that the scientific and medical considerations that justify a ban on human reproductive cloning are applicable to nuclear transplantation to produce stem cells. In view of their potential to generate new treatments for life-threatening diseases and advance biomedical knowledge, the panel recommended that biomedical research using nuclear transplantation to produce stem cells be permitted. Finally, the panel encouraged ongoing national discussion and debate about the range of ethical, societal, and religious issues associated with human cloning research.

On February 14, 2002, the American Association for the Advancement of Science (AAAS; http://archives.aaas.org/docs/documents.php?doc_id=425), the world's largest general scientific organization, affirmed a legally enforceable ban on reproductive cloning; however, the AAAS supported therapeutic or research cloning using nuclear transplantation methods under appropriate government oversight. Similarly, the American Medical Association (AMA; April 6, 2006, http://www.ama-assn.org/ama/pub/category/4560.html), a national physicians' organization, issued a formal public statement against human reproductive cloning. The AMA statement cautioned that human cloning failures could jeopardize promising science and genetic research and prevent biomedical researchers and patients from realizing the potential benefits of therapeutic cell cloning.

On April 10, 2002, President Bush called on the Senate to back legislation banning all types of human cloning (http://www.whitehouse.gov/news/releases/2002/04/20020410-4.html). In his plea to the Senate, Bush said:

Science has set before us decisions of immense consequence. We can pursue medical research with a clear sense of moral purpose or we can travel without an ethical compass into a world we could live to regret. Science now presses forward the issue of human cloning. How we answer the question of human cloning will place us on one path or the other…. Human cloning is deeply troubling to me, and to most Americans. Life is a creation, not a commodity. Our children are gifts to be loved and protected, not products to be designed and manufactured. Allowing cloning would be taking a significant step toward a society in which human beings are grown for spare body parts, and children are engineered to custom specifications; and that's not acceptable…. I believe all human cloning is wrong, and both forms of cloning ought to be banned, for the following reasons. First, anything other than a total ban on human cloning would be unethical. Research cloning would contradict the most fundamental principle of medical ethics, that no human life should be exploited or extinguished for the benefit of another.

On September 25, 2002, Elias Zerhouni, the director of the NIH, testified before the Senate Appropriations Subcommittee on Labor, Health and Human Services, and Education in favor of advancing the field of stem cell research (http://olpa.od.nih.gov/hearings/107/session2/testimonies/stemcelltest.asp). Zerhouni exhorted Congress to continue to fund both human embryonic stem cell research and adult stem cell research simultaneously to learn as much as possible about the potential of both types of cells to treat human disease. He observed that many studies that do not involve human subjects must be performed before any new therapy is tested on human patients. These preclinical studies include tests of the long-term survival and fate of transplanted cells, as well as tests of the safety, toxicity, and effectiveness of the cells in treating specific diseases in animals. Zerhouni promised that trials using human subjects, the clinical research phase, would begin only after the basic foundation had been established. Despite Zerhouni's impassioned plea and subsequent efforts to advance stem cell research, at the close of 2006 U.S. law continued to ban federal funding of any research that might harm human embryos.

Moral and Ethical Objections to Human Cloning

People who oppose human cloning are as varied as the interests and institutions they support. Religious leaders, scientists, politicians, philosophers, and ethicists argue against the morality and acceptability of human cloning. Nearly all objections hinge, to various degrees, on the definition of human life, beliefs about its sanctity, and the potentially adverse consequences for families and society as a whole.

In an effort to stimulate consideration of and debate about this critical issue, the President's Council on Bioethics examined the principal moral and ethical objections to human cloning in Human Cloning and Human Dignity: An Ethical Inquiry (July 2002, http://www.bioethics.gov/reports/cloningreport/fullreport.html). The council's report distinguished between therapeutic and reproductive cloning and outlined key concerns by trying to respond to many as yet unresolved questions about the ethics, morality, and societal consequences of human cloning.

The council determined that the key moral and ethical objections to therapeutic cloning—cloning for biological research—center on the moral status of developing human life. Therapeutic cloning involves the deliberate production, use, and, ultimately, destruction of cloned human embryos. One objection to therapeutic cloning is that cloned embryos produced for research are no different from those that could be used in attempts to create cloned children. Another argument that has been made is that the ends do not justify the means—that research on any human embryo is morally unacceptable, even if this research promises cures for many dreaded diseases. Finally, there are concerns that acceptance of therapeutic cloning will lead society down the "slippery slope" to reproductive cloning, a prospect that is almost universally viewed as unethical and morally unacceptable.

The unacceptability of human reproductive cloning stems from the fact that it challenges the basic nature of human procreation, redefining having children as a form of manufacturing. Human embryos and children may then be viewed as products and commodities rather than as sacred and unique human beings. Furthermore, reproductive cloning might substantially change fundamental issues of human identity and individuality, and allowing parents unprecedented genetic control of their offspring may significantly alter family relationships across generations.

The council concluded that "the right to decide" whether to have a child does not include the right to have a child by any means possible, nor does it include the right to decide the kind of child one is going to have. A societal commitment to freedom does not require use or acceptance of every technological innovation available.

Legislation Aims to Completely Ban Human Cloning

On February 27, 2003, the U.S. House of Representatives voted to outlaw all forms of human cloning. The legislation prohibits the creation of cloned human embryos for medical research as well as the creation of cloned babies. It contains strong sanctions, imposing a maximum penalty of $1 million in civil fines and as many as ten years in jail for violations. The measure did not pass in the Senate, which was closely divided about whether therapeutic cloning should be prohibited along with reproductive cloning. In early February 2003 President Bush issued a policy statement that strongly supported a total ban on cloning. In the Senate two bills were introduced: S. 245 was a complete ban intended to amend the Public Health Service Act to prohibit all human cloning, and S. 303 was a less sweeping measure that also prohibited cloning but protected stem cell research. S. 245 was referred to the Senate Committee on Health, Education, Labor and Pensions and S. 303 was referred to the Senate Committee on the Judiciary. Neither bill, nor any comparable proposed legislation, has emerged from the Senate committees.

Even though nearly all lawmakers concur that Congress should ban reproductive cloning, many disagree about whether legislation should also ban the creation of cloned human embryos that serve as sources of embryonic stem cells. Many legislators agree with scientists that stem cells derived from cloned human embryos have medical and therapeutic advantages over those derived from conventional embryos or adults. Those who oppose the legislation calling for a total ban assert that the aim of allowing research is to relieve the suffering of people with degenerative diseases. They say that the bill's sponsors are effectively thwarting advances in medical treatment and biomedical innovation.

Supporters of the total ban contend that Congress must send an unambiguous message that cloning research and experimentation will not be tolerated. They consider cloning immoral and unethical, fear unintended consequences of cloning, and feel they speak for the public when they assert that it is not justifiable to create human embryos simply for the purpose of experimenting on them and then destroying them.

The fact that there are only about twenty available stem cell lines prompted the introduction of bills during the spring of 2004 that would require funding for human embryonic stem cell research, despite the president's 2001 policy. On April 28, 2004, more than 200 members of the House sent a letter to the president arguing in favor of an expansion of existing policy. Fifty-eight senators sent a similar letter on June 4, 2004. Pleas from patient advocacy groups—along with the death of the former president Ronald Reagan from Alzheimer's disease on June 5, 2004, and Nancy Reagan's appeals to expand the policy—focused considerable media attention on the issue during the summer of 2004, but no legislation was passed that year.

On May 24, 2005, the House passed H.R. 810, the Stem Cell Research Enhancement Act of 2005, which would have permitted federal funding for embryonic stem cell research on cells "derived from human embryos that have been donated from in vitro fertilization clinics, were created for the purposes of fertility treatment, and were in excess of the clinical need of the individuals seeking such treatment." The Senate passed the bill on July 18, 2006, and the following day President Bush vetoed the bill.

State Human Cloning Laws

As of 2006 fifteen states had enacted legislation that addresses human cloning. (See Table 8.1.) California was the first state to ban reproductive cloning in 1997. Since then, twelve other states—Arkansas, Connecticut, Indiana, Iowa, Maryland, Massachusetts, Michigan, Rhode Island, New Jersey, North Dakota, South Dakota, and Virginia—have passed laws prohibiting reproductive cloning. Arizona's and Missouri's legislation addresses the use of public funds for cloning, and Maryland's prohibits the use of state stem cell research funds for reproductive cloning and possibly therapeutic cloning, depending the interpretation of the statute. Louisiana also enacted legislation that prohibited reproductive cloning, but the law expired in July 2003. The laws of Arkansas, Indiana, Iowa, Michigan, North Dakota, and South Dakota also prohibit therapeutic cloning. Virginia's legislation may be interpreted as a complete ban on human cloning; however, it is unclear because the law does not define the term human being, which is used in the definition of human cloning. Rhode Island's law does not prohibit cloning for research, and California's and New Jersey's laws specifically permit cloning for the purpose of research.

California Leads the Way

In 2002 the California state legislature passed a law encouraging therapeutic cloning. Even though there were no provisions for funds in the law, the move was interpreted as support for the research. In 2004 stem cell research advocates offered voters a sweeping ballot measure—Proposition 71—to make public funding available to support stem cell research and therapeutic cloning. Proposition 71 was championed by Robert Klein, a wealthy real estate developer and father of a child with diabetes who might benefit from the research. It also received considerable financial support from the Microsoft founder Bill Gates to finance campaign advertising and lobbying.

On November 2, 2004, Californians approved Proposition 71, a ballot measure with the potential to make the state a leader in human embryonic stem cell research. Proposition 71 enabled the state to establish its own research institute—the California Institute for Regenerative Medicine. The proposition prohibits reproductive cloning but funds human cloning projects designed to create stem cells and allocates $3 billion over ten years in research funds. Those supporting the legislation hoped that stem cell research would become the biggest, most important, and most profitable medical advancement of the twenty-first century. The legislation's supporters intended to use the funds to attract top researchers to the state, making California the epicenter of groundbreaking, lifesaving, and potentially lucrative medical research.

Nicholas Wade reports in "Plans Unveiled for State-Financed Stem Cell Work in California" (New York Times, October 5, 2006) that in October 2006 the California Institute for Regenerative Medicine released its ten-year plan for spending the $3 billion allocated to it. The institute said it will spend $823 million on basic stem cell research, $899 million on applied or preclinical research, and $656 million to advance new treatments through clinical trials. An additional $273 million will enable universities to construct laboratories in which none of the equipment has been purchased with federal funds to ensure that the researchers are not violating the rules that restrict federal money to conduct stem cell research.

Public Opinions about Stem Cell Research and Cloning

According to Gallup poll data, more than 60% of Americans believe using stem cells derived from human embryos in medical research is morally acceptable. Figure 8.5 reveals that the percentage of Americans that considers stem cell research morally acceptable had increased from 52% in 2002 to 61% in 2006.

