Letter of Transmittal to the President

Letter from the President

NBAC Roster

Staff Roster

Acknowledgments

Executive Summary

1. Introduction

2. The Science and Application of Cloning

3. Religious Perspectives

4. Ethical Considerations

5. Legal and Policy Considerations

6. Recommendations of the Commission

A. Glossary

B. List of Speakers

C. List of Commissioned Papers

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CLONING
HUMAN
BEINGS

February 24, 1997
Dr. Harold Shapiro
Chair
National Bioethics
     Advisory Commission
Suite 3C01
6100 Executive Boulevard
Bethesda, Maryland 20892-7508


Dear Dr. Shapiro:


As you know, it was reported today that
researchers have developed techniques to clone
sheep.  This represents a remarkable scientific
discovery, but one that raises important
questions.  While this technological advance
could offer potential benefits in such areas as
medical research and agriculture, it also raises
serious ethical questions, particularly with
respect to the possible use of this technology to
clone human embryos.


Therefore, I request that the National Bioethics
Advisory Commission undertake a thorough
review of the legal and ethical issues associated
with the use of this technology, and report back
to me within ninety days with recommendations
on possible federal actions to prevent its abuse.


Sincerely,


Bill Clinton

National Bioethics Advisory Commission

Harold T. Shapiro, Ph.D. - Chair
President
Princeton University
Princeton, New Jersey

Patricia Backlar Senior Scholar Center for Ethics in Health Care Oregon Health Sciences University Portland, Oregon Senior Research Associate Department of Philosophy Portland State University Portland, Oregon	Ezekiel J. Emanuel, M.D., Ph.D. Associate Professor of Medical Ethics Department of Social Medicine Harvard Medical School Boston, Massachusetts
Arturo Brito, M.D. Assistant Professor of Clinical Pediatrics University of Miami School of Medicine Miami, Florida	Laurie M. Flynn Executive Director National Alliance for the Mentally Ill Arlington, Virginia
Alexander Morgan Capron, LL.B. Henry W. Bruce Professor of Law University Professor of Law and Medicine Co-Director, Pacific Center for Health Policy and Ethics University of Southern California Los Angeles, California	Carol W. Greider, Ph.D. Senior Staff Scientist Cold Spring Harbor Laboratory Cold Spring Harbor, New York
Eric J. Cassell, M.D. Clinical Professor of Public Health Cornell University New York, New York	Steven H. Holtzman Chief Business Officer Millennium Pharmaceuticals Inc. Cambridge, Massachusetts
R. Alta Charo, J.D. Associate Professor of Law and Medical Ethics Schools of Law and Medicine University of Wisconsin Madison, Wisconsin	Bette O. Kramer Founding President Richmond Bioethics Consortium Medical College Richmond, Virginia
James F. Childress, Ph.D. Kyle Professor of Religious Studies Professor of Medical Education Department of Religious Studies University of Virginia Charlottesville, Virginia	Bernard Lo, M.D. Director Program in Medical Ethics University of California at San Francisco San Francisco, California
David R. Cox, M.D., Ph.D. Professor of Genetics and Pediatrics Department of Genetics Stanford University School of Medicine Stanford, California	Lawrence H. Miike, M.D., J.D. Director State Department of Health Honolulu, Hawaii
Rhetaugh Graves Dumas, Ph.D., R.N. Vice Provost for Health Affairs Lucille Cole Professor of Nursing The University of Michigan Ann Arbor, Michigan	Thomas H. Murray, Ph.D. Professor and Director Center for Biomedical Ethics School of Medicine Case Western Reserve University Cleveland, Ohio
Diane Scott-Jones, Ph.D. Professor Department of Psychology Temple University Philadelphia, Pennsylvania

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National Bioethics Advisory Commission
Staff

Executive Director, Acting
William F. Raub, Ph.D

Deputy Executive Director, Acting
Ms. Henrietta D. Hyatt-Knorr, M.A.

Executive Assistant Ms. Patricia Norris	Analysts Ms. Emily Feinstein Mr. E. Randolph Hull, Jr. Mr. Sean Simon Mr. Robert Tanner
Program Director Ms. Margaret C. Quinlan	Senior Secretary Ms. Robin Dorsey
Director, Human Subjects Protections Project William Freeman, M.D., M.P.H.	Secretary Ms. LaShell Gaskins
Senior Analyst (Volunteer) Joel Mangel, J.D.	Senior Consultant Kathi E. Hanna, Ph.D.

---

Acknowledgments

The Commission wishes to express its special gratitude to those scholars who on very short notice and under very tight time constraints prepared at the Commission's request very thoughtful presentations and papers on the issues before the Commission. These scholars include: Lori B. Andrews, Dan W. Brock, Lisa Cahill, Courtney S. Campbell, Robert Mullan Cook-Deegan, Rabbi Elliot Dorff, Nancy Duff, Leon R. Kass, Bartha Maria Knoppers, Ruth Macklin, Gilbert C. Meilaender, Father Albert S. Moraczewski, James L. Nelson, Stuart H. Orkin, John Robertson, Janet Rossant, Abdul Aziz Sachedina, Rabbi Moshe Tendler, and Shirley Tilghman.

The Commission also wishes to thank Kathi E. Hanna, Henrietta Hyatt-Knorr, and the NBAC staff for their unfailing commitment through the ninety days in which this report was formulated and produced. Others who provided superior support include William Raub in his role as Acting Executive Director, Lily Engstrom, Hal Thompson, Damon Thompson, Donna Young, Timothy Morris, Vito Oporto, Janet Miller, Marcia Snowden, and Rosemarie Menz.

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EXECUTIVE SUMMARY

The idea that humans might someday be cloned-created from a single somatic cell without sexual reproduction-moved further away from science fiction and closer to a genuine scientific possibility on February 23, 1997. On that date, The Observer broke the news that Ian Wilmut, a Scottish scientist, and his colleagues at the Roslin Institute were about to announce the successful cloning of a sheep by a new technique which had never before been fully successful in mammals. The technique involved transplanting the genetic material of an adult sheep, apparently obtained from a differentiated somatic cell, into an egg from which the nucleus had been removed. The resulting birth of the sheep, named Dolly, on July 5, 1996, was different from prior attempts to create identical offspring since Dolly contained the genetic material of only one parent, and was, therefore, a "delayed" genetic twin of a single adult sheep.

This cloning technique is an extension of research that had been ongoing for over 40 years using nuclei derived from non-human embryonic and fetal cells. The demonstration that nuclei from cells derived from an adult animal could be "reprogrammed," or that the full genetic complement of such a cell could be reactivated well into the chronological life of the cell, is what sets the results of this experiment apart from prior work. In this report we refer to the technique, first described by Wilmut, of nuclear transplantation using nuclei derived from somatic cells other than those of an embryo or fetus as "somatic cell nuclear transfer."

Within days of the published report of Dolly, President Clinton instituted a ban on federal funding related to attempts to clone human beings in this manner. In addition, the President asked the recently appointed National Bioethics Advisory Commission (NBAC) to address within ninety days the ethical and legal issues that surround the subject of cloning human beings. This provided a welcome opportunity for initiating a thoughtful analysis of the many dimensions of the issue, including a careful consideration of the potential risks and benefits. It also presented an occasion to review the current legal status of cloning and the potential constitutional challenges that might be raised if new legislation were enacted to restrict the creation of a child through somatic cell nuclear transfer cloning.

The Commission began its discussions fully recognizing that any effort in humans to transfer a somatic cell nucleus into an enucleated egg involves the creation of an embryo, with the apparent potential to be implanted in utero and developed to term. Ethical concerns surrounding issues of embryo research have recently received extensive analysis and deliberation in our country. Indeed, federal funding for human embryo research is severely restricted, although there are few restrictions on human embryo research carried out in the private sector. Thus, under current law, the use of somatic cell nuclear transfer to create an embryo solely for research purposes is already restricted in cases involving federal funds. There are, however, no current federal regulations on the use of private funds for this purpose.

The unique prospect, vividly raised by Dolly, is the creation of a new individual genetically identical to an existing (or previously existing) person-a "delayed" genetic twin. This prospect has been the source of the overwhelming public concern about such cloning. While the creation of embryos for research purposes alone always raises serious ethical questions, the use of somatic cell nuclear transfer to create embryos raises no new issues in this respect. The unique and distinctive ethical issues raised by the use of somatic cell nuclear transfer to create children relate to, for example, serious safety concerns, individuality, family integrity, and treating children as objects. Consequently, the Commission focused its attention on the use of such techniques for the purpose of creating an embryo which would then be implanted in a woman's uterus and brought to term. It also expanded its analysis of this particular issue to encompass activities in both the public and private sector.

In its deliberations, NBAC reviewed the scientific developments which preceded the Roslin announcement, as well as those likely to follow in its path. It also considered the many moral concerns raised by the possibility that this technique could be used to clone human beings. Much of the initial reaction to this possibility was negative. Careful assessment of that response revealed fears about harms to the children who may be created in this manner, particularly psychological harms associated with a possibly diminished sense of individuality and personal autonomy. Others expressed concern about a degradation in the quality of parenting and family life.

In addition to concerns about specific harms to children, people have frequently expressed fears that the widespread practice of somatic cell nuclear transfer cloning would undermine important social values by opening the door to a form of eugenics or by tempting some to manipulate others as if they were objects instead of persons. Arrayed against these concerns are other important social values, such as protecting the widest possible sphere of personal choice, particularly in matters pertaining to procreation and child rearing, maintaining privacy and the freedom of scientific inquiry, and encouraging the possible development of new biomedical breakthroughs.

To arrive at its recommendations concerning the use of somatic cell nuclear transfer techniques to create children, NBAC also examined long-standing religious traditions that guide many citizens' responses to new technologies and found that religious positions on human cloning are pluralistic in their premises, modes of argument, and conclusions. Some religious thinkers argue that the use of somatic cell nuclear transfer cloning to create a child would be intrinsically immoral and thus could never be morally justified. Other religious thinkers contend that human cloning to create a child could be morally justified under some circumstances, but hold that it should be strictly regulated in order to prevent abuses.

The public policies recommended with respect to the creation of a child using somatic cell nuclear transfer reflect the Commission's best judgments about both the ethics of attempting such an experiment and our view of traditions regarding limitations on individual actions in the name of the common good. At present, the use of this technique to create a child would be a premature experiment that would expose the fetus and the developing child to unacceptable risks. This in itself might be sufficient to justify a prohibition on cloning human beings at this time, even if such efforts were to be characterized as the exercise of a fundamental right to attempt to procreate.

