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.)