The percentage of Americans that deems stem cell research morally acceptable varies by political affiliation, with support highest among Democrats (68%) and Independents (62%), compared with Republicans (51%). (See Figure 8.6.) According to Lydia Saad in Stem Cell Veto Contrary to Public Opinion (Gallup Poll, July 20, 2006), support also varies by educational attainment—three-quarters (77%) of those with postgraduate degrees consider this research acceptable, compared with 45% of people who had attained a high school education or less.

The Gallup poll also found that most Americans (58%) disapproved of President Bush's July 2006 veto of a bill that would have expanded federal funding for embryonic stem cell research. (See Figure 8.7.) However, Saad notes that just 11% of Americans favor unrestricted government funding of embryonic stem cell research and another 42% support easing current restrictions. Nearly one-quarter (24%) approve of the current funding restrictions and 19% oppose any government funding of this research.

Even though Americans continue to feel that it is morally unacceptable to clone humans, public support for cloning animals increased slightly from 31% in 2001 to 35% in 2005. (See Figure 8.8.) Furthermore, unlike stem cell research, which is favored by more Democrats than Republicans; more Republicans (31%) than Democrats (28%) consider cloning animals morally acceptable. (See Figure 8.9 and Figure 8.10.)

CHAPTER 8
CLONING

The moral issues posed by human cloning are profound and have implications for today and for future generations. Today's overwhelming and bipartisan House action to prohibit human cloning is a strong ethical statement, which I commend. We must advance the promise and cause of science, but must do so in a way that honors and respects life.

—President George W. Bush

We must not say to millions of sick or injured human beings, "go ahead and die and stay paralyzed because we believe …a clump of cells is more important than you are."

—Representative Jerrold Nadler (D-NY)

The Human Genome Project defines three distinct types of cloning. The first is the use of highly specialized deoxyribonucleic acid (DNA) technology to produce multiple, exact copies of a single gene or other segment of DNA to obtain sufficient material to examine for research purposes. This process produces cloned collections of DNA known as clone libraries. The second kind of cloning involves the natural process of cell division to create identical copies of the entire cell. These copies are called a cell line. The third type of cloning, reproductive cloning, is the one that has received the most attention in the mass media. This is the process that generates complete, genetically identical organisms such as Dolly, the famous Scottish sheep cloned in 1996 and named after entertainer Dolly Parton.

Cloning may also be described by the technology used to perform it. For example, the term "recombinant DNA technology" describes the technology and mechanism of DNA cloning. Also known as molecular cloning, or gene cloning, it involves the transfer of a specific DNA fragment of interest to researchers from one organism to a self-replicating genetic element of another species such as a bacterial plasmid. (See Figure 8.1.) The DNA under study may then be reproduced in a host cell. This technology has been in use since the 1970s and is a standard practice in molecular biology laboratories.

Just as GenBank is an online public repository of the human genome sequence, the Clone Registry database is a sort of "public library." Used by genome sequencing centers to record which clones have been selected for sequencing, which sequencing efforts are currently underway, and which are finished and represented by sequence entries in GenBank, the Clone Registry may be freely accessed by scientists worldwide. To effectively coordinate all of this information, a standardized system of naming clones is essential. The nomenclature used is shown in Figure 8.2.

CLONING GENES

Molecular cloning is performed to enable researchers to have many copies of genetic material available in the laboratory for the purpose of experimentation. Cloned genes allow researchers to examine encoded proteins and are used to sequence DNA. Gene cloning also allows researchers to isolate and experiment on the genes of an organism. This is particularly important in terms of human research; in instances where direct experimentation on humans might be dangerous or unethical, experimentation on cloned genes is often practical and feasible.

Cloned genes are also used to produce pharmaceutical drugs, insulin, clotting factors, human growth hormone, and industrial enzymes. Prior to the widespread use of molecular cloning, these proteins were difficult and expensive to manufacture. For example, before recombinant DNA technology, insulin (a pancreatic hormone that regulates blood glucose levels) used by people with diabetes was extracted and purified from cow and pig pancreases. Since the amino acid sequences of insulin from cows and pigs are slightly different than those in human insulin, some patients experienced adverse immune reactions to the nonhuman "foreign insulin." The recombinant human version of insulin is identical to human insulin so it does not produce an immune reaction.

Figure 8.3 shows how a gene is cloned. First, a DNA fragment containing the gene being studied is isolated from chromosomal DNA using restriction enzymes. It is joined with a plasmid (a small ring of DNA found in many bacteria that can carry foreign DNA) that has been cut with the same restriction enzymes. When the fragment of chromosomal DNA is joined with its cloning vector (cloning vectors, such as plasmids and yeast artificial chromosomes, introduce foreign DNA into host cells), it is called a recombinant DNA molecule. Once it has entered into the host cells, the recombinant DNA can be reproduced along with the host cell DNA.

Another molecular cloning technique that is simpler and less expensive than the recombinant cloning method is the polymerase chain reaction (PCR). PCR has also been dubbed "molecular photocopying" because it amplifies DNA without the use of a plasmid. Figure 8.4 shows how PCR is used to generate a virtually unlimited number of copies of a piece of DNA.

REPRODUCTIVE CLONING

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 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.

THERAPEUTIC CLONING

Therapeutic cloning (also termed "embryo cloning") is the creation of embryos for use in biomedical research. The objective of therapeutic cloning is not to create clones but to obtain stem cells. Stem cells are "master cells" capable of differentiating into multiple other cell types. This potential is important to biomedical researchers because stem cells may be used to generate any type of specialized cell, such as nerve, muscle, blood, or brain cells. Many scientists believe that stem cells can not only provide a ready supply of replacement tissue but also may hold the key to developing more effective treatments for common disorders such as heart disease and cancer as well as degenerative diseases such as Alzheimer's and Parkinson's. Some researchers believe that in the foreseeable future it may be possible to induce stem cells to grow into complete organs.

Advocates of therapeutic cloning point to other treatment benefits such as using stem cells to generate bone marrow for transplants. They contend that scientists could use therapeutic cloning to manufacture perfectly matched bone marrow using the patient's own skin or other cells. This would eliminate the problem of rejection of foreign tissue associated with bone marrow transplant and other organ transplantation. Stem cells also have the potential to repair and restore damaged heart and nerve tissue. Further, there is mounting evidence to suggest that stem cells from cloned embryos have greater potential as medical treatments than stem cells harvested from unused embryos at fertility clinics, which are created by in vitro fertilization and are now the major source of stem cells for research. These prospective benefits are among the most compelling arguments in favor of cloning to obtain embryonic stem cells.

Stem cells used in research are harvested from the blastocyst after it has divided for five days, during the earliest stage of embryonic development. Harvesting stem cells does, however, destroy the embryo. Many people regard human embryos as human beings or at least potential human beings and consider their destruction to be immoral or unethical.

In November 2001 ACT researchers announced that they had created a cloned human embryo, and, unlike groups that had claimed to have done this before, the ACT team published its results (Jose B. Cibelli et al., "Somatic Cell Nuclear Transfer in Humans: Pronuclear and Early Embryonic Development," e-biomed: The Journal of Regenerative Medicine, vol. 2, November 25, 2001). The biotechnology firm's press release boasted that this achievement offered "the first proof that reprogrammed human cells can supply tissue" and asserted that this accomplishment was a vital first step toward the objective of therapeutic cloning—using cloned embryos to harvest embryonic stem cells able to grow into replacement tissue perfectly matched to individual patients. To clone the human embryos, investigators collected women's eggs and painstakingly removed the genetic material from the eggs with a thin needle. A skin cell was inserted inside each of eight enucleated eggs, which were then chemically stimulated to divide. Just three of the eight eggs began dividing, and only one reached six cells before cell division ceased.

The same year investigators at the South Australian Research and Development Institute used lambs to experiment with therapeutic cloning. The goal was to replace cells stricken with Parkinson's disease with healthy ones derived from a cloned embryo. In 2003 researchers in Italy reported successfully using adult stem cells to cure mice that had a form of multiple sclerosis. The scientists injected the diseased mice with stem cells that had been extracted from the brains of adult mice reproduced in the laboratory. Postmortem examination of the mice showed that the stem cells had migrated to and then repaired damaged areas of the nerves and brain.

In August 2003 a Chinese research team led by Huizhen Sheng, an American-trained scientist working at the Shanghai Second Medical University, reported that it had made human embryonic stem cells by combining human skin cells with rabbit eggs. Their accomplishment was published in the Chinese scientific journal Cell Research, a peer-reviewed publication of the Shanghai Institute of Cell Biology and the Chinese Academy of Sciences. The researchers removed the rabbit eggs' DNA and injected human skin cells inside them. The eggs then grew to form embryos containing human genetic material. After several days the embryos were dissected to extract their stem cells.

In February 2004 scientists at Seoul National University in Korea reported in the journal Science that they had successfully cloned healthy human embryos, removed embryonic stem cells, and grown them in mice. Scientists in England sought permission from their government to perform similar research, and a team of Harvard scientists sought and obtained permission from their university's ethics board to create cloned human embryos for medical research.

In February 2005 Professor Ian Wilmut, who had cloned Dolly the sheep, was granted a license by the British government to clone human embryos to generate stem-cell lines to study motor neuron disease (MND). Wilmut and his colleagues planned to clone embryos to generate stem cells that would in turn become motor neurons with MND-causing gene defects. By observing the stem cells grow into neurons, the researchers hoped to discover what causes the cells to degenerate. They planned to compare the stem cells with healthy and diseased cells from MND patients to gain a better understanding of the illness and to test potential drug treatments.

Human reproductive cloning remains illegal in Britain but therapeutic cloning—creating embryos as a source of stem cells to cure diseases—is allowed on an approved basis. The license granted to Wilmut and his colleagues is the second one granted by Britain's Human Fertilisation and Embryology Authority.

On March 14, 2005, Dr. Wilmut was awarded Germany's most prestigious medical award—the Paul Ehrlich and Ludwig Darmstaedter Prize—despite opposition from some members of the German Finance Ministry, which partly funds the award. In response, Wilmut vowed to spend the $134,000 (U.S.) prize on projects to help patients suffering from ailments such as Parkinson's disease (Angelika Brecht-Levy, "Dolly the Sheep's Creator Gets Award," Associated Press, March 14, 2005).

In 2004 Hans S. Keirstead, an assistant professor at the University of California at Irvine, used human embryonic stem cells to enable paralyzed rats to walk. He intended to begin clinical trials of this therapy to treat people with recent spinal cord injuries in 2005. Dr. Keirstead campaigned alongside the late Christopher Reeve, the paralyzed actor who championed stem cell therapy, to encourage Californians to vote to approve Proposition 71, a ballot measure allocating $3 billion of the state's money to embryonic stem cell research over the next decade. The measure passed in November 2004, and in 2005 plans were underway to distribute the funds.