Beyond the issue of the safety of the procedure, however, NBAC found that concerns relating to the potential psychological harms to children and effects on the moral, religious, and cultural values of society merited further reflection and deliberation. Whether upon such further deliberation our nation will conclude that the use of cloning techniques to create children should be allowed or permanently banned is, for the moment, an open question. Time is an ally in this regard, allowing for the accrual of further data from animal experimentation, enabling an assessment of the prospective safety and efficacy of the procedure in humans, as well as granting a period of fuller national debate on ethical and social concerns. The Commission therefore concluded that there should be imposed a period of time in which no attempt is made to create a child using somatic cell nuclear transfer.1

Within this overall framework the Commission came to the following conclusions and recommendations. I. The Commission concludes that at this time it is morally unacceptable for anyone in the public or private sector, whether in a research or clinical setting, to attempt to create a child using somatic cell nuclear transfer cloning. We have reached a consensus on this point because current scientific information indicates that this technique is not safe to use in humans at this point. Indeed, we believe it would violate important ethical obligations were clinicians or researchers to attempt to create a child using these particular technologies, which are likely to involve unacceptable risks to the fetus and/or potential child. Moreover, in addition to safety concerns, many other serious ethical concerns have been identified, which require much more widespread and careful public deliberation before this technology may be used.

The Commission, therefore, recommends the following for immediate action:

• A continuation of the current moratorium on the use of federal funding in support of any attempt to create a child by somatic cell nuclear transfer.
• An immediate request to all firms, clinicians, investigators, and professional societies in the private and non-federally funded sectors to comply voluntarily with the intent of the federal moratorium. Professional and scientific societies should make clear that any attempt to create a child by somatic cell nuclear transfer and implantation into a woman's body would at this time be an irresponsible, unethical, and unprofessional act.

II. The Commission further recommends that:

• Federal legislation should be enacted to prohibit anyone from attempting, whether in a research or clinical setting, to create a child through somatic cell nuclear transfer cloning. It is critical, however, that such legislation include a sunset clause to ensure that Congress will review the issue after a specified time period (three to five years) in order to decide whether the prohibition continues to be needed. If state legislation is enacted, it should also contain such a sunset provision. Any such legislation or associated regulation also ought to require that at some point prior to the expiration of the sunset period, an appropriate oversight body will evaluate and report on the current status of somatic cell nuclear transfer technology and on the ethical and social issues that its potential use to create human beings would raise in light of public understandings at that time.

III. The Commission also concludes that:

• Any regulatory or legislative actions undertaken to effect the foregoing prohibition on creating a child by somatic cell nuclear transfer should be carefully written so as not to interfere with other important areas of scientific research. In particular, no new regulations are required regarding the cloning of human DNA sequences and cell lines, since neither activity raises the scientific and ethical issues that arise from the attempt to create children through somatic cell nuclear transfer, and these fields of research have already provided important scientific and biomedical advances. Likewise, research on cloning animals by somatic cell nuclear transfer does not raise the issues implicated in attempting to use this technique for human cloning, and its continuation should only be subject to existing regulations regarding the humane use of animals and review by institution-based animal protection committees.
• If a legislative ban is not enacted, or if a legislative ban is ever lifted, clinical use of somatic cell nuclear transfer techniques to create a child should be preceded by research trials that are governed by the twin protections of independent review and informed consent, consistent with existing norms of human subjects protection.
• The United States Government should cooperate with other nations and international organizations to enforce any common aspects of their respective policies on the cloning of human beings.

IV. The Commission also concludes that different ethical and religious perspectives and traditions are divided on many of the important moral issues that surround any attempt to create a child using somatic cell nuclear transfer techniques. Therefore, we recommend that:

• The federal government, and all interested and concerned parties, encourage widespread and continuing deliberation on these issues in order to further our understanding of the ethical and social implications of this technology and to enable society to produce appropriate long-term policies regarding this technology should the time come when present concerns about safety have been addressed.

V. Finally, because scientific knowledge is essential for all citizens to participate in a full and informed fashion in the governance of our complex society, the Commission recommends that:

• Federal departments and agencies concerned with science should cooperate in seeking out and supporting opportunities to provide information and education to the public in the area of genetics, and on other developments in the biomedical sciences, especially where these affect important cultural practices, values, and beliefs.

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Chapter One
INTRODUCTION

The idea that humans might someday be cloned-created from a single somatic cell without sexual reproduction-moved further away from science fiction and closer to a genuine scientific possibility on February 23, 1997. On that date, The Observer broke the news that Ian Wilmut, a Scottish scientist, and his colleagues at the Roslin Institute were about to announce the successful cloning of a sheep by a new technique. The technique involved transplanting the genetic material of an adult sheep, apparently obtained from a differentiated somatic cell 2, into an egg from which the nucleus had been removed. The resulting birth of the sheep, named Dolly, on July 5, 1996 appears to mark yet another milestone in our ability to control, refine, and amplify the forces of nature.

The Scottish sheep experiment was different from prior attempts to create identical offspring from a pair of adult animals. It used a cloning technique to produce an animal that was a genetic twin of an adult sheep. Put another way, Dolly contained the genetic material of only one parent. This technique of transferring a nucleus from a somatic cell into an egg is an extension of research that had been ongoing for over 40 years using nuclei derived from non-human embryonic and fetal cells. The demonstration that nuclei from cells derived from an adult animal could be "reprogrammed," or that the full genetic complement of such a cell could be reactivated well into the chronological life of the cell, is what sets the results of this experiment apart from prior work. In this report we refer to the technique, first reported by Wilmut, of nuclear transplantation using nuclei derived from somatic cells other than those of an embryo or fetus as "somatic cell nuclear transfer."

For some time, scientific evidence has suggested that the genetic material contained in differentiated somatic cells may retain the potential to direct the development of healthy fertile adult animals, but its capacity to do so remained unproved (Di Bernadino, 1997). The Roslin experiment, therefore, was a significant scientific event with potentially profound implications since it brings us closer to the possibility of developing a capacity to create clone human beings in an asexual manner. Although for the past ten years scientists have routinely cloned sheep and cows from embryo cells, this was the first successful experiment using the nucleus of a somatic cell from an adult animal to clone an animal that matured to a fully developed state.

The issues surrounding the cloning of human beings have long been the subject of periodic concern and debate among philosophers, scientists, ethicists, and others, particularly following the publication of Joshua Lederberg's 1966 article on cloning in the American Naturalist (Lederberg, 1966). Nevertheless, the impact of these most recent developments on our national psyche has been quite remarkable. Some commentators have suggested that the furor aroused by the new possibility for cloning is out of proportion to most of the ethical, legal, and moral issues it raises, since these same issues have been raised by previous developments and are simply emerging again in a novel and striking form. Nevertheless, it is important to acknowledge that the possibilities raised by this new technique certainly would be unprecedented and that some would consider its use to be a truly radical step. This type of cloning would involve three novel developments: the replacement of sexual procreation with asexual replication of an existing set of genes; the ability to predetermine the genes of a child; and the ability to create many genetically identical offspring.

Some scientists were surprised that the technical barriers of cell differentiation and development seemingly could be so easily overcome when using somatic cells as the source for nuclear transfer. The public-including many members of the scientific community-responded to Dolly with a combination of fascination, hope for useful new understandings of human biology, and profound concern-even alarm-about the prospect of being able to create whole humans from a single somatic cell via nuclear transfer cloning techniques. Although much of the initial public reaction was one of fear, concern, and serious moral reservations about the potential use or abuse of this new technological capacity, a few voices were heard cautiously suggesting that a better understanding of cell dynamics in humans and animals might enable us to develop new cures for various diseases. Thus, it is important to reflect not only on the dangers and ethical reservations but also on the potential human benefits from the use of this type of cloning that might arise in such areas as treating particular infertility problems, transplanting cells or tissues, or preventing certain genetically transmitted harms to offspring.

A few of the initial objections to this new type of cloning were either speculative or based on simple misunderstandings, such as, that cloning would allow for the instantaneous creation of a fully grown adult from the cells of an individual. Other fears stemmed from the incorrect idea that an exact copy, although much younger, of an existing person could be made. This fear reflects an erroneous belief that one's genes bear a simple relationship to the physical and psychological traits that make up a person. Although genes provide the building blocks for each individual, it is the interaction among a person's genetic inheritance, the physical and cultural environment, and the process of learning that result in the uniqueness of each individual human. Thus, the idea that nuclear transplantation cloning could be used to re-create exemplary or evil people has no scientific basis and is simply false.

Other objections to nuclear transplantation cloning, however, are based on carefully articulated philosophical ideals, deep cultural commitments, or religious beliefs, and these deserve continuing and careful consideration. These objections reflect deeply held beliefs about the value of human individuality and personal autonomy, the meaning of family and the value of a child, respect for human life and the natural world, and the preservation of the integrity of the human species.

Many public leaders in the United States responded to the announcement about Dolly with immediate and strong condemnation of any attempt to clone human beings in this new manner. The reasons ranged from frightening science fiction imagery to the judgment that cloning of human beings is a serious violation of basic human rights and human dignity. The reaction abroad was similar, with many nations seemingly ready-indirectly or directly-to prohibit cloning human beings in this fashion. Indeed, many international organizations such as UNESCO and the Council of Europe have a long-established and well-articulated concern that research and clinical applications in biology and genetics remain consistent with a fundamental commitment to human dignity and human rights. To date, at least Argentina, Australia, Great Britain, Denmark, Germany, and Spain have enacted laws banning cloning human beings. Unfortunately, some of the deep concerns supporting such views and associated legislation are stated in vague or overly broad terms. The widespread public discomfort, even revulsion, about cloning human beings deserves the best articulation possible, a task that takes time and requires the considered reflections of diverse groups within American society and abroad.

Within days of the published report of the apparently successful cloning of a sheep in this new manner, President Clinton instituted a ban on federal funding for research related to cloning of human beings. In addition, the President asked the recently appointed National Bioethics Advisory Commission (NBAC) to address within ninety days the ethical and legal issues that surround the subject of cloning human beings. This provided a welcome opportunity for initiating a thoughtful analysis of the many dimensions of the issue, including a careful consideration of the potential risks and benefits. It also presented an occasion to review the current legal status of cloning and the potential constitutional challenges that might be raised if new legislation were enacted to restrict the creation of a child through somatic cell nuclear transfer.

The Commission began its discussions fully recognizing that any effort in humans to transfer a somatic cell nucleus into an enucleated egg involves the creation of an embryo, with the apparent potential to be implanted in utero and developed to term. Ethical concerns surrounding issues of embryo research have recently received extensive analysis and deliberation in our country. Indeed, federal funding for human embryo research is severely restricted, although there are few restrictions on human embryo research carried out in the private sector. Thus, under current law, the use of somatic cell nuclear transfer to create an embryo solely for research purposes is already restricted in cases involving federal funds. There are, however, no current regulations on the use of private funds for this purpose.

The unique prospect, vividly raised by Dolly, is the creation of a new individual genetically identical to an existing (or previously existing) person-a "delayed" genetic twin. This prospect has been the source of the overwhelming public concern about such cloning. While the creation of embryos for research purposes alone always raises serious ethical questions, the use of somatic cell nuclear transfer to create embryos raises no new issues in this respect. The unique and distinctive ethical issues raised by the use of somatic cell nuclear transfer to create children relate to, for example, serious safety concerns, individuality, family integrity, and treating children as objects. Consequently, the Commission focused its attention on the use of such techniques for the purpose of creating an embryo which would then be implanted in a woman's uterus and brought to term. It also expanded its analysis of this particular issue to encompass activities in both the public and private sector.