Research Promises Therapeutic Benefits without Cloning

In "Homologous Recombination in Human Embryonic Stem Cells" (Nature Biotechnology, vol. 21, no. 3, February 2003), Thomas Zwaka and James Thomson reported that they had used human embryonic stem cells to splice out individual genes and substituted different genes in their place. Zwaka and Thomson's accomplishment was heralded as a first step toward the goal of regenerating parts of the human body by transplanting either stem cells or tissues grown from stem cells into patients. The researchers used electrical charges and chemicals to make the cells' membranes permeable; the cells allowed the customized genes to enter, and they then found and replaced their counterparts in the cells' DNA.

The ability to make precise genetic changes in human stem cells could be used to boost their therapeutic potential or make them more compatible with patients' immune systems. Some researchers assert that the success of this bioengineering feat might eliminate the need to pursue the hotly debated practice of therapeutic cloning, but others caution that such research could heighten concerns among those who fear that stem cell technology will lead to the creation of "designer babies," bred for specific characteristics such as appearance, intelligence, or athletic prowess.

In May 2003 University of Pennsylvania researchers Hans Schoeler and Karin Huebner reported another historic first: They transformed ordinary mouse embryo cells into egg cells in laboratory dishes (ScienceDaily, May 2, 2003). Schoeler and Huebner selected from a population of stem cells the ones that bore certain genetic traits suggesting the potential to become eggs. They then isolated those in laboratory dishes. After a while, the cells morphed into two kinds of cells, including young egg cells. The eggs matured normally and appeared to be healthy in terms of their appearance, size, and gene expression. When cultured for a few days, the eggs also underwent spontaneous division and formed structures resembling embryos, a process called parthenogenesis. This finding implies that the eggs were fully functional and likely can be fertilized with sperm.

Once refined, this technology could be applied to produce egg cells in the laboratory that would enable scientists to engineer traits into animals and help conservationists rebuild populations of endangered species. It offers researchers the chance to observe mammalian egg cells as they mature, a process that occurs unseen within the ovary. The technology also offers an unparalleled opportunity to learn about meiosis (reduction division), the process of cell division during which an egg or sperm disgorges half of its genes so it can join with a gamete of the opposite sex. There are many potential medical benefits as well. For example, women who cannot make healthy eggs could use this technology to ensure healthy offspring.

Like many new technologies, transforming cells into eggs simultaneously resolves existing ethical issues and creates new ones. For example, since the embryonic stem cells spontaneously transformed themselves into eggs, this procedure overcomes many of the ethical objections to cloning, which involves creating offspring from a single parent. On the other hand, it paves the way for the creation of "designer eggs" from scratch and, if performed with human cells, could redefine the biological definitions of mothers and fathers.

In September 2003 efforts to transform stem cells into sperm were successful. Toshiaki Noce and his colleagues in Tokyo observed male mouse embryonic stem cells that developed spontaneously, with some cells actually becoming germ cells. When the researchers transplanted the germ cells into testicular tissue, the cells underwent meiosis and formed sperm cells. One possible medical application of this technology would be to assist couples who are infertile because the male cannot produce healthy sperm. One of the ethical issues that might arise would be the potential for two men to both be biological fathers of a child. Another is the potential to generate a human being who never had any parents using two laboratory-grown stem cells, one transformed into a sperm and the other into an egg. Many ethicists advise consideration of such issues before permitting human experimentation.

In November 2004 researchers from the University of Pennsylvania School of Veterinary Medicine used cells from mice to grow sperm progenitor cells in a laboratory culture. Known as spermatogonial stem cells, the progenitor cells are incapable of fertilizing egg cells but give rise to cells that develop into sperm. The researchers transplanted the cells into infertile mice, which were then able to produce sperm and father offspring that were genetically related to the donor mice.

This breakthrough has many potential applications, including developing new treatments for male infertility and extending the reproductive lives of endangered species. Researchers also will attempt to genetically manipulate the sperm cells grown in a culture medium and then implant the cells into animals. In this way they could introduce new traits into laboratory animals and livestock, such as disease resistance. The culture technique offers researchers additional opportunities to investigate the potential of spermatogonial stem cells as a source for adult stem cells to replace diseased or injured tissue.

OPINIONS SHAPE PUBLIC POLICY

The difficulty and low success rate of much animal reproductive cloning (an average of just one or two viable offspring result from every 100 attempts) and the as-yet-inadequate understanding about reproductive cloning have prompted many scientists and physicians to deem it unethical to attempt to clone humans. Many attempts to clone mammals have failed, and about one-third of clones born alive suffer from anatomical, physiological, or developmental abnormalities that are often debilitating. Some cloned animals have died prematurely from infections and other complications at rates higher than conventionally bred animals, and some researchers anticipate comparable outcomes from human cloning. Furthermore, scientists cannot yet describe or characterize how cloning influences intellectual and emotional development. While the attributes of intelligence, temperament, and personality may not be as important for cattle or other primates, they are vital for the health and well-being of humans. Without considering the myriad religious, spiritual, social, and other ethical concerns, the presence of so many unanswered questions about the science of reproductive cloning has prompted many investigators to consider any attempts to clone humans as scientifically irresponsible, unacceptably risky, and morally unallowable.

On August 9, 2001, President George W. Bush announced his decision to allow federal funds to be used for research on existing human embryonic stem cell lines as long as the derivation process (which begins with the removal of the inner cell mass from the blastocyst) had already been initiated and the embryo from which the stem cell line was derived no longer had the possibility of development as a human being. The president established the following criteria that research studies must meet to qualify for federal funding:

• The stem cells must have been drawn from an embryo created for reproductive purposes that was no longer needed for these purposes.
• Informed consent must have been obtained for the donation of the embryo and no financial inducements provided for donation of the embryo.

In January 2002 the Panel on Scientific and Medical Aspects of Human Cloning was convened by the National Academy of Sciences; the National Academy of Engineering; the Institute of Medicine Committee on Science, Engineering, and Public Policy; and the National Research Council, Division on Earth and Life Studies Board on Life Sciences. Following the panel, a report was issued that called for a ban on human reproductive cloning. The report concluded that human reproductive cloning would be dangerous for the woman, fetus, and newborn, and was likely to fail. It cited as an example of potential harm the observation that since many eggs are needed for human reproductive cloning attempts, human experimentation might expose more women to health risks from high levels of hormones used to stimulate egg production or from the surgical procedures used to extract eggs, which are not risk-free.

The study panel did not address the issue of whether human reproductive cloning would be acceptable to society even if it became medically feasible and safe. The panel recommended a legally enforceable ban with substantial penalties as the best way to discourage human reproductive cloning experiments in both the public and private sectors. It cautioned that a voluntary measure might be ineffective because many of the technologies needed to accomplish human reproductive cloning are widely accessible in private clinics and other organizations that are not subject to federal regulations.

The panel did not, however, conclude that the scientific and medical considerations that justify a ban on human reproductive cloning are applicable to nuclear transplantation to produce stem cells. In view of their potential to generate new treatments for life-threatening diseases and advance biomedical knowledge, the panel recommended that biomedical research using nuclear transplantation to produce stem cells be permitted. Finally, the panel encouraged ongoing national discussion and debate about the range of ethical, societal, and religious issues associated with human cloning research.

On February 14, 2002, the world's largest general scientific organization, the American Association for the Advancement of Science (AAAS) affirmed a legally enforceable ban on reproductive cloning; however, the AAAS supported therapeutic or research cloning using nuclear transplantation methods under appropriate government oversight. Similarly, the American Medical Association (AMA), a national physicians' organization, issued a formal public statement against human reproductive cloning. The AMA statement cautioned that human cloning failures could jeopardize promising science and genetic research and prevent biomedical researchers and patients from realizing the potential benefits of therapeutic cell cloning.

On April 10, 2002, President Bush called on the Senate to back legislation banning all types of human cloning. In his plea to the Senate, Bush said:

Science has set before us decisions of immense consequence. We can pursue medical research with a clear sense of moral purpose or we can travel without an ethical compass into a world we could live to regret. Science now presses forward the issue of human cloning. How we answer the question of human cloning will place us on one path or the other.… Human cloning is deeply troubling to me, and to most Americans. Life is a creation, not a commodity. Our children are gifts to be loved and protected, not products to be designed and manufactured. Allowing cloning would be taking a significant step toward a society in which human beings are grown for spare body parts, and children are engineered to custom specifications; and that's not acceptable.… I believe all human cloning is wrong, and both forms of cloning ought to be banned, for the following reasons. First, anything other than a total ban on human cloning would be unethical. Research cloning would contradict the most fundamental principle of medical ethics, that no human life should be exploited or extinguished for the benefit of another.

On September 25, 2002, Elias Zerhouni, the director of the National Institutes of Health (NIH), testified before the Senate Appropriations Subcommittee on Labor, Health and Human Services, and Education in favor of advancing the field of stem cell research. Zerhouni exhorted Congress to continue to fund both human embryonic stem cell research and adult stem cell research simultaneously in order to learn as much as possible about the potential of both types of cells to treat human disease. He observed that many studies that do not involve human subjects must be performed before any new therapy is tested on human patients. These preclinical studies include tests of the long-term survival and fate of transplanted cells, as well as tests of the safety, toxicity, and effectiveness of the cells in treating specific diseases in animals. Zerhouni promised that trials using human subjects, the clinical research phase, would begin only after the basic foundation had been established.

Zerhouni explained NIH plans to increase the number of stem cell researchers by making this research attractive to most talented research scientists and soliciting grant applications to support training courses to teach investigators how best to grow stem cells into useful lines. He also described NIH efforts to address issues that restrict widespread availability of these stem cell sources, such as NIH agreements with four stem cell providers to allow researchers access to their cells.

Moral and Ethical Objections to Human Cloning

People who argue against human cloning are as varied as the interests and institutions they support. Religious leaders, scientists, politicians, philosophers, and ethicists have argued against the morality and acceptability of human cloning. Nearly all objections hinge, to various degrees, on the definition of human life, beliefs about its sanctity, and the potentially adverse consequences for families and society as a whole.

In an effort to stimulate consideration of and debate about this critical issue, the President's Council on Bioethics examined the principal moral and ethical objections to human cloning in Human Cloning and Human Dignity: An Ethical Inquiry (Washington, DC: 2002). The Council's report distinguished between therapeutic and reproductive cloning and outlined key concerns by posing and endeavoring to respond to many as yet unresolved questions about the ethics, morality, and societal consequences of human cloning.

The Council determined that the key moral and ethical objections to therapeutic cloning—cloning for biological research—center on the moral status of developing human life. Therapeutic cloning involves the deliberate production, use, and ultimately, destruction of cloned human embryos. One reason opponents object to therapeutic cloning is that cloned embryos produced for research are no different from those that could be used in attempts to create cloned children. Another argument that has been made is that the means do not justify the ends—that research on any human embryo is morally unacceptable, even if this research promises cures for many dreaded diseases. Finally, there are concerns that acceptance of therapeutic cloning will lead society down the "slippery slope" to reproductive cloning, a prospect that is almost universally viewed as unethical and morally unacceptable.

The unacceptability of human reproductive cloning stems from the fact that it challenges the basic nature of human procreation, redefining having children as a form of manufacturing. Human embryos and children may then be viewed as products and commodities rather than sacred and unique lives. Further, reproductive cloning might substantially change fundamental issues of human identity and individuality, and by allowing parents unprecedented genetic control of their offspring, has the potential to significantly alter family relationships across generations.

The Council concluded that "the right to decide" whether to have a child does not include the right to have a child by any means possible, nor does it include the right to decide the kind of child one is going to have. A societal commitment to freedom does not require use or acceptance of every technological innovation available.

Legislation Aims to Completely Ban Human Cloning

On February 27, 2003, the House of Representatives voted to outlaw all forms of human cloning. The legislation, which passed with a vote count of 241 to 155, prohibits the creation of cloned human embryos for medical research as well as the creation of cloned babies. It contains strong sanctions, imposing a maximum penalty of $1 million in civil fines and as many as ten years in jail for violations. The measure did not pass in the Senate, which is closely divided about whether therapeutic cloning should be prohibited along with reproductive cloning. In early February 2003 President Bush issued a policy statement that strongly supported a total ban on cloning. In the Senate two bills were introduced: S.245 was a complete ban intended to amend the Public Health Service Act to prohibit all human cloning, and S.303 was a less sweeping measure that also prohibited cloning but protected stem cell research.

In 2003 a total of five bills were introduced in the House and two in the Senate. The House did not hold any hearings, although it passed H.R. 534, the Human Cloning Prohibition Act of 2003. H.R. 534 would prohibit both reproductive and therapeutic cloning and institute a criminal penalty of up to ten years in prison for violations. The Senate held three hearings on cloning in 2003. Two were held by the Senate Commerce, Science, and Transportation Subcommittee on Science, Technology, and Space and one by the Senate Committee on the Judiciary.

While nearly all lawmakers concur that Congress should ban reproductive cloning, many disagree about whether legislation should also ban the creation of cloned human embryos that serve as sources of embryonic stem cells. Many legislators agree with scientists that stem cells derived from cloned human embryos have medical and therapeutic advantages over those derived from conventional embryos or adults. Those who oppose the legislation calling for a total ban assert that the aim of allowing research is to relieve the suffering of people with degenerative diseases. They say that the bill's sponsors are effectively thwarting advances in medical treatment and biomedical innovation.

Supporters of the total ban contend that Congress must send an unambiguous message that cloning research and experimentation will not be tolerated. They consider cloning immoral and unethical, fear unintended consequences of cloning, and feel they speak for the public when they assert that it is not justifiable to create human embryos simply for the purpose of experimenting on them and then destroying them.

The range of positions on cloning in Congress is reflected in the sweeping bans already enacted in Iowa and Michigan, as well as the California prohibition against reproductive cloning. Several states impose civil penalties for violations, while Michigan has instituted criminal penalties.

On March 11, 2003, the AAAS held a workshop to discuss the legal and scientific considerations of regulatory issues governing human cloning initiatives. In Regulating Human Cloning, a report summarizing the event, the AAAS described a range of ethical and operational issues, including:

• Concerns about egg donation—the sources of donor eggs and the mechanisms to prevent conflicts of interest among physicians, researchers, research sites, and fertility clinics
• Research procedures—development of and consensus about stringent guidelines for responsible conduct of research cloning, including provisions that embryos may not be allowed to develop beyond fourteen days
• Risk assessment—the role of existing regulatory agencies in preventing errors, misuse of technology, and illicit reproductive cloning
• Access and delivery of products—determining who will gain access to new or unique therapies and whether the Food and Drug Administration would have to approve each derived stem cell line
• Regulatory structure—centralized or collaborative agency oversight and development of entirely new regulatory agencies

The fact that there are only about twenty available stem cell lines prompted the introduction of bills during the spring of 2004 that would require funding for human embryonic stem cell research, despite the President's 2001 policy. On April 28, 2004, more than 200 members of the House and Senate sent letters to the President arguing in favor of an expansion of existing policy. Pleas from patient advocacy groups—along with the death of former President Ronald Reagan from Alzheimer's disease on June 5, 2004, and Nancy Reagan's appeals to expand the policy—focused considerable media attention on the issue during the summer of 2004. On June 9, 2004, H.R. 4531, the Ronald Reagan Memorial Stem Cell Research Act of 2004, was introduced. It required that:

• The Secretary of Health and Human Services, acting through the Director of NIH, conduct and support research using human embryonic stem cells
• Research be conducted in accordance with the guidelines published in 2000; this requirement would apply regardless of any federal administrative policies established after the publication of such guidelines, including restrictions on the sources of human embryonic stem cells
• The amount of $87 million in FY 2005 and such sums as may be necessary thereafter be appropriated to fund the research

In addition to H.R. 4531, on March 11, 2004, the House introduced H.R. 3960, the Stem Cell Replenishment Act of 2004, which would permit federal funds to be used for research on human embryonic stem cells and require the NIH to revise the guidelines published in 2000 to ensure the availability of not less than sixty stem cell lines for research purposes. In June 2004 H.R. 4682, the Stem Cell Research Enhancement Act of 2004, was introduced. H.R. 4682 would support research with human embryonic stem cells that meets the following criteria:

• The stem cells must be derived from embryos that were created for fertility purposes, but not used, and donated from in vitro fertilization clinics.
• Prior to consideration of embryo donation, it must be determined that the embryos will never be implanted in a woman and would otherwise be discarded.
• Donation must be made with written informed consent and without any financial or other inducements.

The following month H.R. 4812, the Stem Cell Discovery through Diversity Act, was introduced. H.R. 4812 required the director of the NIH to conduct and support research using human embryonic stem cells. H.R. 4812 would prohibit the use of federal funds to derive such stem cells, establish an office within the Office of the Director of NIH (the Ronald Reagan Office of Human Stem Cell Research) to coordinate human embryonic stem cell research, and require the director of the NIH to ensure that the program includes donations from a significant number of individuals who are members of racial or ethnic minority groups. By the spring of 2005 no further action had been taken on any of the legislation introduced in 2004.

State Human Cloning Laws

As of 2005, ten states had enacted legislation that addresses human cloning. California was the first state to ban reproductive cloning in 1997. Since then, eight other states—Arkansas, Iowa, Michigan, Rhode Island, North Dakota, Virginia, New Jersey, and South Dakota—have passed laws prohibiting reproductive cloning. Missouri forbids the use of public funds for human cloning research. Louisiana also enacted legislation that prohibited reproductive cloning, but the law expired in July 2003. Arkansas, Iowa, Michigan, North Dakota, and South Dakota laws also prohibit therapeutic cloning. The Rhode Island law does not prohibit cloning for research, and California and New Jersey human cloning laws specifically permit cloning for the purpose of research. (See Table 8.1.)

California Leads the Way

In 2002 the California State Legislature passed a law encouraging therapeutic cloning. Despite the fact that there were no provisions for funds in the law, the move was interpreted as support for the research. The following year a bill to fund the research failed, so in 2004 stem cell research advocates offered voters a sweeping ballot measure—Proposition 71—to make public funding available to support stem cell research and therapeutic cloning. Proposition 71 was championed by Robert Klein, a wealthy real estate developer and father of a child with diabetes who might benefit from the research. It also received considerable financial support from Microsoft founder Bill Gates to finance campaign advertising and lobbying.

On November 2, 2004, Californians voted in Proposition 71, a ballot measure with the potential to make the state a leader in human embryonic stem cell research. Proposition 71 enabled the state to establish its own research institute—The California Institute for Regenerative Medicine—which prohibits reproductive cloning but will fund human cloning projects designed to create stem cells and allocate $3 billion over ten years in research funds that the Bush administration has to date refused to provide. Californians voted in favor of stem cell research in the hope that it will become the biggest, most important, and most profitable medical advancement of the twenty-first century. The legislation's supporters hoped to use these funds to attract top researchers and become the epicenter of groundbreaking, lifesaving, and potentially lucrative medical research.

A number of organizational and ethical questions about California's plan to publicly fund human cloning

projects for medical research remained unresolved in the spring of 2005. Among them is how to obtain the thousands of eggs needed to conduct the research. Concern about donor egg procurement has been expressed by a variety of Christian groups that consider cloning an immoral act that wantonly creates and destroys life for scientific purposes. Women's rights organizations also expressed concern, asserting that the potential for exploitation of poor women exists when profit-driven companies in need of donor eggs offer to pay women to take fertility drugs and harvest their eggs. They fear that some women may experience long-term adverse health consequences as a result of using fertility drugs. Testifying before a California State Legislative committee on March 9, 2005, Francine Coeytaux of the Pro-Choice Alliance for Responsible Research said, "This new technology will require eggs from thousands of women. Women will be the first human subjects of Proposition 71" (Paul Elias, "Cloning Sparks Concern over Egg Donors," Associated Press, March 10, 2005, http://news.yahoo.com/news?tmpl=story&cid=541&u=/ap/20050311/ap_on_he_me/stem_cells_donors&printer=1).

Opposing Viewpoints about Nuclear
Transplantation Research

The AAAS report summarized the arguments for and against nuclear transplantation research, the technology that is used for cloning. Those who favor this technology include scientists, patient advocacy groups, and the biotechnology industry. They perceive the debate about the moral and legal status of human embryos as relatively unimportant when compared to the prospect of cures arising from research using nuclear transplantation. They contend that a ban on implantation of the product of nuclear transplantation would be no more difficult to enforce than a ban on nuclear transplantation itself. They also fear that imposing criminal sanctions on scientific research would discourage innovation, limit research efforts, and effectively impede medical progress.

Opponents include religious conservatives, who assert that human embryos must be treated as human beings and as such should not be harmed or destroyed, even for the purpose of research. They contend that permitting nuclear transplantation would inevitably lead to reproductive cloning, because a ban on implantation would be nearly impossible to enforce. In an unusual alliance, religious conservatives are united in this stance with medical ethicists and environmental and women's rights activists, who may support nuclear transplantation but believe that it should be completely banned until its safety and effectiveness are ensured.