Controlling Nature

Humankind's efforts to control nature date back as far as recorded history. In particular, domesticated plants and animals have been the mainstay of our agricultural heritage. Over time human mastery over nature often has been met, quite understandably, with opposition and concern, and frequently has been considered by some to be an affront to the natural order of things or by others to be at odds with interpretations of God's revealed word. Indeed many myths and legends, ancient as well as modern, deal directly with humankind's on-going struggle to ensure that the benefits of our new technological capacities clearly outweigh the harms-both expected and unexpected. The idea that our growing technological mastery is filled with moral ambiguity and capable of both vast good and catastrophic evil is deeply embedded in many cultural traditions.

A prime example is the mythology of the Argo, the first ship, in classical Greek culture. The Greeks see the initial act of shipbuilding as both the origin of culture and the origin of decline. While sailing enables one to encounter other persons and other possibilities, it also brings marauders and war, and its very existence bespeaks the danger of unlimited human desire. Thus, the ability to build and sail boats is both a boon and a curse. Euripides' Medea starts with a lament about the trees that were cut down to build the Argo and the other troubles that followed:

Would that the Argo had never winged its way to the land of Colchis... Would that pine trees had never been felled in the glens of Mount Pelion and furnished oars for the hands of the heroes who at Pelias' command set forth in quest of the Golden Fleece.

Concern about our tools and technology has been greatly accelerated with the coming of modern industrialized societies. Is it possible, some now wonder, that our confidence in human competence and technology may be just another myth? How, some are now asking, can we find some moral compass or moral limit to our desire to master everything and possess all? Only such limits, many would say, can save us from the moral ambiguity of our own cleverness.

In recent years, concern about humankind's control over nature has been particularly acute in relation to the new moral choices created by the stunning developments in the biomedical sciences, especially in the area of human reproduction. Although personal reproductive health is considered to be, in most cases, a private matter, ongoing controversies regarding the moral standing of human genetic material and particular human interventions in procreation have focused public attention on the ethical and legal implications of new reproductive techniques. In many cases, initial fears give way to cautious acceptance, but a wariness lingers that is easily reawakened with each new advance.

Artificial insemination by donor, for example, was considered a form of adultery when first introduced in the 1940s. It is now a widely used and accepted practice in the treatment of infertility, although some continue to have serious reservations. When prenatal diagnosis was introduced in the late 1960s, the public simultaneously welcomed the opportunity to prevent lethal disease in newborns but worried about the use of such techniques to select "vanity" characteristics or nonmedical traits in offspring. The birth of Louise Brown, conceived via in vitro fertilization, in 1978 was another dramatic event, providing a new and controversial means to parenthood. With all of these technical advances, there has been a continuing debate about safety, legality, ethical acceptability, and the government's right to intervene in private matters.

Research itself, not just its clinical application, has often sparked debate. For example, research involving human fetuses has been a subject of intense national debate and disagreement for over two decades (Institute of Medicine, 1994). Federal research in this area continues to be restricted to that which has potential therapeutic benefit to the fetus, or involves no more than minimum risk to the fetus even if potential benefit to the mother can be demonstrated. Restrictions also remain regarding embryo research. Despite the recommendations of the National Institutes of Health Human Embryo Research Panel (1994), that certain targeted and carefully regulated research using early human embryos be eligible for federal funds, in December 1994 the President directed NIH not to allocate federal funds for research programs that involved the creation of human embryos solely for research purposes. This issue was also addressed by Congress, which inserted language in the FY96 and FY97 appropriations bills that widened the presidential ban to prohibit virtually all human embryo research conducted with federal funds. Work in this area continues in the United States, but it is largely limited to the private sector, and thus takes place without any federal regulation.

Recombinant DNA research represents another example of controversy and intense debate. In the 1970s, concerns about the safety of unintended release of recombinant organisms led to a voluntary research moratorium in the scientific community and the development of guidelines (Fredrickson, 1991). Similarly, all experiments involving gene therapy (treatment of specific diseases by inserting human genes into human patients) are subject to review and approval by a federal body.

As segments of human DNA or human cells became the focus of study and the objects of manipulation, their use as research materials raised increasingly important ethical issues about how these materials are obtained, transformed, and, in some cases, used to develop commercial products (Office of Technology Assessment, 1987). Such research with human genetic material generates questions about respect for persons and the human body, and the value and moral status to be placed on cells and tissues.

Genetic and reproductive technologies also cause concern because of the specter of eugenics and of real or imagined social control through manipulation of human genes. Genetic control suggests broken taboos, and, in the words of Henry David Thoreau, implies that "men have become the tools of their tools"(Blank, 1981). While these concerns are often set against and partly attributable to a backdrop of fiction, fantasy, and misunderstanding, they are, more importantly, related to profound concerns regarding the nature of humankind and its relationship to other aspects of the natural world. 3 When the bizarre and fantastic scenarios are removed, we are left with a myriad of reactions: sincere expressions of opposition; serious moral concerns; new hope for a better understanding of human biology and the prospect of combating currently untreatable afflictions; calls for more study; and guarded statements about the need for some measure of control (Macklin, 1994; 1997).

Controlling Science

With some notable exceptions, the scientific community has enjoyed for centuries a great deal of autonomy in directing and regulating its research agenda. Since mid-century, however, demands for external regulation have increased, in part because much research, particularly in the biological sciences, is publicly funded and therefore requires some additional measure of accountability. More importantly, society has become more sensitive to concerns about the dangers-particularly to human participants-of the research itself and its future consequences. Thus, our evolving moral sensibilities together with the spectacular advances in biomedical science have generated new ethical concerns. As Bernard Davis of Harvard Medical School and others have noted, society sometimes seeks to regulate or restrict research when it poses the specters of dangerous or unfamiliar products, powers, or ideas (Davis, 1980).

The regulation of science has thus become part of the landscape, particularly for those who receive federal funds (Office of Technology Assessment, 1986). In addition to environmental, health, occupational, and safety regulations, scientists must also comply with animal welfare and human subjects protections and abide by restrictions and moratoria on specific types of research. Because science is both a public and social enterprise and its application can have profound impact, society recognizes that the freedom of scientific inquiry is not an absolute right and scientists are expected to conduct their research according to widely held ethical principles. There are times when limits on scientific freedom must be imposed, even if such limits are perceived as an impediment by an individual scientist. Moreover, appropriate ethical constraints are a matter for both scientists and the broader public to formulate and implement. At the same time, limits on freedom of inquiry must be justified, and impositions on such freedom should satisfy certain conditions-for example, that the limits are not arbitrary, that they emerge from the thoughtful balancing of costs and benefits, that they are not unnecessarily oppressive, that they do not lightly impinge on long established rights and freedoms, that there is some continuing public discourse with those affected by the ban, and that such limitations be open to reconsideration in the light of new information and new understanding.

Consideration of Ethical and Religious Perspectives

When the President asked NBAC to take up the issue of the cloning of human beings he admonished that "any discovery that touches upon human creation is not simply a matter of scientific inquiry, it is a matter of morality and spirituality as well." Although well aware that the United States Constitution prohibits the establishment of policies that are solely motivated by religious beliefs, NBAC shared the President's concern and sought out testimony about the cloning of human beings from leading scholars from a variety of religious traditions. In the same spirit NBAC also commissioned a background paper on the positions a number of religious traditions have taken or are considering on the cloning of human beings.

NBAC felt this was especially important because religious traditions influence and shape the moral views of many U.S. citizens and religious teachings over the centuries have provided an important source of ideas and inspiration. Although in a pluralistic society particular religious views cannot be determinative for public policy decisions that bind everyone, policy makers should understand and show respect for diverse moral ideas regarding the acceptability of cloning of human beings in this new manner.

Although some religious responses to the cloning of human beings through somatic cell nuclear transfer are tied tightly to particular scriptural texts or other faith commitments, often these ideas can be stated forcefully in terms understandable and persuasive to all persons, irrespective of specific religious beliefs. For example, appeal may be made to a view of human nature or of human reason, rather than exclusively to a religious source of knowledge such as scripture or revelation.

NBAC also wanted to determine whether various religious traditions, despite their distinctive sources of authority and argumentation, reach similar conclusions about this type of human cloning. A convergence of views across these traditions, as well as across secular traditions, would be instructive, even if not necessarily determinative, for public policy.

While many Americans look to their religious faiths for moral guidance on issues, other sources of moral knowledge and insight are also important. Many moral considerations that would be widely acknowledged as legitimate do not depend for their force on particular religious commitments or a specific philosophical outlook. For example, the conviction that it is wrong to harm a child is broadly shared among Americans. If you inquire why it is wrong to harm a child, people may give different answers. Some may refer to their religious convictions that a child is a gift from God. Others may say that it is always wrong to harm an innocent person without some compelling reason. To many people, this is a bedrock principle of ethics, even if it has no single, universally acknowledged foundation in a specific religious or philosophical tradition. Rather, it finds its foundation in many different understandings of morality, some religious, some secular. Moral ideas such as the obligation not to inflict harm on others are accessible to all Americans and, therefore, can provide a robust foundation for public policy.

America has a vibrant tradition of ethical dialogue in which all are invited to participate. What moral considerations deserve our attention and which are the most important in responding to a particular issue? These are questions that arise with every new controversy. Whether one's ethical beliefs come from theological commitments, philosophical arguments, or from hard-won life experience, all voices should be welcome to the conversation, and all thoughtful views are entitled to a respectful hearing. While tolerance is a widely accepted virtue in American it is important to remind ourselves that it is built on the idea of mutual respect and the capacity to accept, whenever possible, the moral worth of others with whom one may disagree. Tolerance, therefore, means both agreeing to disagree and accepting the challenge of sustaining a community where moral authority will, to some extent, always be contested.

Policy makers, therefore, need to consider a range of moral views when they try to determine whether a particular policy is ethically justifiable as well as politically feasible. A particular policy may not be politically feasible, for instance, if it evokes thoughtful, widespread and vigorous moral opposition. In such circumstances its social costs may outweigh its putative benefits, and additional education and deliberation may be required before new policies are put in place.

Consideration of Law and Public Policy

The public policy chosen with respect to the cloning of human beings via somatic cell nuclear transfer should reflect a keen knowledge of the science, our best judgments about the ethics of attempting such an experiment, and our traditions regarding limitations on individual actions in the name of the common good. Americans in this era, relative to earlier generations, have a wide interest in and substantial knowledge of science. Nevertheless, in the weeks following the report of Dolly, the public, the media, and even some scientists demonstrated a surprising lack of understanding of the science involved in cloning. NBAC believes that public debate about issues such as human cloning requires an even more educated populace. Science policy has become public policy, which can be decided wisely only by an informed nation.

American tradition has been to avoid prohibiting or regulating personal activities, absent a compelling reason related to effects on others or society as a whole. Where the individual actions are expressions of fundamental rights, such as the right to free speech or the right to privacy, the reasons for limitation must be compelling, and the limitations made as minimal as possible.