Changing Views about Cloning

An ABC News/Beliefnet Poll, conducted by telephone in August 2001, found that while 63% of Americans surveyed favored stem cell research, the majority opposed any form of cloning. Three-fifths (63%) opposed therapeutic cloning, and even more (87%) think human cloning should be against the law. Religion seemingly plays a part in such opinions—while 79% of evangelical Protestants and 65% of Catholics felt therapeutic cloning should be illegal, smaller numbers of nonevangelical Protestants (53%) and those who listed no religion (46%) felt the same way.

The December 2001 Gallup Poll survey "Americans Oppose Idea of Human Cloning," conducted following the Senate's failed attempt to impose a six-month moratorium on human embryo cloning, reported that opposition to reproductive cloning was overwhelming but that a majority of Americans (54%) supported therapeutic cloning for purposes of medical research or treatment. Americans opposed cloning for a variety of reasons: they felt it was at odds with their religious beliefs; they believed it interfered with distinctiveness and individuality; they feared it may be used for questionable purposes; and they were concerned that the technology used to clone may be dangerous.

The same analysis found that men were more supportive of therapeutic cloning than women were, and younger Americans were more supportive than were older Americans. Of Americans under age fifty, 60% supported therapeutic cloning, compared with 46% of those ages fifty and above. There were only slight differences in support according to political party, but those who described themselves as liberals (64%) and moderates (62%) were more supportive than those who called themselves conservatives (44%).

Interestingly, the February 2001 Time/CNN Poll asked Americans about specific circumstances in which human cloning would be justified. The greatest support (28%) was for producing copies of vital human organs to help save lives. About one in five respondents felt cloning would be justified either to save the life of the person being cloned or to help infertile couples to have children. The poll also found that most Americans do not expect that cloning will be possible or commonplace in the near future. Less than half (45%) of Americans felt it would be possible to create human clones in the next ten years, and 15% of respondents said it would never be possible to clone humans.

A May 2002 Gallup Poll found a subtle shift in public opinions about cloning. Although there was still resounding opposition to reproductive cloning—90% of those surveyed opposed it—there was far less opposition to therapeutic cloning. Only 37% of survey participants opposed cloning human organs or body parts for use in medical transplantation, and less than half (44%) opposed cloning human cells for use in medical research. Those who attended church regularly and those living in the Midwest and the South tended to disapprove of cloning more strongly. As expected, there was also a relationship between attitudes about abortion and about cloning, with 50% of Americans who described themselves as "pro-choice" favoring the cloning of human embryos and three-quarters of self-defined "pro-life" Americans opposing it.

Although the majority of all Americans staunchly opposed cloning for the purposes of creating a human being, reproductive cloning was favored by three times as many men as women. Similarly, more men than women favored using technology to clone human cells from adults for use in medical research.

The May 2002 Gallup Poll revealed that the overwhelming majority of Americans persisted in their belief that human and animal cloning are morally wrong, though there was somewhat more support for animal cloning than for human cloning. Americans objected not only to human cloning, but also to cloning pet animals, and the majority also opposed the cloning of endangered species to keep them from becoming extinct.

The December 27, 2002, announcement that a private firm had allegedly cloned a human baby sparked renewed public debate about cloning. A January 2003 Gallup Poll found that Americans remained strongly opposed to legalizing human cloning. In the January 14, 2003, Gallup Organization briefing "Americans View a Brave New World of Cloning," correspondent Deborah Jordan Brooks concluded that "the public is not, however, universally opposed to all kinds of cloning efforts. Many distinguish between cloning human cells for medical research and organs and body parts for medical transplants, and that designed to result in the actual birth of a human being."

In May 2004 another Gallup Poll found that slightly more Americans felt that cloning animals was acceptable than in the previous year, but the moral acceptability of cloning humans remained about the same—7% in 2001 and 2002 versus 9% in 2004. (See Table 8.2 and Table 8.3.) Still, the gap between the perceived moral acceptability of cloning animals and humans looms large. Twice as many Americans feel it is morally wrong to clone animals (64% versus 32%), while 88% see human cloning as morally wrong; just 9% believe it is morally acceptable.

Similarly, Americans' views about stem cell research were essentially unchanged from 2002 to 2004. In 2004 a scant 2% more respondents deemed medical research using stem cells as morally acceptable. (See Table 8.4.) Slightly more than half (54%) felt stem cell research was acceptable, while 37% believed it was morally wrong. (See Table 8.4.) Interestingly, despite their largely Republican political affiliations, affluent Americans tend to hold more liberal views about stem cell research and cloning than less well-to-do Americans. Forty-two percent of wealthier respondents believed it was morally acceptable to clone animals compared with 27% of less affluent respondents; 9% more affluent respondents than nonaffluent respondents felt it was morally acceptable to conduct embryonic stem cell research. (See Figure 8.6.)

TEENS' VIEWS ABOUT THE MORALITY OF CLONING.

An August 2003 Gallup Youth Survey asked teens whether they believed cloning animals and humans is

morally acceptable or morally wrong. The majority of teens said that cloning animals and humans is morally wrong. Just 20% of the teens surveyed felt that cloning humans is morally acceptable. (See Figure 8.7.)

Girls were less likely than boys to find cloning acceptable. Twice as many boys (43%) as girls (20%) said they believed that cloning animals is morally acceptable, and three times as many boys (30%) as girls (10%) felt that cloning humans is morally acceptable. (See Figure 8.8.) Not unexpectedly, attitudes varied widely among teens who attended church or synagogue regularly and those who did not. Far fewer churchgoing teens found cloning animals to be morally acceptable (23% compared with 39%). The gap was even greater when it came to cloning humans—just 9% of churchgoing teens deemed it morally acceptable compared with 29% of nonchurchgoers. (See Figure 8.9.)

GLOBAL POLICIES ON HUMAN CLONING

In many parts of the world there are laws prohibiting reproductive cloning and pending legislation banning therapeutic cloning, experimentation on embryos, and other types of genetic manipulation. The information in this section was largely drawn from research and materials prepared by Global Lawyers and Physicians, a nonprofit, nongovernmental organization that focuses on health and human rights issues.

In North America Canada's 1995 Moratorium on New Reproductive and Genetic Technologies was reaffirmed with the March 29, 2004, introduction of Bill C-6—An Act Respecting Assisted Human Reproduction—which stipulates that "no person shall knowingly create a human clone, or transplant a human clone into a human being."

In the United States the President's Council on Bioethics issued a report on July 10, 2002, endorsing the prohibition of reproductive cloning and a moratorium on therapeutic cloning. In 2004 President Bush called on the Senate to adopt legislation to ban both reproductive and therapeutic cloning.

Mexico's 1997 General Health Law, which implicitly prohibits human cloning, was under review in 2005, and the Mexican government was debating a bill originally introduced in 2002 that bans manipulation of an embryo's genetic code. On January 15, 2004, Panama enacted a law prohibiting human cloning. Throughout South America there are comparable laws prohibiting cloning, although Brazil's legislation permits intervention in human genetic material for the treatment of genetic defects.

The Council of Europe's January 1998 Convention on Human Rights and Biomedicine strictly prohibited efforts to create a human being genetically identical to another human being and permitted interventions to modify the human genome only for preventive, diagnostic, or therapeutic purposes and only when its aim is not to modify the genome of any descendants. Austrian law does not ban the cloning of human beings but limits research on human embryos. The law stipulates that embryos can be used only for implantation in the donor and may not be used for other purposes, and the donation of embryos or gametes is prohibited. Belgian law prohibits reproductive cloning but does permit research on embryos under stringent conditions. Legislation in Finland, France, the Republic of Georgia, Hungary, and the Netherlands prohibits modifying the germ line but permits research performed to cure or prevent hereditary diseases.

In February 2004 Italy passed the "Assisted Medical Procreation Law," which prohibits "selection, manipulation, or any other procedure directed at altering the genetic patrimony/heritage of the embryo or the gamete, or to predetermine their genetic characteristics, with the exception of diagnostic and therapeutic purposes." The law also forbids "cloning interventions by means of nuclear transfer or early embryo splitting whether for reproductive or therapeutic purposes."

In December 2001 Sweden moved toward enacting legislation affirming that "creating embryos through somatic cell nuclear transfer for therapeutic purposes can be ethically defensible." Among other stipulations, Switzerland's Federal Order of December 1998 on the Revision of the Federal Constitution states that "the Confederation shall legislate on the use of the human germ-line and genetic heritage. In doing so, it shall ensure that human dignity, personhood, and the family are protected." In November 2004 Switzerland approved by referendum the Federal Act on Research on Surplus Embryos and Embryonic Stem Cells, which prohibits both the creation of embryos for research purposes (therapeutic cloning) and cloning for reproductive purposes.

In the United Kingdom therapeutic cloning is governed by the Human Fertilisation and Embryology Act of 1990, which was amended to permit therapeutic cloning research on January 31, 2001. In February 2005 Professor Ian Wilmut, the scientist who cloned Dolly the sheep, and his colleagues Dr. Paul de Sousa and Professor Christopher Shaw were granted a license to clone human embryos for medical research.

In January 2004 the Ukraine instituted a ban on human reproductive cloning, but cloning for research or therapeutic purposes was not prohibited in the Ban on Human Reproductive Cloning Bill.

Japan, China, and Singapore maintain less than a complete ban on human cloning. In effect since 2001, the Japanese Law Concerning Regulation Relating to Human Cloning Techniques and Other Similar Techniques prohibits the transfer of embryos created by techniques of human cloning, but it permits the application of such for research purposes as long as the embryo created is not allowed to be transplanted into a human or an animal. On July 18, 2002, Singapore approved legislation permitting therapeutic cloning under strict regulations, but the Human Cloning and Other Prohibited Practices Bill of September 2, 2004, clearly prohibits human reproductive cloning, including the following stipulations:

• No person shall place any human embryo clone in the body of a human or the body of an animal.
• No person shall develop any human embryo, that is created by a process other than the fertilization of a human egg by human sperm, for a period more than fourteen days, excluding any period when the development of the embryo is suspended.
• Prohibition against developing a human embryo outside the body of a woman for more than fourteen days.
• Prohibition against collecting viable human embryos from the body of a woman.
• Prohibition against placing prohibited embryos in the body of a woman.
• Prohibition against importing and exporting prohibited embryos.
• Prohibition against commercial trading in human eggs, human sperm, and human embryos

In August 2003 China's Ministry of Health issued its "Ethical Principles on Assisted Reproductive Technologies for Human Beings and Human Sperm Bank," which permits cloning for research and therapeutic purposes. In January 2004 the Chinese Ministry of Science and Technology and Ministry of Health issued "Ethical Guidelines on Human Embryonic Stem Cells," which prohibited research for human reproductive cloning.

In the Middle East only Israel has legislation governing genetic interventions. Its 1998 prohibition introduced a five-year moratorium on human reproductive cloning and germ line engineering. The purpose of the moratorium was to "determine a prescribed period of five years during which no kind of genetic intervention shall be performed on human beings in order to examine the moral, legal, social, and scientific aspects of such kinds of intervention and the implications of such for human dignity." Israel's Law 5759–1999—Prohibition of Genetic Intervention (Human Cloning and Genetic Manipulation of Reproductive Cells) was amended in March 2004 to strictly prohibit reproductive cloning and genetic intervention such as germ line gene therapy.

South Africa's Law on Human Tissue 1983 bans the cloning of human cells; however, it has been amended to read that gene modification of the human germ line should not yet be attempted, offering the possibility of sanctioning future research efforts. Australia reinforced its anti-cloning stance with the January 7, 2003, enactment of the Prohibition of Human Cloning Act No. 144–2002, which "prohibits human cloning and other unacceptable practices associated with reproductive technology and for related purposes." In 2004 New Zealand enacted its Human Assisted Reproductive Technology Act No. 92, which prohibits:

• Artificially forming for reproductive purposes a cloned embryo. For the purposes of this item, a cloned embryo is not formed by splitting, on one or more occasions, an embryo that has been formed by the fusion of gametes.
• Artificially forming for reproductive purposes a hybrid embryo.
• Implanting into a human being a cloned embryo; an animal gamete or embryo; a hybrid embryo; a genetically modified gamete, human embryo, or hybrid embryo; gametes derived from a fetus, or an embryo that has been formed from a gamete or gametes derived from a fetus.
• Implanting into an animal a human gamete, human embryo, or a hybrid embryo.

The United Nations Addresses Human Cloning

In November 2004 the United Nations General Assembly set up an informal group to endeavor to negotiate a nonbinding statement to guide countries on cloning and embryonic stem cell research. The United States and a group of mostly developing nations were agitating for stricter policies, while European countries and Japan sought greater laxity for scientific research.

A draft guideline introduced by Belgium and supported by more than twenty countries—including Japan and many European nations—would ban reproductive cloning and allow governments to determine whether to allow some stem cell and other research. The rival draft guideline, supported by the United States, Costa Rica, and more than sixty other countries—mainly developing nations—would ban all human cloning in all countries that ratified it.

In view of the divisiveness of this issue and the disparate viewpoints, it is not surprising that the UN diplomats failed to reach agreement on a nonbinding declaration that would encourage governments to adopt laws on human cloning that would be acceptable to both advocates and opponents of stem cell research. In February 2005 the bitterly divided UN General Assembly committee adopted a nonbinding declaration calling on governments to prohibit all forms of human cloning, including techniques used in research on human stem cells. The resolution calls on member states to enact legislation "to prohibit all forms of human cloning in as much as they are incompatible with human dignity and the protection of human life, adopt the measures necessary to prohibit the application of genetic engineering techniques that may be contrary to human dignity, and to take measures to prevent the exploitation of women in the application of life sciences."

Although the resolution is nonbinding and serves only as a recommendation as opposed to a legal requirement, the United States and other countries seeking to ban all forms of human cloning considered the UN declaration a victory.

Cloning Genes

Gene cloning, or molecular cloning, has several different meanings to a molecular biologist. A clone is an exact copy, or replica, of something. In the literal sense, cloning a gene means to make many exact copies of a segment of a DNA molecule that encodes a gene. This is in marked contrast to cloning an entire organism—regenerating a genetically identical copy of the organism—which is technically much more difficult (with animals) and can involve ethical ramifications not associated with gene cloning. Molecular biologists exploit the replicative ability of cultured cells to clone genes.

Purposes of Gene Cloning

To study genes in the laboratory, it is necessary to have many copies on hand to use as samples for different experiments. Such experiments include Southern or Northern blots, in which genes labeled with radioactive or fluorescent chemicals are used as probes for detecting specific genes that may be present in complex mixtures of DNA.

Cloned genes also make it easier to study the proteins they encode. Because the genetic code of bacteria is identical to that of eukaryotes , a cloned animal or plant gene that has been introduced into a bacterium can often direct the bacterium to produce its protein product, which can then be purified and used for biochemical experimentation. Cloned genes can also be used for DNA sequencing, which is the determination of the precise order of all the base pairs in the gene. All of these applications require many copies of the DNA molecule that is being studied.

Gene cloning also enables scientists to manipulate and study genes in isolation from the organism they came from. This allows researchers to conduct many experiments that would be impossible without cloned genes. For research on humans, this is clearly a major advantage, as direct experimentation on humans has many technical, financial, and ethical limitations.

Cloning Techniques

Cloning genes is now a technically straightforward process. Usually, cloning uses recombinant DNA techniques, which were developed in the early 1970s by Paul Berg, of Stanford University, and, independently, by Stanley Cohen and Herbert Boyer, of Stanford and the University of California. These researchers devised methods for excising genes from DNA at precise positions, using restriction enzymes and then using the enzyme known as DNA ligase to splice the resulting gene-containing fragment into a plasmid vector .

Plasmids are small, circular DNA molecules that occur naturally in many species of bacteria. The plasmids naturally replicate and are passed on to future generations of bacterial cells. To replicate, all plasmids must contain a sequence, called an origin of replication, which directs the bacterial DNApolymerase to replicate the DNA molecule. In addition, recombinant plasmids contain one or more selectable markers. A selectable marker is a gene that confers on the bacterium harboring the plasmid the ability to survive under conditions in which bacteria lacking the plasmid would otherwise die. Usually, such genes encode enzymes that enable the bacterium to live and grow despite the presence of an antibiotic drug.

The recombinant plasmid is then introduced into a host cell, such as an Escherichia coli bacterium, by a process called transformation, and the cell is allowed to multiply and form a large population of cells. Each of these cells harbors many identical copies of the recombinant plasmid. The cells are then cultured in growth media containing the antibiotic to which the plasmid confers resistance. This ensures that only cells containing the recombinant plasmid will survive and replicate. A researcher then harvests the cells and can extract and purify many copies of the plasmid.

Another method to produce many copies of a DNA molecule, which is even simpler than traditional recombinant cloning methods, is the polymerase chain reaction (PCR). PCR amplifies the DNA in a reaction tube without the need for a plasmid to be grown in bacteria.

Importance for Medicine and Industry

The ability to clone a gene is not only valuable for conducting biological research. Many important pharmaceutical drugs and industrial enzymes are produced from cloned genes. For example, insulin, clotting factors, human growth hormone, cytokines (cell growth stimulants), and several anticancer drugs in use are produced from cloned genes.

Before the advent of gene cloning, these proteins had to be purified from their natural tissue sources, a difficult, expensive, and inefficient process. Using recombinant methods, biomedical companies can prepare these important proteins more easily and inexpensively than they previously could. In addition, in many cases the product that is produced is more effective and more highly purified. For example, before the hormone insulin, which many diabetes patients must inject, became available as a recombinant human protein, it was purified from pig and cow pancreases. However, pig and cow insulin has a slightly different amino acid sequence than the human hormone. This sometimes led to immune reactions in patients. The recombinant human version of the hormone is identical to the natural human version, so it causes no immune reaction.

Gene cloning is also used to produce many of the molecular tools used to study genes. Even restriction enzymes, DNA ligase, DNA polymerases, and many of the other enzymes used for recombinant DNA methods are themselves, in most cases, produced from cloned genes, as are enzymes used in many other industrial processes.

Genomic Versus cDNA Clones

A gene can take varying forms, and so can gene clones. The proteincoding regions of most eukaryotic genes are interrupted by noncoding sequences called introns, which are ultimately excluded from the mature messenger RNA (mRNA) after the gene is transcribed. In addition to the protein-coding sequences, all genes contain "upstream" and "downstream" regulatory sequences that control when, in which tissues, and under what circumstances the gene is transcribed. A clone containing the entire region of a gene as it exists on the chromosome, including introns and nontranscribed regulatory sequences, is called a genomic clone because it is derived directly from genomic, or chromosomal, DNA.

It is also possible to clone a gene directly from its messenger RNA transcript, from which all introns have been removed. This type of clone, called a complementary DNA or cDNA clone, includes only the protein-coding sequences and upstream and downstream sequences that do not code for amino acids but that may control how the mRNA transcript gets translated to protein.

To prepare cDNA a researcher starts with mRNA and then makes a complementary single-stranded DNA copy using the enzyme reverse transcriptase. Reverse transcriptase is a DNA polymerase that synthesizes DNA based on an RNA template that is produced by retroviruses. After the mRNA strand is digested away by another enzyme, called RNase H, DNA polymerase can synthesize a second DNA strand by using the newly made first strand cDNA as a template.

Because cDNAs lack introns, the protein-coding region in a cDNA molecule is contained in a single uninterrupted sequence, called an open reading frame, or ORF. This makes cDNA clones extremely useful for predicting the amino acid sequence of the protein that a gene encodes. It also makes it possible to direct protein synthesis from a eukaryotic cDNA clone in a bacterium, which cannot splice introns. With introns still present in a cloned gene, the bacteria will misinterpret the intron sequences as protein-encoding sequences. The resulting incorrect messanger RNA will encode a protein with an incorrect amino acid.

"Gene Cloning" Usually Means "Gene Identification"

When researchers report in a scientific journal that they have "cloned a gene" they are not referring to the rather mundane process of amplifying copies of a DNA molecule. What they are really talking about is the molecular identification of a previously unknown gene, and determination of its precise position on a chromosome. There are many different methods that can be used to identify a gene. Two of the most common approaches are discussed below.

A gene can be defined in several ways. In fact, the concept of the gene is undergoing a re-evaluation as scientists are analyzing the complete genomes of more and more organisms and finding that many sequences encode more than one protein product. Gregor Mendel identified genes—for example, he identified the factor that made peas either yellow or green—long before he or anyone else knew that genes were encoded on segments of the DNA that made up chromosomes. Studying genetics in the fruit fly, Drosophila melanogaster, Morgan and Sturtevant demonstrated that genes are entities that reside at measurable locations, or loci, on chromosomes, although they did not yet understand the biochemical nature of genes.

Modern geneticists often use the same methods as Mendel and Morgan to identify genes by physical traits, or phenotypes, that mutations in them can cause in an organism. But today we can go even further. Using a broad range of molecular biology techniques, including gene cloning, researchers can now determine the precise DNA coding sequence that corresponds to a particular phenotype . This capability is tremendously powerful, because discovering the gene responsible for a trait can help humankind understand the cellular and biochemical processes underlying the trait. For example, geneticists have learned a great deal about the basis of cancer by identifying genes that, when mutated, contribute to cancer. By studying these genes, researchers now know that many of them control when cells divide (e.g., proto-oncogenes and tumor suppressor genes) or when they die (e.g., the apoptosis genes). Under some circumstances, when such genes are damaged by mutation, cells divide when they shouldn't, or don't die when they should, leading to cancer.