The possibility of cloning human beings in this new fashion appears to raise concerns about direct physical harms to the children who may result. This in itself is sufficient to justify a prohibition on such attempts at this time, even if such efforts were to be characterized as the exercise of a fundamental right to procreate. More speculative psychological harms to the child, and effects on the moral, religious, and cultural values of society may be enough to justify continued prohibitions in the future, but more time is needed for discussion and evaluation of these concerns.

In its discussion of potential policy options, NBAC considered the relative benefits of achieving an immediate prohibition through federal legislation on cloning human beings using somatic cell nuclear transfer techniques. It also considered more indirect means to deter such experiments.

Indirect, non-legislative options considered by NBAC include cooperation by the private sector, both research and clinical, in a moratorium on such experiments and/or clinical practice, and the continued prohibition of the use of federal funds to support such experiments. The American Medical Association, the World Medical Association, and the World Health Organization, for example, have already called for such a moratorium on clinical activities.

NBAC also weighed, in terms of nuclear transplantation cloning, the potential impact of a possible legislative measure to extend basic human subjects protections to all research conducted in the United States. This would insure that any research efforts to clone a human in this manner would, along with all other research using human subjects, be covered by the twin protections of informed consent and appropriate scientific review to insure an ethically acceptable balance between risks and benefits. In light of the early state of animal research in this area, such protections should prevent such cloning research from going forward at this time.

Finally, NBAC recognized that cooperation with other governments in the enforcement of any common elements of our respective policies could strengthen any of the measures adopted by the United States. Because science is a global endeavor, international cooperation would ensure consistency across borders and enhance public confidence in scientific research generally.

Process of NBAC and Organization of the Report

The results of NBAC's 90-day analysis are presented in this report. In its deliberations, NBAC focused its discussion on the science of the cloning of human beings using the somatic cell nuclear transfer technique, and the ethical, religious, legal, and regulatory implications of cloning human beings in this manner. To aid in these tasks NBAC invited testimony from an array of scientists, scientific societies, ethicists, theologians, and legal experts, and heard from a wide variety of interested parties during the public comment session at each meeting. In addition, it commissioned numerous background papers from recognized experts to inform its work.

This report consists of five chapters in addition to this one. Chapter Two describes the scientific developments that preceded and made possible the cloning of Dolly and speculates on potential applications of this and related technologies. Chapter Three presents some of the key themes in religious interpretations and evaluations of human cloning. Chapter Four outlines the numerous ethical concerns raised by the prospect of cloning human beings via somatic cell nuclear transfer. Chapter Five discusses the legal and policy issues considered by the NBAC as it pondered various recommendations. The final section, Chapter Six, presents the recommendations made by NBAC in response to the President's request.

In many instances, NBAC found itself moving at a rapid pace in only partly charted waters. In those times it relied on its individual and collective wisdom, judgment, and moral foundations, and the advice of others. NBAC argued and debated the issues as it searched for appropriate formulations of the problem and for the wisdom to suggest useful policy options. While the members of NBAC learned a great deal during its deliberations, we could not reach a resolution on all of the issues before us. Nevertheless, it was able to accomplish two things. First, it developed a set of recommendations, which are set out in Chapter Six. Second, it agreed that it was important to take a number of steps to ensure the continuation of an informed national discussion of these issues and other developments in the biomedical sciences and clinical practices that have an impact on our moral lives and cultural traditions.

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References

Blank, R.H., The Political Implications of Human Genetic Technology (Boulder, CO: Westview Press, 1981).

Davis, B.D., "Three specters: Dangerous products, powers, or ideas," in Genetics and the Law II, A. Milunsky and G.J. Annas (eds.) (New York: Plenum Press, 1980).

Di Bernadino, M.A., Genomic Potential of Differentiated Cells (New York: Columbia University Press, 1997).

Fredrickson, D.S., "Asilomar and recombinant DNA: The end of the beginning," in Biomedical Politics, K.E. Hanna (ed.) (Washington, D.C.: National Academy Press, 1991).

Institute of Medicine, Fetal Research and Applications: A Conference Summary (Washington, D.C.: National Academy Press, 1994).

Lederberg, J., "Experimental genetics and human evolution," The American Naturalist 100:519- 531, 1966.

Macklin, R., "Splitting embryos on the slippery slope: Ethics and public policy," Kennedy Institute of Ethics Journal 4(3)209-225, 1994.

Macklin, R., Testimony before the National Bioethics Advisory Committee, Washington, D.C., March 14, 1997.

National Institutes of Health, Report of the Human Embryo Research Panel (Bethesda, MD: National Institutes of Health, 1994).

Office of Technology Assessment, New Developments in Biotechnology: Ownership of Human Tissues and Cells, OTA-BA-337 (Washington, D.C.: U.S. Government Printing Office, 1987).

Office of Technology Assessment, The Regulatory Environment for Science (Washington, D.C.: U.S. Government Printing Office, 1986).

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Chapter Two
THE SCIENCE AND APPLICATION OF CLONING4

The report in February 1997 that scientists in Scotland had cloned a sheep, Dolly, led to much public discussion of "cloning" of animals and speculation about the possibility of cloning humans. The term "cloning" is used by scientists to describe many different processes that involve making duplicates of biological material. In most cases isolated genes or cells are duplicated for scientific study, and no new animal results. This type of cloning, using genes and cells, has led to many medical advances such as providing insulin to treat diabetes and therapies for hemophilia. The sheep experiment was different; it used a cloning technique called "somatic cell nuclear transfer" and resulted in an animal that was a genetic twin-although delayed in time-of an adult sheep. This technique of transferring a nucleus from a somatic cell into an egg that produced Dolly was an extension of experiments that had been ongoing for over 40 years. These experiments were aimed at understanding how development of an animal from a single fertilized egg is carried out. In recent years the agricultural industry has been trying to improve nuclear transplantation cloning to facilitate the breeding of desirable livestock and some biotechnology companies are exploring ways to use nuclear transfer cloning to improve the production of therapeutic drugs. In addition to drug production, understanding the details of nuclear transplantation cloning might lead to new therapies to treat human disease. For instance it might be possible to grow human cells and tissues for transplantation and grafts that would not be rejected after transfer, as they often are today. These kinds of benefits are currently only hypothetical and much additional research will be needed in animal systems. Although the birth of Dolly was lauded as an amazing success, in fact the procedure is not perfected. Only one sheep was produced from over two hundred nuclear transfers. In addition, it is not yet clear whether Dolly is normal or whether she could have subtle problems that might lead to serious diseases. Using this technique to produce a human child might result in, for example, malformations or disease due to problems inherent in the technique. Thus, while using animals to understand the biological process that produced Dolly holds great promise for future medical advances, there is no current scientific justification for attempting to produce a human child at this time with this technique.

What is Cloning?

The word clone is used in many different contexts in biological research but in its most simple and strict sense, it refers to a precise genetic copy of a molecule, cell, plant, animal, or human being. In some of these contexts, cloning refers to established technologies that have been part of agricultural practice for a very long time and currently form an important part of the foundations of modern biological research.

Indeed, genetically identical copies of whole organisms are commonplace in the plant breeding world and are commonly referred to as "varieties" rather than clones. Many valuable horticultural or agricultural strains are maintained solely by vegetative propagation from an original plant, reflecting the ease with which it is possible to regenerate a complete plant from a small cutting. The developmental process in animals does not usually permit cloning as easily as in plants. Many simpler invertebrate species, however, such as certain kinds of worms, are capable of regenerating a whole organism from a small piece, even though this is not necessarily their usual mode of reproduction. Vertebrates have lost this ability entirely, although regeneration of certain limbs, organs, or tissues can occur to varying degrees in some animals.

Although a single adult vertebrate cannot generate another whole organism, cloning of vertebrates does occur in nature, in a limited way, through multiple births, primarily with the formation of identical twins. However, twins occur by chance in humans and other mammals with the separation of a single embryo into halves at an early stage of development. The resulting offspring are genetically identical, having been derived from one zygote, which resulted from the fertilization of one egg by one sperm.

At the molecular and cellular level, scientists have been cloning human and animal cells and genes for several decades. The scientific justification for such cloning is that it provides greater quantities of identical cells or genes for study; each cell or molecule is identical to the others.

At the simplest level, molecular biologists routinely make clones of deoxyribonucleic acid (DNA), the molecular basis of genes. DNA fragments containing genes are copied and amplified in a host cell, usually a bacterium. The availability of large quantities of identical DNA makes possible many scientific experiments. This process, often called molecular cloning, is the mainstay of recombinant DNA technology and has led to the production of such important medicines as insulin to treat diabetes, tissue plasminogen activator (tPA) to dissolve clots after a heart attack, and erythropoietin (EPO) to treat anemia associated with dialysis for kidney disease.

Another type of cloning is conducted at the cellular level. In cellular cloning copies are made of cells derived from the soma, or body, by growing these cells in culture in a laboratory. The genetic makeup of the resulting cloned cells, called a cell line, is identical to that of the original cell. This, too, is a highly reliable procedure, which is also used to test and sometimes to produce new medicines such as those listed above. Since molecular and cellular cloning of this sort does not involve germ cells (eggs or sperm), the cloned cells are not capable of developing into a baby.

The third type of cloning aims to reproduce genetically identical animals. Cloning of animals can typically be divided into two distinct processes, blastomere separation and nuclear transplantation cloning.

In blastomere separation, the developing embryo is split very soon after fertilization when it is composed of two to eight cells (see figure 1). Each cell, called a blastomere, is able to produce a new individual organism. These blastomeres are considered to be totipotent, that is they possess the total potential to make an entire new organism. This totipotency allows scientists to split animal embryos into several cells to produce multiple organisms that are genetically identical. This capability has tremendous relevance to breeding cattle and other livestock.

In the early 1980s, a more sophisticated form of cloning animals was developed, known as nuclear transplantation cloning. The nucleus of somatic cells is diploid - that is, it contains two sets of genes, one from the mother and one from the father. Germ cells, however, contain a haploid nucleus, with only the maternal or paternal genes. In nuclear transplantation cloning, the nucleus is removed from an egg and replaced with the diploid nucleus of a somatic cell. In such nuclear transplantation cloning there is a single genetic "parent," unlike sexual reproduction where a new organism is formed when the genetic material of the egg and sperm fuse (see figure 2). The first experiments of this type were successful only when the donor cell was derived from an early embryo. In theory, large numbers of genetically identical animals could be produced through such nuclear transplantation cloning. In practice, the nuclei from embryos which have developed beyond a certain number of cells seem to lose their totipotency, limiting the number of animals that can be produced in a given period of time from a single, originating embryo.

The new development in the experiments that Wilmut and colleagues carried out to produce Dolly was the use of much more developed somatic cells isolated from adult sheep as the source of the donor nuclei. This achievement of gestation and live birth of a sheep using an adult cell donor nucleus was stunning evidence that cell differentiation and specialization are reversible. Given the fact that cells develop and divide after fertilization and differentiate into specific tissue (e.g., muscle, bone, neurons), the development of a viable adult sheep from a differentiated adult cell nucleus provided surprising evidence that the pattern of gene expression can be reprogrammed. Until this experiment many biologists believed that reactivation of the genetic material of mammalian somatic cells would not be complete enough to allow for the production of a viable adult mammal from nuclear transfer cloning.