Positional Cloning

Positional cloning starts with the classical methods developed at the turn of the twentieth century by Thomas Hunt Morgan, Alfred Sturtevant, and their colleagues, of genetically mapping a particular phenotype to a region of a chromosome. A detailed discussion of genetic mapping is beyond the scope of this section, but, in general, it is based on conducting genetic crosses between individuals with two different mutant traits and analyzing how often the traits occur together in the progeny of subsequent generations.

Genetic mapping provides a general idea of where a gene is located on a particular chromosome, but it does not identify the precise DNA sequence that encodes the gene. The next step is to locate the gene on what is called the physical map of the chromosome. A physical map is a high-resolution map of all the DNA sequences that make up a chromosome. One type of physical map is a restriction map, which depicts the order of DNA fragments produced when a large DNA molecule is cut with restriction endonucleases (restriction enzymes).

Restriction maps have been made for the complete genomes of several model genetic organisms, such as the fruit fly (Drosophila melanogaster ), and the roundworm, (Caenorhabditis elegans ). For these organisms, individual large DNA fragments—on the order of forty to one hundred thousand base pairs from the whole genome—have been cloned in bacterial plasmid vectors to make a "library" of the genome. Each fragment is mapped to a known position, but the identify of the gene or genes it contains is originally unknown. To identify the genes, a cloned fragment is introduced into a mutant fly or roundworm.

To pinpoint the location of a particular gene, a researcher can introduce one or several of the plasmid clones from the physical map that are in the general vicinity of the region on the genetic map where the gene is thought to lie into a mutant that is defective in the gene of interest. If the introduced DNA corrects the mutant's defect, that DNA probably contains a normal copy of the defective gene. But these large clones usually contain several genes. By further "trimming" the DNA into smaller subfragments and testing the ability of each subfragment to rescue mutants, the researcher can eventually home in on the gene. As further confirmation that this gene is the cause of the mutant phenotype, the researcher can isolate the corresponding gene from the mutant and determine its DNA sequence to see if it contains a mutation (a DNA sequence alteration) relative to the normal gene sequence.

Expression Cloning

In some cases, a researcher becomes interested in studying a gene not because mutations in it cause an interesting phenotype but because the protein it encodes has interesting properties. A prominent example is beta-amyloid protein, which accumulates in the brains of Alzheimer's disease patients.

Expression cloning is a method of isolating a gene by looking for the protein it encodes. If the protein of interest is an enzyme, it can be found by testing for its biochemical activity. A very common method for identifying a particular protein is by using antibodies, or immunoglobulins, that bind specifically to that protein. Expression cloning usually uses a cDNA library, in which protein-coding sequences are uninterrupted by introns. Each cDNA is inserted into an "expression vector," which contains all the necessary signals for the DNA to be transcribed into mRNA. The mRNA can then be translated into protein. Thus the host cell harboring the clone will produce the gene's protein product, and the protein can then be detected by biochemical or immunologic methods. Once the cell making the protein is found, the cDNA can be re-isolated and the gene sequenced by standard means.

Gene cloning techniques continue to advance rapidly, aided by the Human Genome Project and bioinformatics. It is likely that positional cloning will take on a secondary role, and that bioinformatics and proteomics methods will begin to contribute more, as more progress in these fields is made.

see also Bioinformatics; Blotting; Chromosomes, Artificial; Cloning Organisms; Cloning: Ethical Issues; DNA Libraries; Gene; Gene Discovery; Human Genome Project; Linkage and Recombination; Marker Systems; Morgan, Thomas Hunt; Plasmid; Polymerase Chain Reaction; Recombinant DNA; Restriction Enzymes; Reverse Transcriptase; RNA Processing; Sequencing DNA; Transformation.

Paul J. Muhlrad

Bibliography

Alberts, Bruce, et al. Molecular Biology of the Cell, 4th ed. New York: Garland Science, 2002.

Lodish, Harvey, et al. Molecular Cell Biology, 4th ed. New York: W. H. Freeman and Company, 2000.

Micklos, David A., and Greg A. Freyer. DNA Science: A First Course in Recombinant DNA Technology. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1990.

Watson, James D., et al. Recombinant DNA, 2nd ed. New York: Scientific American Books, 1992.

Cloning

Cloning hit the news headlines in 1997 when scientists in Scotland announced they had successfully cloned a sheep, named Dolly, in 1996. Although several other animal species had been cloned in the previous 20 years, it was Dolly that caught the public's attention. Suddenly, the possibility that humans might soon be cloned jumped from the pages of science fiction stories into the mainstream press. Dolly was the first adult mammal ever cloned.

Cloning is the science of using artificial methods to create clones. A clone is a single cell, a group of cells, or an organism produced in a laboratory without sexual reproduction. In effect, the clone is an exact genetic copy of the original source, much like identical twins. There are two types of cloning. Blastomere separation, also called "twinning" after the naturally occurring process that creates identical twins, involves splitting a developing embryo soon after the egg is fertilized by sperm. The result is identical twins with DNA from both parents. The second cloning type, called nuclear transfer, is what scientists used to create Dolly. In cloning Dolly, scientists transferred genetic material from an adult female sheep to an egg in which the nucleus containing its genetic material had been removed.

Simple methods of cloning plants, such as grafting and stem cutting, have been used for more than 2,000 years. The modern era of laboratory cloning began in 1958 when the English-American plant physiologist Frederick C. Steward cloned carrot plants from mature single cells placed in a nutrient culture containing hormones, chemicals that play various and significant roles in the body.

The first cloning of animal cells occurred in 1964. In the first step of the experiment, biologist John B. Gurdon destroyed with ultraviolet light the genetic information stored in a group of unfertilized toad eggs. He then removed the nuclei (the part of an animal cell that contains the genes) from intestinal cells of toad tadpoles and injected them into those eggs. When the eggs were incubated (placed in an environment that promotes growth and development), Gurdon found that 1–2% of the eggs developed into fertile, adult toads.

The first successful cloning of mammals was achieved nearly 20 years later. Scientists in both Switzerland and the United States successfully cloned mice using a method similar to that of Gurdon. However, the Swiss and American methods required one extra step. After the nuclei were taken from the embryos of one type of mouse, they were transferred into the embryos of another type of mouse. The second type of mouse served as a substitute mother that went through the birthing process to create the cloned mice. The cloning of cattle livestock was achieved in 1988 when embryos from cows were transplanted to unfertilized cow eggs whose own nuclei had been removed.

Since Dolly, the pace and scope of cloning mammals has greatly intensified. In February 2002, scientists at Texas A&M University announced they had cloned a cat, the first cloning of a common domestic pet. Named "CC" (for carbon copy or copycat), the cat is an exact genetic duplicate of a two–year–old calico cat. Scientists cloned CC in December 2001 using the nuclear transfer method. In April 2002, a team of French scientists announced they had cloned rabbits using the nuclear transfer process. Out of hundreds of embryos used in the experiment, six rabbits were produced, four that developed normally and two that died. Two of the cloned rabbits mated naturally and produced separate litters of seven and eight babies

The first human embryos were cloned in 1993 using the blastomere technique that placed individual embryonic cells (blastomeres) in a nutrient culture where the cells then divided into 48 new embryos. These experiments were conducted as part of some studies on in vitro (out of the body) fertilization aimed at developing fertilized eggs in test tubes that could then be implanted into the wombs of women having difficulty becoming pregnant. However, these fertilized eggs did not develop to a stage that was suitable for transplantation into a human uterus.

Research into cloning humans also picked up greatly following the success of Dolly. An Italian physician said in April 2002 that a woman was pregnant with what would be the world's first cloned human baby. The doctor, Severino Antinori, operates a fertility clinic near the Vatican in Rome. In March 2002, a Chinese researcher said she had cloned a human embryo to the blastocyst stage, the point at which stem cells can be harvested. Scientists in several other countries also are believed conducting human cloning experiments.

The cloning of cells promises to produce many benefits in farming, medicine, and basic research. In farming, the goal is to clone plants that contain specific traits that make them superior to naturally occurring plants. For example, field tests have been conducted using clones of plants whose genes have been altered in the laboratory by genetic engineering to produce resistance to insects, viruses, and bacteria. New strains of plants resulting from the cloning of specific traits have led to fruits and vegetables with improved nutritional qualities, longer shelf lives, and new strains of plants that can grow in poor soil or even under water.

A cloning technique known as twinning could induce livestock to give birth to twins or even triplets, thus reducing the amount of feed needed to produce meat. Cloning also holds promise for saving certain rare breeds of animals from extinction , such as the giant panda .

In medicine, gene cloning has been used to produce vaccines and hormones. Cloning techniques have already led to the inexpensive production of the hormone insulin for treating diabetes and of growth hormones for children who do not produce enough hormones for normal growth. The use of monoclonal antibodies in disease treatment and research involves combining two different kinds of cells (such as mouse and human cancer cells) to produce large quantities of specific antibodies. These antibodies are produced by the immune system to fight off disease. When injected into the blood stream, the cloned antibodies seek out and attack disease–causing cells anywhere in the body.

Despite the benefits of cloning and its many promising avenues of research, certain moral, religious, and ethical questions concerning the possible abuse of cloning have been raised. At the heart of these questions is the idea of humans tampering with life in a way that could harm society, either morally or in a real physical sense. Some people object to cloning because it allows scientists to "act like God" in manipulating living organisms.

The cloning of Dolly and the fact that some scientists are attempting to clone humans raised the debate over this practice to an entirely new level. A person could choose to make two or 10 or 100 copies of himself or herself by the same techniques used with Dolly. This realization has stirred an active debate about the morality of cloning humans. Some people see benefits from the practice, such as providing a way for parents to produce a new child to replace one dying of a terminal disease. Other people worry about humans taking into their own hands the future of the human race.

Another controversial aspect of cloning deals not with the future but the past. Could Abraham Lincoln or Albert Einstein be recreated using DNA from a bone, hair, or tissue sample? If so, perplexing questions arise about whether this is morally or ethically acceptable? Some scientists say that while it might be possible to do this, the clone might be identical in appearance and in some traits, it would not have the same personality as the original Lincoln. This is because Lincoln, like all people, was greatly shaped from birth by his environment and personal experiences in addition to his genetic coding. Although a duplicate of her mother, CC, the cloned calico cat, has a different color pattern on her fur. This is because environmental factors strongly influence her development in the womb.

Also, since the movie "Jurassic Park" was released in 1993, there has been considerable public discussion about the possibility of cloning dinosaurs and other prehistoric or extinct species. In 1999, the Australian Museum in Sydney, Australia , announced scientists were attempting to clone a thylacine (a meat–eating marsupial related to kangaroos and opossums). It has been extinct since 1932 but the museum has the body of a baby thylacine that has been preserved for 136 years. The problem is that today's cloning techniques are possible only with living tissue. Even the head of the project has doubts, saying the chance of cloning a living thylacine is 30% over the next 200 years.

[Ken R. Wells ]

RESOURCES

BOOKS

Cefrey, Holly. Cloning and Genetic Engineering (Life in the Future). New York: Children's Press, 2002.

Pence, Gregory E. Who's Afraid of Human Cloning? Lanham, MD: Rowman & Littlefield Publishers, 1998.

PERIODICALS

Gibbs, Nancy. "Baby, It–s You! And You, And You..." Time (Feb. 11, 2001).

Hobson, Katherine. "Pets of the Future." U.S. News & World Report (March 11, 2002): p. 46.

Masibay, Kim Y. "Copy Cat." Science World (March 25, 2002): p. 6–7.

McGovern, Celeste. "Brave New World." The Report Newsmagazine (April 29, 2002).

Pistoi, Sergio. "Father of the Impossible Children." Scientific American (April 2002): p. 38–40.

"The Clone Wars.' Business Week (March 25, 2002): p. 94.

Weidensaul, Scott. "Raising the Dead." Audubon (May–June 2002): p.
58–67.

ORGANIZATIONS

The Human Cloning Foundation, <http://www.humancloning.org>

Society for Developmental Biology, 9650 Rockville Pike, Bethesda, MD USA 20814 301–571–0647, Fax: 301–571–5704, Email: ichow@faseb.org, <http://www.sdb.bio.purdue.edu>

Cloning Organisms

There are two distinct types of cloning: molecular and organismal. Molecular cloning is the removal of a stretch of DNA, usually a gene, from an organism, and its insertion into another piece of DNA, such as a plasmid , to form a substance called recombinant DNA. This recombinant DNA may then be expressed in, or simply carried passively by, another organism, such as bacteria. Organismal cloning, the subject of this entry, is the production of genetically identical organisms and, as such, can be used to produce genetically identical copies of livestock or may be used to produce new members of endangered or even extinct species. It may be especially cost-effective to clone animals that produce therapeutic proteins such as blood clotting factors, thus combining both types of cloning. Cloning is controversial, however, because our understanding of the procedures needed to clone mammals may be applied to human cloning, which gives rise to profound ethical issues.

The History of Cloning

Cloning has a long history. Animals that reproduce sexually produce clones whenever identical twins are born. These twins are genetically indistinguishable, and are formed when a fertilized egg separates at a very early stage of development. Clones are also the natural product of asexual reproduction, although in this case perfect clones cannot be maintained through an infinite number of generations, because spontaneous mutations can and do occur. Lastly, clones can be produced by regeneration in both plants and animals. For example, plant cuttings will regenerate roots and, ultimately, an entire "new" plant, and some invertebrates, such as planaria, can regenerate two identical animals if the adult is cut in half. In these forms, cloning has been with us for a very long time.

Since the mid-1960s, scientists have been able to culture plant cells, that is, grow cells from plants such as tobacco and carrots in a petri dish, to get thousands of genetically identical cells. From such cultured cells an unlimited quantity of cloned plants can then be grown. These cultured cells can be modified to contain recombinant, or cloned, DNA as well.

Cloning Amphibians

The first cloning of a vertebrate by nuclear transfer was reported by John Gurdon of the University of Cambridge in the 1950s. In nuclear transplantation, the nucleus of an unfertilized donor egg is either mechanically removed or it is destroyed by ultraviolet light in a process called enucleation. The original nucleus is then replaced by a nucleus containing a full set of genes that has been taken from a body cell of an organism. This procedure eliminates the need for the fertilization of an egg by a sperm.

The most successful nuclear transplants have been achieved after serially transferring donor intestinal nuclei, that is, putting an adult nucleus from an intestinal cell into an egg whose nucleus was destroyed, allowing the egg to divide only a certain number of times, removing nuclei from these cells, and repeating this process several times before allowing the embryo to complete development. Eventually, transplantation of nuclei from albino embryonic frog cells into enucleated eggs from a dark green female frog led to the production of adult albino frog clones, demonstrating that a properly treated adult nucleus could support the full development of an egg into an adult clone. Later experiments demonstrated that nuclei from cells of other tissues, even quiescent cells such as blood cells, could also be used if properly treated. Despite these successes, no adult frog has been cloned when a nucleus from an adult cell was used without serial transfer. Without serial transfer of the nuclei, the animals would only develop to the tadpole stage, and then they would die.

Cloning of Mammals: Dolly

Nuclear transplantation has also been successful in producing mammalian clones, most notably of sheep, cattle, pigs, and mice. The most famous cloned mammal is a sheep named "Dolly," the first animal to be cloned directly from an adult cell. Experiments leading to the birth of Dolly were done at the Roslin Institute with collaborators at Pharmaceutical Proteins Limited, both in Scotland. This group had earlier produced Megan and Morag, the first mammals to be cloned from cultured cells. These two sheep were produced from embryonic cells, however, not from cells of an adult animal.

Dolly was born in the summer of 1996, the product of a nucleus from the mammary gland of a six-year-old female Finn-Dorsett sheep and an egg from a Scottish Blackface female. Mammary gland cells were grown in a petri dish and were deprived of nutrients so that they would stop dividing, just like an unfertilized egg. Donor eggs were taken from sheep soon after ovulation , and nuclei were mechanically removed from them. These enucleated eggs were then fused with the cultured mammary gland cells so that a mammary gland nucleus would be inside an unfertilized egg. Two hundred and seventy-seven such embryos were constructed and temporarily allowed to divide in a petri dish, and then all of them were transferred into the oviduct of a temporary surrogate mother. Of the original 247 embryos, only 29 developed further, and these were transferred to 13 hormonally treated surrogate mothers.

Only one surrogate mother became pregnant, and she only had one live lamb, named Dolly. The success rate was very low, but Dolly has been proven to be a true clone: She has all the characteristics of a Finn-Dorsett sheep. Independent scientists used a technique called DNA fingerprinting to show that Dolly's DNA matched the donor mammary cells but did not match that of other sheep in the Finn-Dorsett flock, nor did her DNA match that of her surrogate mother or the egg donor. Similar results have been obtained by Ryuzo Yanagimachi at the University of Hawaii, who worked with several generations of cloned mice.

In 1997 Polly, a sheep created with a combination of both molecular and organismal cloning techniques, was born. Polly was derived from a fetal sheep cell that had been engineered to contain the human gene that makes coagulation factor IX. Factor IX is missing in people with a disease called hemophilia type B. Polly and two other sheep were engineered to produce factor IX in their milk, thus providing people with hemophilia access to a safer and less expensive source of clotting factor than was previously available. Because Polly was made from more easily cultured and, therefore, more easily engineered embryonic cells, it is thought that this type of cloning technology holds the most promise for the future of pharmaceutical production of proteins that cannot be made in bacteria.

In January 2001, the first cloned member of an endangered species was born. This was a gaur, a wild ox native to India and southeast Asia, which the researchers named Noah. The gaur was chosen by Advanced Cell Technology as a candidate for cloning after the company had successfully cloned domestic cattle, which are related to the gaur species.

The embryo from which Noah developed was created from the nuclei of frozen skin cells that had been taken from an adult male gaur that had died eight years earlier. Skin cell nuclei were fused with enucleated domestic cow eggs to produce forty embryos. One of these forty was carried to full term in a surrogate cow mother. Unfortunately, Noah died of an infection two days after his birth (the infection is thought to be unrelated to his origin as a cloned animal). Despite Noah's death, it is likely that cloning will eventually be used to aid the conservation of endangered species. In the future, scientists may attempt to clone a recently extinct species, should intact DNA for an extinct species be obtained.

Problems with Cloning

In general, the success rate of mammalian cloning is low, with less than 0.1 to 2.0 percent of transplanted nuclei yielding a live birth. The vast majority of transplants fail to divide or to develop normally, indicating there is much we still do not understand about reprogramming an adult nucleus to support embryonic development. One thing that is clear, however, is that having both the donor cell and host egg cell in a nondividing state is essential for success.

What might be both the most vexing and most interesting problem with cloning is related to aging. Chromosomes "show their age" by a shortening in their tips, or telomeres , a process that occurs every time the cell they are in divides. This telomere shortening occurs in all cells except eggs, sperm, and most cancer cells, and shortened telomeres are correlated with the aging of organisms. Since the nuclear DNA in most cloned animals is taken from an adult, the chromosomes of cloned animals are expected to have shorter telomeres than animals of the same birth age that are produced by sexual reproduction, causing researchers to wonder whether cloned animals will age prematurely. Shorter telomeres have been found in Dolly and other cloned sheep, but telomeres are reported not to be shorter in cloned mice or cattle. Underlying reasons for the different results may include differences between cell types or species used.

The Myth of the Perfect Clone

Cloned animals are not 100 percent identical to their "parents." Whenever nuclear transplantation is used to produce cloned organisms, the offspring display some differences from the organism that donated the nuclei. The egg donor contributes mitochondria, the energy producers of eukaryotic cells, and these mitochondria have their own small amount of DNA-containing genes used for energy metabolism. Since mitochondria are inherited only with egg cytoplasm, they will not match the mitochondria of the animal from which the nucleus was taken. In addition, maternally derived gene products, both mRNA (messenger RNA) and protein, which serve to begin embryonic development, will differ from that of the nuclear donor, as will the uterine environment and the external environment. Thus, for example, clones produced by nuclear transplantation will be significantly less identical than will clones produced by twinning.

see also Cloning: Ethical Issues; Cloning Genes; Conservation Biology: Genetic Approaches; Hemophilia; Mitochondrial Genome; Reproductive Technology; Telomere; Transgenic Animals; Twins.

Elizabeth A. De Stasio

Bibliography

Gurdon, J. B., and Alan Colman. "The Future of Cloning." Nature 402 (1999): 743.

Lanza, Robert P., Betsy L. Dresser, and Philip Damiani. "Cloning Noah's Ark." Scientific American (Nov., 2000): 84-89.

Wilmut, Ian. "Cloning for Medicine." Scientific American (Dec., 1998): 58-63.

Wilmut, Ian, Keith Campbell, and Colin Tudge. The Second Creation: Dolly and the Age of Biological Control. Cambridge, MA: Harvard University Press, 2000.