The Science That Led to Dolly

Until the birth of Dolly, developmental and molecular biologists focused their efforts on understanding the processes of cellular differentiation, the regulation of genes during this process, the factors that stimulate differentiation, and the reversibility of this process. Biologists have investigated whether, once cellular differentiation occurs, the process is reversible. These questions have by no means been fully answered by the appearance of Dolly. If anything, the existence of Dolly stimulates even more speculation and inquiry. This section describes the background of the science that led to the birth of the cloned sheep, including early studies of differentiation and development, research on regulation of gene expression, experiments using nuclear transfer in animals, and studies of cell programming and division.

Early Studies of Differentiation and Development

Nearly every cell contains a spheroid organelle called the nucleus which houses nearly all the genes of the organism. Genes are composed of DNA, which serve as a set of instructions to the cell to produce particular proteins. Although all somatic cells contains the same genes in the nucleus, the particular genes that are activated vary by the type of cell. For example, a differentiated somatic cell, such as a neuron, must keep a set of neural-specific genes active and silence those genes specific to the development and functioning of other types of cells such as muscle or liver cells.

Investigations which began over 40 years ago sought to determine whether a differentiated somatic cell still contained all genes, even those it did not express. Early experiments in frogs and toads by Gurdon (1962) and by Briggs and King (1952) provided strong evidence that the expression potential of the genes in differentiated cells is essentially unchanged from that of the early embryo. Nuclei from donor differentiated cells were injected into recipient eggs in which the nucleus had been inactivated (figure 3). The first series of experiments used cells from tadpoles as the source of donor nuclei (Gurdon, 1962) and adult frogs were produced, albeit at a very low efficiency. Although the cells used were highly specialized, they were not derived from the adult frog, so the cells might not have been fully differentiated.

In these experiments, because isolated nuclei were used, other cellular components were not transferred to the recipient egg. Among those other cellular components is an organelle called the mitochondrion, the energy-producing component of the cell. Although most of the genes specifying this cellular component reside in the nucleus, the mitochondrion itself houses some of its own genes. Thus, in somatic cell nuclear transfer, mitochondrial genes are not transferred to the enucleated egg along with the nuclear genes. Because there are some serious diseases associated with mitochondrial genes, nuclear transplantation could allow an embryo to develop with new, healthy mitochondria from a donor.

Gurdon and colleagues performed another carefully controlled series of experiments in which they used nuclei from adult frog skin cells for transfer to an enucleated egg (Gurdon, et al., 1975). Four percent of the nuclei transferred eventually gave rise to fully developed tadpoles. These experiments provided evidence that the genes contained in the nuclei of differentiated cells could be reactivated by the cytoplasm of the egg and thus direct normal development, but only up to a certain stage. No viable adult frog ever developed from these tadpoles and there was a decrease in the number of tadpoles born as the age of the transferred nucleus increased. This left open the possibility that complete reactivation of the adult nucleus was prevented by some irreversible change in the genetic material, and that there was a progressive decline in nuclear potential with age.

Careful analysis, however, suggested that the major reason for developmental failure of the transplanted embryos appeared to be chromosomal abnormalities that occurred during the process of nuclear transplantation itself. The rate of cell division of adult cells is much slower than that of the cells of the early frog embryo. Thus, in reality, for this technique to work it would be necessary that the transplanted adult nucleus reprogram its gene expression, replicate its DNA, and enter the normal embryonic cell division cycle within an hour of nuclear transfer. It is remarkable, given the mechanics and timing of the process, that any nuclei from adult somatic cells were successful in generating an embryo. Although they did not produce normal adult animals, the amphibian nuclear transfer experiments of Gurdon and others succeeded in demonstrating that the differentiated state of adult somatic cells do not involve major irreversible changes in their DNA.

Regulation of Gene Expression

In recent years, it has been determined that most patterns of differentiated gene expression are maintained by active control mechanisms, in which particular genes are turned on or off by regulatory proteins (Blau, 1992). Further studies suggested that it might be possible to reprogram the gene expression of somatic cells so that they perform a different task. The role of a particular cell type (e.g., muscle, liver, or skin) depends on the combination of regulatory proteins it expresses. While in certain specialized cells, such as white blood cells, actual rearrangements and deletions of DNA occur, for the most part, however, gene expression is not regulated by the loss of DNA but by the turning off of specific genes. Thus, it should be possible to activate or inactivate almost any gene in a cell, given the right cellular environment containing the appropriate regulatory molecules.

To reprogram the gene expression of a somatic cell it is not essential to fuse it with an egg; in some cases re-programming can occur through fusion of two adult cells. Cell fusion experiments, in which different somatic cell types are fused, have demonstrated that extensive reprogramming of differentiated nuclei can occur. For example, when muscle cells are fused with non-muscle cells of various sorts, muscle-specific genes are activated in the non-muscle cells (Blau et al., 1985), and, similarly, genes that code for hemoglobin can be activated in many cell types after fusion with red blood cells (Baron and Maniatis, 1986). These and other kinds of experiments have led to the isolation of specific factors that regulate cell differentiation, such as the gene that regulates the formation of muscle cells (Weintraub, 1993).

These studies have further demonstrated that the stability of the differentiated state is not absolute. Thus, given the appropriate regulatory molecules and enough time to reprogram an adult nucleus, somatic cells can re-initiate earlier programs of differentiation.

Nuclear Transfer in Mammals

Experiments in mammals have also suggested that is possible to reprogram adult somatic cells. Following success in the nuclear transfer experiments in frogs, scientists attempted to repeat the experiments in mice. It was known that early development occurs at a considerably slower rate in mammals than amphibians, giving hope that reprogramming of the donor nucleus would occur more efficiently. For example, the first cell division in mice occurs about a day after fertilization, giving ample time, it was thought, for the reprogramming of gene expression and adjustment of the cell division cycle. This proved not to be the case. Early experiments showed that nuclei from somatic cells fused with fertilized eggs did not undergo nuclear division (Graham, 1969).

However, a series of experiments in mice in the mid 1980s showed that nuclei could be successfully exchanged between fertilized eggs, with 90 percent reaching the blastocyst stage of embryonic development and beyond (McGrath and Solter, 1984). Nuclei recovered and transplanted from embryos at the two-cell stage could direct development to the blastocyst stage. Nuclei transferred from embryos at later stages, however, could not successfully recapitulate development. In fact, in mice, nuclei show less totipotency than whole cells. Many experiments have shown that blastomeres up to the early blastocyst stage are still totipotent when combined with other embryonic cells (Rossant and Pedersen, 1986). This means that the failure of nuclear reprogramming has to be the result of something other than irreversible changes to the genetic material of the cells. In 1986, Willadsen reported experiments with sheep. Unlike the situation in mice, enucleated eggs from sheep could be fused with blastomeres taken from embryos at the eight-cell stage to provide donor nuclei and viable offspring were produced (Willadsen, 1986).

Recent experiments have used nuclear transfer into enucleated unfertilized eggs (figure 4). Using these very early stage eggs prolongs the period of possible reprogramming before the donor nucleus has to undergo the first division. And the advent in the last few years of electrofusion for both fusion of cells and activation of the egg has been another major advance, because activation and fusion occur simultaneously. Because these experiments use fusion of two cells and not simple injection of an isolated nucleus, all of the cellular components are transferred. Thus, the mitochondria, which contain some genes of their own, are transferred along with the nucleus. Because an enucleated egg also contains mitochondria, the result of a fusion experiment is a cell with a mixture of mitochondria from both the donor and the recipient. Since the mitochondrial genes represent an extremely small proportion of the total number of mammalian genes, mixing of mitochondria per se is not expected to have any major effects on the cell. However, if the nucleus donor suffers from a mitochondrial disease, and the egg donor does not, then mixture of the mitochondria may significantly alleviate the disease.

Over the past ten years or so there have been numerous reports of successful nuclear transfer experiments in mammals, nearly all of them using cells taken directly from early embryos. The oldest embryonic nucleus that can successfully support development differs among species. Four-cell blastomere nuclei have been successfully used in pigs (Prather, et al., 1989). In mice, no nucleus older than the eight-cell stage has been used successfully (Cheong, et al., 1993). In rabbits, 32- to 64-cell early embryos can be used as nuclear donors (Yang, et al., 1992). In cows and sheep, cells from what is called the inner cell mass (ICM) of the 120-cell blastocyst stage (see figure 1) have been used successfully (Collas and Barnes, 1994; Smith and Wilmut, 1989). Indeed, in both cows and sheep, cell lines have been made from these ICM cells and nuclei from these cells have been used to reprogram development after transfer into enucleated unfertilized eggs.

In the first experiments of this sort by Sims and First (1994), cow cells derived from embryos were grown in the laboratory for up to 28 days, and then used as nuclear donors, without any attempt at synchronization of the cell division cycle of the donor cells. Of those successfully fused with eggs, 24 percent developed to the blastocyst stage, and 4/34 (12 percent) of the blastocysts transferred to recipient cows developed into normal calves. This success rate compares favorably with those seen using earlier blastomeres and suggests that it might be possible to achieve successful nuclear transfer from permanent cell lines established from early embryos.

Reprogramming of Nuclei and Synchronization of the Cell Division Cycle

There has been some study of the events that occur once a transferred nucleus is exposed to the cytoplasm of the egg and some, but not all, of the parameters that affect success of nuclear transfer are known (Fulka, et al., 1996). Enucleated eggs used for fusion only proceed to division after activation by some artificial signal, such as the electrical current used in the electrofusion technique. When donor nuclei are introduced into the enucleated egg, they usually undergo DNA replication, nuclear envelope breakdown, and chromosome condensation. After activation of the egg the nuclear envelope is reformed around the donor chromosomes. The nucleus now takes on the appearance of a typical egg nucleus at this stage, which is large and swollen. It is assumed that this process begins the reprogramming of the transferred donor nucleus by exposing the chromosomes to the egg cytoplasm and beginning the exchange of egg-derived proteins for the donor nucleus' own proteins (Prather and First, 1990).

It is not clear whether exposure to proteins found in the earliest stages of development and/or nuclear swelling is a prerequisite for reprogramming for later development. Experiments in a number of species have shown that, when nuclei are fused with eggs that have been activated some hours prior to fusion, no DNA replication, chromosome condensation, or nuclear swelling occurs, but normal development can transpire (Campbell, et al., 1994; Stice, et al., 1994).

Once again, it is not obvious which of the processes described above are required for normal development. In rabbits, cows, sheep, and mice (Cheong, et al., 1993; Collas, et al., 1992) experiments have shown that nuclei from cells in the early phases of the cell division cycle do better than cells in later stages. In the first phase of the cell cycle, termed G1 (for Gap phase 1), cells contain only one complete set of chromosomes and are relatively quiescent. They then enter a period of DNA synthesis or replication, called S-phase, followed by a rest phase, called G2 (Gap phase 2), at which time they each have a duplicate copy of each chromosome. This doubling of the chromosomes is in preparation for cell division where an equal number will be divided between the two daughter cells. Because DNA replication is induced after nuclear transfer, any nucleus that has initiated replication before transfer will end up with too much DNA, which will likely result in chromosome anomalies. Thus, the need to transfer nuclei in the G1 phase before replication is initiated, is likely to be important to avoid chromosome damage that will prevent development of the embryo into a viable offspring.

Changes in Technique that Allowed for the Birth of Dolly

In background work that preceded the birth of Dolly, Wilmut and colleagues established cell lines from sheep early embryos, or blastocysts, and used these cells as nuclear donors (Campbell, et al., 1996). In an attempt to avoid the problems of nuclear transfer of non-G1 nuclei into activated eggs, they starved the donor cell line by removing all nutrients from the medium prior to nuclear transfer. Under these starvation conditions, the cells exit the cell cycle and enter the so-called "G0" state (Gap phase 0), similar to the G1 phase in which chromosomes have not replicated. Fusion of G0 nuclei with eggs ensures that the donor chromosomes have not initiated replication prior to fusion. It was also suggested that the G0 state might actually increase the capacity of the nucleus to be reprogrammed by the egg cytoplasm. However, there is currently no direct evidence to support this, nor to conclude that nuclei synchronized in the G0 stage are any better than nuclei synchronized in G1. For Wilmut and colleagues, approximately 14 percent of fusions resulted in development of blastocysts, and 4/34 (12 percent) embryos transferred developed into live lambs. Two died shortly after birth. The success rate in sheep and cow experiments was almost identical, and suggests that division of cells in culture for many days does not inhibit the ability of their nuclei to be reprogrammed by the egg environment. Could the same be true of nuclei from fully differentiated somatic cells?

All of this background work led up to Dolly (Wilmut, et al., 1997). Wilmut and colleagues took late embryo, fetal cell cultures, and cell cultures derived from the mammary gland of an adult sheep and applied the same approach of synchronizing the cells in the G0 stage prior to nuclear transfer. They reported successful production of live offspring from all three cell types, although only 29 of 277 (11 percent) of successful fusions between adult mammary gland nuclei and enucleated oocytes developed to the blastocyst stage, and only 1 of 29 (3 percent) blastocysts transferred developed into a live lamb. This experiment was, in fact, the first time any fully developed animal had been born following transfer of a somatic cell nucleus, since the earlier frog experiments only generated tadpoles.

It should be noted, however, that the amount of new information regarding the stability of the differentiated state derived from this experiment is small, as no attempt was made to document that the donor cells were fully differentiated cells, the genes of which expressed specialized mammary gland proteins. In the earlier experiments with frogs, the fact that the donor cells were fully differentiated was documented in such a manner. In the present case, Dolly could have been derived from a less-differentiated cell in the population, such as a mammary stem cell.

Remaining Scientific Uncertainties

Several important questions remain unanswered about the feasibility in mammals of nuclear transfer cloning using adult cells as the source of nuclei:

First, can the procedure that produced Dolly be carried out successfully in other cases? Only one animal has been produced to date. Thus, it is not clear that this technique is reproducible even in sheep.

Second, are there true species differences in the ability to achieve successful nuclear transfer? It has been shown that nuclear transfer in mice is much less successful than in larger domestic animals. Part of this difference may reflect the intensity of research in this area in the last ten years; agricultural interests have meant that more nuclear transfer work has been performed in domestic animals than in mice. But part of the species differences may be real and not simply reflect the greater recent effort in livestock. For example, in order for a differentiated nucleus to redirect development in the environment of the egg, its constellation of regulatory proteins must be replaced by those of the egg in time for the embryo to use the donor nucleus to direct normal development of the embryo. The species difference may be the result of the different times of embryonic gene activation.

In mammals, unlike many other species, the early embryo rapidly activates its genes and cannot survive on the components stored in the egg. The time at which embryonic gene activation occurs varies between species-the late 2-cell stage in mice (Schultz, 1993), the 4-8 cell stage in humans (Braude, et al., 1988) and the 8-16 cell stage in sheep. The later onset of embryonic gene activation and transcription in sheep provides an additional round or two of cell divisions during which nuclear reprogramming can occur, unlike the rapid genome activation in the mouse. Further cross-species comparisons are needed to assess the importance of this difference in the time of genome activation for the success of nuclear transfer experiments. In humans, for example, the time period before gene activation is very short, which might not permit the proper reprogramming of genes after nuclear transfer to allow for subsequent normal development.

Third, will the phenomenon of genetic imprinting affect the ability of nuclei from later stages to reprogram development? In mammals imprinting refers to the fact that the genes inherited on the chromosomes from the father (paternal genes) and those from the mother (maternal genes) are not equivalent in their effects on the developing embryo (Solter, 1988). Some heritable imprint is established on the chromosomes during the development of the egg and the sperm such that certain genes are expressed only when inherited from the father or mother. Imprinting explains why parthenogenetic embryos, with only maternally inherited genes, and androgenetic embryos, with only paternally inherited genes, fail to complete development (Fundele and Surani, 1994). Nuclei transferred from diploid cells, whether embryonic or adult, should contain maternal and paternal copies of the genome, and thus not have an imbalance between the maternally and paternally derived genes.

The successful generation of an adult sheep from a somatic cell nucleus suggests that the imprint can be stable, but it is possible that some instability of the imprint, particularly in cells in culture, could limit the efficiency of nuclear transfer from somatic cells. It is known that disturbances in imprinting lead to growth abnormalities in mice and are associated with cancer and rare genetic conditions in children.

Fourth, will cellular aging affect the ability of somatic cell nuclei to program normal development? As somatic cells divide they progressively age and there is normally a defined number of cell divisions that they can undergo before senescence. Part of this aging process involves the progressive shortening of the ends of the chromosomes, the telomeres, and other genetic changes. Germ cells (eggs and sperm) evade telomere shortening by expressing an enzyme, telomerase, that can keep telomeres full length. It seems likely that returning an adult mammalian nucleus to the egg environment will expose it to sufficient telomerase activity to reset telomere length, since oocytes have been found to be potent sources of telomerase activity (Mantell and Greider, 1994).

Fifth, will the mutations that accumulate in somatic cells affect nuclear transfer efficiency and lead to cancer and other diseases in the offspring? As cells divide and organisms age, mistakes and alterations (mutations) in the DNA will inevitably occur and will accumulate with time. If these mistakes occur in the sperm or the egg, the mutation will be inherited in the offspring. Normally mutations that occur in somatic cells affect only that cell and its descendants which are ultimately dispensable. Nevertheless, such mutations are not necessarily harmless. Sporadic somatic mutations in a variety of genes can predispose a cell to become cancerous. Transfer of a nucleus from a somatic cell carrying such a mutation into an egg would transform a sporadic somatic mutation into a germline mutation that is transmitted to all of the cells of the body. If this mutation were present in all cells may lead to a genetic disease or cancer. The risks of such events occurring following nuclear transfer are difficult to estimate.

Why Pursue Animal Cloning Research?

Research on nuclear transfer cloning in animals may provide information that will be useful in biotechnology, medicine, and basic science. Some of the immediate goals of this research are:

• to generate groups of genetically identical animals for research purposes
• to rapidly propagate desirable animal stocks
• to improve the efficiency of generating and propagating transgenic livestock
• to produce targeted genetic alterations in domestic animals
• to pursue basic knowledge about cell differentiation

Cloning Animals for Research Purposes

Inbred strains of mice have been a mainstay of biological research for years because they are essentially genetically identical and homozygous (i.e., both copies of each gene inherited from the mother and father are identical). Experimental analysis is simplified because differences in genetic background that often lead to experimental variation are eliminated. Generating such homozygous inbred lines in larger animals is difficult and time consuming because of the long gestation times and small numbers of offspring. The concept of generating small groups of identical animals by nuclear transfer has been proposed as an alternative strategy to obtaining a genetically identical group of animals, and apparently underlies a recent report from Oregon on successful nuclear transfer from early embryonic nuclei in rhesus macaque monkeys (Weiss and Schwartz, 1997).

Repeated cycles of nuclear transfer can expand the number of individual animals derived from one donor nucleus, allowing more identical animals to be generated. The first nuclear transfer embryo is allowed to divide to early blastomere stages and then those cells are used as donor nuclei for another series of transfers. This process can be carried on indefinitely, in theory, although practice suggests that successful fusion rates decline with each cycle of transfer. One experiment in cows, for example, produced 54 early embryos after three cycles of transfer from a single blastomere nucleus from one initial embryo (Stice and Keefer, 1993). Viable calves were produced from all three cycles of nuclear transfer.

This approach is likely to be limited in its usefulness, however. A group of cloned animals derived from nuclear transfer from an individual animal is self-limited. Unless they are derived from an inbred stock initially, each clone derived from one individual will differ genetically from a clone derived from another individual. Once a cloned animal is mated to produce offspring, the offspring will no longer be identical due to the natural processes which shuffle or recombine genes during development of eggs and sperm. Thus each member of a clone has to be made for each experiment by nuclear transfer, and generation of a large enough number of cloned animals to be useful as experimental groups is likely to be prohibitively expensive in most animals.

Advantages of Nuclear Transfer Cloning for Breeding Livestock

In animal breeding, the rapid spread of certain traits within stocks of domestic animals is of obvious commercial importance and has very long historical standing. Artificial insemination and embryo transfer can increase the effective reproductive output of individual elite male and female animals and are widely used in the livestock industry. Nuclear transfer cloning, especially from somatic cell nuclei, could provide an additional means of expanding the number of chosen livestock. The ability to make identical copies of adult prize cows, sheep, and pigs is a feature unique to nuclear transfer technologies, and may well be used in livestock production, if the efficiencies of adult nuclear transfer can be improved. The net effect of multiplying chosen animals by cloning will be to reduce the overall genetic diversity in a given livestock line, likely with severe adverse long-term consequences. If this technique became widespread, efforts would have to be made to ensure a pool of genetically diverse animals for future livestock maintenance.

Improved Generation and Propagation of Transgenic Livestock

There is considerable interest in being able to genetically alter farm animals by introduction and expression of genes from other species, such as humans. So-called "transgenic animals" were first developed using mice, by microinjection of DNA into the nucleus of the egg. This ability to add genes to an organism has been a major research tool for understanding gene regulation and for using the mouse as a model in studies of certain human diseases. It has also been applied to other species including livestock. Proposed applications of this technology to livestock improvement include the possible introduction of growth-enhancing genes, genes that affect milk quality or wool fibers, or disease-resistance genes (Ward and Nancarrow, 1995). But progress has been slow. Initial results of the manipulation of meat production by expression of excess growth hormone in pigs led to undesirable side effects (Pursel, et al., 1989).

Currently, the major activity in livestock transgenesis is focused on pharmaceutical and medical applications. The milk of livestock animals can be modified to contain large amounts of pharmaceutically important proteins such as insulin or factor VIII for treatment of human disease by expressing human genes in the mammary gland (Houdebine, 1994). In sheep greater than 50 percent of the proteins in milk can be the product of a human gene (Colman, 1996). The milk of even transgenic mice can yield large (milligram) quantities of recombinant proteins. Since many such proteins are active at very low concentrations, it is estimated that production of human drugs from transgenic animals could be considerably more cost-effective than current methods.

Another major area of interest is the use of transgenic animals for organ transplantation into humans. Pig organs, in many cases, are similar enough to humans to be potentially useful in organ transplants, if problems of rejection could be overcome. Rejection can already be partly overcome by the expression of human complement (a component of the immune system) regulatory proteins in transgenic pigs. Further transgenic manipulation such as the expression of human antigens in pigs could alleviate organ shortages by minimizing or eliminating the rejection of pig organs transplanted into humans, although other barriers, such as the possible transmission of viruses from pigs to humans, must be overcome.

However the current method of directly injecting genes into fertilized eggs is inefficient. Not all injected eggs will develop into transgenic animals, and then not all transgenic animals will express the added gene in the desired manner. The production of transgenic livestock is slow and expensive. Nuclear transfer would speed up the expansion of a successful transgenic line, but, perhaps more importantly, it would allow more efficient generation of transgenic animals in the first place. Foreign DNA, such as a human gene, could be introduced into cell lines in culture and cells expressing the transgene could be characterized and used as a source of donor nuclei for cloning, and all offspring would likely express the human gene. This, in fact, was the motivation behind the experiments that led to the production of Dolly. If a human gene such as that for insulin could be expressed in the mammary gland, the milk of the sheep would be an excellent source of insulin to treat diabetes.

Generating Targeted Gene Alterations

The most powerful technology for gene replacement in mammals was developed in mice. This technique adds manipulated or foreign DNA to cells in culture to replace the DNA present in the genome of the cells. Thus mutations or other alterations that would be useful in medical research can be introduced into an animal in a directed and controlled manner and their effects studied, a process called gene targeting (Capecchi, 1989). This technology would be of limited use, however, without some means of taking the changes generated in cultured cells and reintroducing them into animals. In mice, this can be achieved by the use of embryonic stem (ES) cells that are capable of being cultured indefinitely in the undifferentiated state. ES cells retain the potential to form all cells of the animal, including the germ cells, when returned to the environment of the early embryo (figure 5). As the technique is currently used in mice, the first generation of animals generated from the ES cells are "chimeric," that is they are made up of a mixture of cells from two different animals. These mice must then be bred one more time to transmit altered genes to the next generation. Using this technique, any genetic alteration made in the embryonic stem cells in culture can be introduced back into mice (Robertson, 1986).

This use of gene replacement and embryonic stem cell technology has been responsible for the explosion in the generation of "knock-out" mice, in which specific genes have been deleted from the genome. These mice have been invaluable in current studies to understand normal gene function and to allow the generation of accurate models of human genetic disease. Gene targeting approaches can also be used to ensure correct tissue-specific expression of foreign genes and to suppress the expression of genes in inappropriate tissues. If applied to domestic animals, this technology could increase the efficiency of the expression of foreign genes by targeting the introduced genes to appropriate regions of the chromosome. It could also be used to directly alter the normal genes of the organism, which could influence animal health and productivity or to help develop transgenic organs that are less likely to be rejected upon transplantation. However, to date, there are no fully validated embryonic stem cell lines in domestic animals. Nuclear transfer from somatic cell lines into an egg, as reported by Wilmut and colleagues, provides a possible alternative to the embryonic stem cell route for introduction of targeted gene alterations into the germ line of animals.

Apart from the fact that embryonic stem cell lines have not yet been produced from farm animals, the other argument for using nuclear transfer to introduce germ line genetic alterations in farm animals is that it eliminates one generation of breeding from the initial chimeric animals. This is an important time and cost saving factor in farm animals with long generation times and small litter size. However this factor might not be as important as once thought. In mice, it turns out, embryonic stem cells can also be used to generate cloned animals carrying gene alterations directly without the initial generation of chimeric animals. When 'tetraploid' embryos that are not themselves capable of developing normally are used as the host cells, the entire mouse fetus can be derived directly from the normal diploid ES cells (figure 6)(Nagy, et al., 1993). Although this procedure is not yet very efficient, it illustrates the remarkable properties of ES cells and suggests that similar approaches could be applied in other species such as farm animals.

Basic Research on Cell Differentiation

The basic cellular processes that allowed the birth of Dolly by nuclear transfer using the nucleus from an adult somatic donor cell are not well understood. If indeed the donor cell was a fully differentiated cell and not a rare, less differentiated stem cell that resulted in this cloned sheep, there will be many questions to ask about how this process occurred. How the specialized cell from the mammary gland was reprogrammed to allow the expression of a complete developmental program will be a fascinating area of study. Developmental biologists will want to know which genes are reprogrammed, when they are expressed, and in what order. This might shed light on the still poorly understood process of sequential specialization that must occur during development of all organisms.

Molecular biologists will also likely learn much from studying how reprogramming and reactivation occurred. What regulatory proteins in the host egg participated in the reprogramming? How did these proteins interact with each other and the DNA so that inactive genes from the mammary gland cells might be activated again? Answers to these kinds of questions will contribute to our overall understanding of how cells grow, divide, and become specialized.

Basic research into these fundamental processes may also lead to the development of new therapies to treat human disease. It is not possible to predict from where the essential new discoveries will come. However, already the birth of Dolly has sparked ideas about potential benefits that might be realized. To explore the possibility of these new therapies, extensive basic research is needed.

Much of this basic research will likely be done in the mouse as this animal is widely used by developmental biologists, and thus a great deal is already known about its development. However, as described above, the use of cloning in other animals-such as cows, pigs and sheep-by agricultural and biotechnology companies will also contribute to understanding of the basic processes involved. The study of nuclear transplantation cloning in a wide variety of animals will be very useful. Although many of the basic cellular mechanisms underlying animal development are the same in all mammals, there are subtle developmental variations that often lead to major technical differences in working with a particular species. Because a technique is often perfected in one species before being applied to another, knowing which parts of the techniques are widely applicable and which might need to be perfected for the given species will be of great value. This body of research into animal systems will answer many questions about the feasibility of various new therapeutic applications being proposed for human cells. New innovations in treating human disease can be tested in animal systems to determine if the basic foundation of the idea is sound before experiments using human cells would be required. Thus the path to testing the potential therapies to treat human disease, described below, should initially go through testing in animal models before progressing to human cell research.

Potential Therapeutic Applications of Nuclear Transfer Cloning

The demonstration that, in mammals as in frogs, the nucleus of a somatic cell can be reprogrammed by the egg environment provides further impetus to studies on how to reactivate embryonic programs of development in adult cells. These studies have exciting prospects for regeneration and repair of diseased or damaged human tissues and organs, and may provide clues as to how to reprogram adult differentiated cells directly without the need for oocyte fusion. In addition, the use of nuclear transfer has potential application in the field of assisted reproduction.

Potential Applications in Organ and Tissue Transplantation

Many human diseases, when they are severe enough, are treated effectively by organ or tissue transplantation, including some leukemias, liver failure, heart and kidney disease. In some instances the organ required is non-vital, that is, it can be taken from the donor without great risk (e.g., bone marrow, blood, kidney). In other cases, the organ is obviously vital and required for the survival of the individual, such as the heart. All transplantation is imperfect, with the exception of that which occurs between identical twins, because transplantation of organs between individuals requires genetic compatibility.

In principle, the application of nuclear transfer cloning to humans could provide a potential source of organs or tissues of a predetermined genetic background. The notion of using human cloning to produce individuals for use solely as organ donors is repugnant, almost unimaginable, and morally unacceptable. A morally more acceptable and potentially feasible approach is to direct differentiation along a specific path to produce specific tissues (e.g., muscle or nerve) for therapeutic transplantation rather than to produce an entire individual. Given current uncertainties about the feasibility of this, however, much research would be needed in animal systems before it would be scientifically sound, and therefore potentially morally acceptable, to go forward with this approach.

Potential Applications in Cell-based Therapies

Another possibility raised by cloning is transplantation of cells or tissues not from an individual donor but from an early embryo or embryonic stem cells; the primitive, undifferentiated cells from the embryo that are still totipotent. This potential application would not require the generation and birth of a cloned individual. Embryonic stem cells provide an interesting model for such studies, since they represent the precursors of all cell lineages in the body. Mouse embryonic stem cells can be stimulated to differentiate in vitro into precursors of the blood, neuronal and muscle cell lineages, among others (Weiss and Orkin, 1995), and they thus provide a potential source of stem cells for regeneration of all tissues of the body.

It might be possible to take a cell from an early blastomere and treat it in such a manner as to direct its differentiation along a specific path. By this procedure it might be possible to generate in the laboratory sufficient numbers of specialized cells, for example bone marrow stem cells, liver cells, or pancreatic beta-cells (which produce insulin) for transplantation. If even a single tissue type could be generated from early embryonic cells by these methods and used clinically, it would constitute a major advance in transplantation medicine by providing cells that are genetically identical to the recipient.

One could imagine the prospect of nuclear transfer from a somatic cell to generate an early embryo and from it an embryonic stem cell line for each individual human, which would be ideally tissue-matched for later transplant purposes. This might be a rather expensive and far-fetched scenario. An alternative scenario would involve the generation of a few, widely used and well characterized human embryonic stem cell lines, genetically altered to prevent graft rejection in all possible recipients.

The preceding scenarios depend on using cells of early human embryos, generated either by in vitro fertilization or nuclear transfer into an egg. Because of ethical and moral concerns raised by the use of embryos for research purposes it would be far more desirable to explore the direct use of human cells of adult origin to produce specialized cells or tissues for transplantation into patients. It may not be necessary to reprogram terminally differentiated cells but rather to stimulate proliferation and differentiation of the quiescent stem cells which are known to exist in many adult tissues, including even the nervous system (Gage, et al., 1995). Experiments in this area are likely to focus more on the conditions required for direct stimulation of the stem cells in specific tissues, than actual use of nuclear transfer to activate novel developmental programs. These approaches to cellular repair using adult stem cells will be greatly aided by an understanding of how stem cells are established during embryogenesis.

Another strategy for cell-based therapies would be to identify methods by which somatic cells could be "de-differentiated" and then "re-differentiated" along a particular path. This would eliminate the need to use cells obtained from embryos. Such an approach would permit the growth of specialized cells compatible with a specific individual person for transplantation. Although at the current time this strategy is highly speculative, ongoing research in animal systems may identify new approaches or new molecular targets that might make this approach feasible.

It will be of great importance to understand through experiments in animals how the environment of the egg reprograms a somatic cell nucleus. What cellular mechanisms can be elucidated? What components are involved in these processes? Can we direct cells along particular developmental pathways in the laboratory and use these cells for therapy? The capacity to grow human cells of different lineages in culture would also dramatically improve prospects for effective somatic gene therapy.

Assisted Reproduction

Another area of medicine where the knowledge gained from animal work has potential application is in the area of assisted reproduction. Assisted reproduction technologies are already widely used and encompass a variety of parental and biological situations, that is, donor and recipient relationships. In most cases, an infertile couple seeks remedy through either artificial insemination or in vitro fertilization using sperm from either the male or an anonymous donor, an egg from the woman or a donor, and in some cases surrogacy. In those instances where both individuals of a couple are infertile or the prospective father has non-functional sperm, one might envision using cloning of one member of the couple's nuclei to produce a child.

Although this constitutes an extension of current clinical practice, aside from the serious, moral, and ethical issues surrounding this approach, there are significant technical and medical causes for caution, some of which were described in the research questions enumerated above.

In most situations of assisted reproduction, other than the intentional union of the gametes by in vitro techniques, the fertilized egg and initial cells of the early embryo are not otherwise manipulated. In some rare cases, such as preimplantation genetic diagnosis, the embryo is manipulated by the removal of one of the identical cells of the blastomere to test its genetic status. In contrast, if nuclear transfer were to be used as a reproductive option, it would entail substantially more invasive manipulation. Thus far, the animal cloning of Dolly is a singular success, one seemingly normal animal produced from 277 nuclear transfers. Until the experiment is replicated the efficiency, and even the validity, of the procedure cannot be fully determined. It is likely that the mere act of manipulating a nucleus and transferring it into an egg could decrease the percentage of eggs that go on to develop and implant normally, as well as increase the rate of birth defects.

Cloning and Genetic Determinism

The announcement of Dolly sparked widespread speculation about a human child being created using somatic cell nuclear transfer. Much of the perceived fear that greeted this announcement centered around the misperception that a child or many children could be produced who would be identical to an already existing person.

This fear reflects an erroneous belief that a person's genes bear a simple relationship to the physical and psychological traits that compose that individual. This belief, that genes alone determine all aspects of an individual, is called "genetic determinism." Although genes play an essential role in the formation of physical and behavioral characteristics, each individual is, in fact, the result of a complex interaction between his or her genes and the environment within which they develop, beginning at the time of fertilization and continuing throughout life. As social and biological beings we are creatures of our biological, physical, social, political, historical, and psychological environments. Indeed, the great lesson of modern molecular genetics is the profound complexity of both gene-gene interactions and gene-environment interactions in the determination of whether a specific trait or characteristic is expressed. In other words, there will never be another you.

While the concept of complete genetic determinism is wrong and overly simplistic, genes do play a major role in determining biological characteristics including a predisposition to certain diseases. Moreover, the existence of families in which many members are affected by these diseases suggest that there is a single gene that is passed down with each generation that causes the disease. When such a disease gene is identified, scientists often say they have "cloned the gene for" breast cancer, for instance, implying a direct cause and effect of gene and disease. Indeed, the recent efforts of the Human Genome Project have led to the isolation of a large number of genes that are mutated in specific diseases, such as Duchenne Muscular Dystrophy, and certain types of breast and colon cancer.

However, recent scientific findings have revealed that a "one-gene, one-disease" approach is far too simplistic. Even in the relatively small list of genes currently associated with a specific disease, knowing the complete DNA sequence of the gene does not allow a scientist to predict if a given person will get the disease. For example, in breast cancer there can be many different changes in the DNA, and for some specific mutations there is a calculated risk of developing the disease, while for other changes the risk is unknown. Even when a specific genetic change is identified that "causes" the disease in some people, others may be found who have the same change but do not get the disease. This is because other factors, either genetic or environmental, are altered that mask or compensate for "the" disease gene. Thus even with the most sophisticated understanding of genes, one cannot determine with certainty what will happen to a given person with a single change in a single gene.

Once again, the reason rigid genetic determinism is false is that genes interact with each other and with the environment in extremely complex ways. For example, the likelihood of developing colon cancer, a disease with a strong hereditary component and for which researchers have identified a single "causative" gene, is also strongly influenced by diet. When one considers a human trait that is determined by multiple genes, the situation becomes even more complex. The number of interactions between genes and environment increases dramatically. In fact, the ability to predict what a person will be like knowing only their genes becomes virtually impossible because it is not possible to know how the environment and chance factors will influence the outcome.

Thus the idea that one could make through somatic cell nuclear transfer a team of Michael Jordans, a physics department of Albert Einsteins, or an opera chorus of Pavarottis, is simply false. Knowing the complete genetic makeup of an individual would not tell you what kind of person that individual would become. Even identical twins who grow up together and thus share the same genes and a similar home environment have different likes and dislikes, and can have very different talents. The increasingly sophisticated studies coming out of human genetics research are showing that the better we understand gene function, the less likely it is we will ever be able to produce at will a person with any given complex trait.

Conclusions

The term "clone" has many meanings but in its simplest and most scientific sense it means the making of identical copies of molecules, cells, tissues, and even entire animals. The latest news about cloning Dolly the sheep involved somatic cell nuclear transplant cloning. In this process the nucleus from an adult somatic cell is transplanted into an enucleated ovum to produce a developing animal that is a "delayed" genetic twin of the adult.

There are many applications that nuclear transfer cloning might have for biotechnology, livestock production, and new medical approaches. Work with embryonic stem cells and genetic manipulation of early embryos in animal species (including nuclear transfer) is already providing unparalleled insights into fundamental biological processes and promises to provide great practical benefit in terms of improved livestock, improved means of producing pharmaceutical proteins, and prospects for regeneration and repair of human tissues.

However, the possibility of using human cloning for the purposes of creating a new individual entails significant scientific uncertainty and medical risk at this time. Potential risks include those known to be associated with the manipulation of nuclei and eggs and those yet unknown, such as the effects of aging, somatic mutation, and improper imprinting. These effects could result in high rates of failed attempts at pregnancy as well as the increased likelihood of developmentally and genetically abnormal embryos.

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References

Baron, M.H., and T. Maniatis, "Rapid reprogramming of globin gene expression in transient heterokaryons," Cell 46:591-602, 1986.

Blau, H.M., "Differentiation requires continuous active control," Annual Review of Biochemistry 61:1213-1230, 1992.

Blau, H.M., Pavlath, G.K., Hardeman, E.C., Chiu, C.P., Silberstein, L., Webster, S.G., Miller, S.C., and C. Webster, "Plasticity of the differentiated state," Science 230:758-766, 1985.

Braude, P., Bolton, V., and S. Moore, "Human gene expression first occurs between the four- and eight-cell stages of preimplantation development," Nature 332:459-461, 1988.

Briggs, R., and T.J. King, "Transplantation of living nuclei from blastula cells into enucleated frog's eggs," Proceedings of the National Academy of Sciences (USA) 38:455-463, 1952.

Campbell, K.H., Loi, P., Cappai, P., and I. Wilmut, "Improved development to blastocyst of ovine nuclear transfer embryos reconstructed during the presumptive S-phase of enucleated activated oocytes," Biology of Reproduction 50:1385-1393, 1994.

Campbell, K.H., McWhir, J., Ritchie, W.A., and I. Wilmut, "Sheep cloned by nuclear transfer from a cultured cell line," Nature 380:64-66, 1996.

Capecchi, M. R., "The new mouse genetics: altering the genome by gene targeting," Trends in Genetics 5:70-76, 1989.

Cheong, H.T., Takahashi, Y., and H. Kanagawa, "Birth of mice after transplantation of early cell-cycle-stage embryonic nuclei into enucleated oocytes," Biology of Reproduction 48:958-963, 1993.

Chiu, C.P., and C.B. Harley, "Replicative senescence and cell immortality: The role of telomeres and telomerase," Proceedings of the Society for Experimental Biology and Medicine 214:99-106, 1997.

Collas, P., and F.L. Barnes, "Nuclear transplantation by microinjection of inner cell mass and granulosa cell nuclei," Molecular Reproduction and Development 38:264-267, 1994.

Collas, P., Balise, J. J., and J.M. Robl, "Influence of cell cycle stage of the donor nucleus on development of nuclear transplant rabbit embryos," Biology of Reproduction 46:492-500, 1992.

Colman, A., "Production of proteins in the milk of transgenic livestock: Problems, solutions, and successes," American Journal of Clinical Nutrition 63:639S-645S, 1996.

Fundele, R.H., and M.A. Surani, "Experimental embryological analysis of genetic imprinting in mouse development," Developmental Genetics 15:515-522, 1994.

Fulka, J., First, N.L., and R.M. Moor, "Nuclear transplantation in mammals: remodeling of transplanted nuclei under the influence of maturation promoting factor," BioEssays 18:835-840, 1996.

Graham, C.F. "The fusion of cells with one- and two-cell mouse embryos," Wistar Institute Symposium Monographs 9:19-35, 1969.

Gage, F.H., Ray, J., and L.J. Fisher, "Isolation, characterization, and use of stem cells from the CNS," Annual Review of Neuroscience 18:159-192, 1995.

Gurdon, J.B. "The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles," Journal of Embryology and Experimental Morphology 10, 622-640, 1962.

Gurdon, J.B., Laskey, R.A., and O. R. Reeves, "The developmental capacity of nuclei transplanted from keratinized skin cells of adult frogs," Journal of Embryology and Experimental Morphology 34:93-112, 1975.

Houdebine, L.M., "Production of pharmaceutical proteins from transgenic animals," Journal of Biotechnology 34:269-287, 1994.

Latham, K.E., Garrels, J.I., and D. Solter, "Alterations in protein synthesis following transplantation of mouse 8- cell stage nuclei to enucleated 1- cell embryos," Developmental Biology 163:341-350, 1994.

Mantell, L.L., and C.W. Greider, "Telomerase activity in germline and embryonic cells of Xenopus," EMBO Journal 13:3211-3217, 1994.

McGrath, J., and D. Solter, "Inability of mouse blastomere nuclei transferred to enucleated zygotes to support development in vitro," Science 226:1317-1319, 1984.

Nagy, A., Rossant, J., Nagy, R., Abramow-Newerley, W., and J.C. Roder, "Derivation of completely cell culture-derived mice from early passage embryonic stem cells," Proceedings of the National Academy of Sciences (USA)90:8424-8428, 1993.

Prather, R.S., Sims, M.M., and N.L. First, "Nuclear transplantation in early pig embryos," Biology of Reproduction 41:414-418, 1989.

Prather, R.S., and N.L. First, "Cloning embryos by nuclear transfer," Journal of Reproduction and Fertility. Supplement 41:125-134, 1990.

Pursel, V.G., Pinkert, C.A., Miller, K.F., Bolt, D.J., Campbell, R.G., Palmiter, R.D., Brinster, R.L., and R.E. Hammer, "Genetic engineering of livestock," Science 244: 1281-1288, 1989.

Robertson, E.J., "Pluripotential stem cell lines as a route into the mouse germ line," Trends in Genetics 2:9-13, 1986.

Rossant J., and R.A. Pederson, Experimental Approaches to Mammalian Embryonic Development (Cambridge: Cambridge University Press, 1986). Schultz, R.M., "Regulation of zygotic gene activation in the mouse," BioEssays 15:531-538, 1993.

Sims, M., and N.L. First, "Production of calves by transfer of nuclei from cultured inner cell mass cells," Proceedings of the National Academy of Sciences (USA)91:614