Technical Hurdles in Cloning
Therapeutic Cloning
Human Clones
A clone can be defined as an individual or group of individuals that descend, through asexual reproduction, from a single individual. In other words, a clone is an exact copy of the original individual. Humans have practiced cloning of plant species for thousands of years. A leaf, a piece of stem, or root of a certain plant placed in a pot with soil or in a petri dish with tissue culture media can regenerate a new individual, genetically identical (clone) to the plant from which the leaf, stem, or root piece was taken. Today, cloning is a common agricultural practice used in many species that can easily reproduce asexually, such as sugar cane, banana, citrus, potato, strawberry, many grasses, roses, and many tree crops.
Cloning is based on two principles:
All cells of any organism contain the complete genetic makeup of the species.
Totipotence, the ability of one cell to differentiate and regenerate a completely new individual.
Although the regeneration of a complete plant from a somatic tissue (leaf, root, stem, etc.) is an ancient practice, it was only in the 1950s that biologists discovered the principles behind regeneration of whole individuals from a single cell. Unlike animal cells, most plant cells retain their potential to express any of their genes and therefore are able to repeat the developmental processes involved in regenerating complete individuals. Cloning of plants offers the possibility of developing millions of individuals exactly identical to the original source of the regenerated cells. This is a common method of reproduction in asexual plant species.
Most animal cells do not have that same capability of naturally regenerating a complete individual from a cell. In animals, this potential is lost during cell specialization. A specific class of cells called stem cells is the only cell type known to retain their totipotence. Stem cells can be found in marrow tissue, fat tissue, and developing embryos. These types of cells have been the focus of animal cloning efforts. In animals, cloning can be accomplished using the technique called nuclear transplant. The technique has been used for many years in animal cloning using embryonic cells for amphibians such as toads. Animal embryonic cells maintain their totipotence after the first few cellular divisions. As the embryo continues its development, the cells lose their ability to differentiate into other cells and, consequently, the capability for complete regeneration ceases quickly. Contrary to the relative ease of nuclear cell transfer in amphibians, this process is much more complex in mammals. Although cloning of toads was accomplished for the first time in 1952, cloning of mice using the same technique was not accomplished until 1977.
Cloning using nuclear transfer involves the manipulation of two cells. The recipient cell is usually a nonfertilized egg from a female taken soon after ovulation. Harvesting of these eggs is done by laparoscopy or by transvaginal suction. The donor cell, which is the one providing the genetic material for regenerating the clone, is collected from the individual to be copied. Any somatic cell could be used for the purpose, including cells from the skin, mammary glands, or mucous membranes. Under a microscope, the recipient cell (egg) is held, by suction, at the end of a pipette. With an extremely fine micropipette, the chromosomes are removed. At this point the nucleus from the donor cell is then fused with the recipient egg previously deprived of its chromosomes. Some of the cells, if implanted into the uterus of a surrogate mother, start developing into an embryo and eventually a fetus. The procedure involves the removal or destruction of the chromosomes from the recipient egg cell, and the subsequent introduction of the chromosomes from the donor cell. The egg, with the newly introduced genetic material, begins the developmental process in the uterus of a surrogate mother to form a complete individual, genetically identical to the donor that supplied the nucleus. This technique has been used with success for cloning sheep, cattle, mice, monkeys, and other mammals.
The first cloned mammal from somatic cells of an adult donor was the sheep Dolly, born in February 1997. Dolly was cloned using mammary cells from an adult sheep. This widely covered event occurred at the Roslin Institute in Scotland, and the lead scientist was Dr. Ian Wilmut (Figure 6-1).
The main players in animal cloning are Geron Corporation, Advanced Cell Technology (ACT), PPL Therapeutics, and Infigen. Geron (http://www.geron.com) is a biopharmaceutical company focused on developing and commercializing therapeutic and diagnostic products for applications in oncology and regenerative medicine, and research tools for drug discovery. It is researching embryonic stem cells for treating disease. ACT (http://www.advancedcell.com) was the first to clone a human embryo for therapeutic purposes. ACT is involved in nuclear transfer for human therapeutics and animal cloning. PPL Therapeutics (http://www.ppl-therapeutics.com) applies transgenic technology to the production of human proteins for therapeutic and nutritional use. PPL Therapeutics, in partnership with the Roslin Institute, cloned the first mammal from an adult cell, Dolly. Infigen (http://www.infigen.com) commercializes applications of cloning technologies and genetic testing in the cattle breeding, pharmaceutical, and nutraceutical fields.
There are two major problems or limitations found in the cloning of mammals. First, following the introduction of the donor's nucleus into the egg, it must be reimplanted into a gestating surrogate mother. Most of the implanted eggs abort, forcing scientists to perform several implantations, in the hopes that at least one of the females will have normal gestation. In the case of amphibians (e.g., toads), the development of the embryo occurs outside of the adult's body, thereby facilitating the development of the fetus.
The second major challenge in animal cloning is the size of the fetus at birth. Most of the surrogate mothers have to deliver via Cesarean section. This is especially true in bovines, as the clones tend to be about twice as big as normal newborn calves. The large size of the fetus during gestation can represent a substantial risk for the surrogate mother. Additionally, clones tend to have a high incidence of birth defects, and many clones die in the first hours following birth. Common abnormalities observed in cloned animals include failures of the kidney, heart, circulatory system, liver, and lungs. In addition, the placenta of the surrogate mother does not always function properly during gestation.
The causes of the high abortion rate and abnormalities in clones are still not completely understood, but it is suspected that they are at least partially the result of the complexity of the genetic reprogramming that takes place in the genes from the donor that are inserted into the egg. If a gene is expressed inadequately or it is not expressed at a critical point in development, the result can be a developmental defect. Genetic reprogramming involves the regulation of thousands of genes in a systematic and orderly way. Any asynchrony in the expression of the genes can contribute to defects in the fetus or even result in abortion. Additionally, when cloning is done with nuclei from somatic cells, they bear any preexisting mutations that might have occurred after the cells had differentiated into specialized cells. These mutations would have otherwise been screened out in gametogenesis.
With the current knowledge and technology, mammalian cloning is still a highly unsafe and inefficient procedure. The expectation is that, as new knowledge is generated from more experience, the main limitations in cloning will be at least partially solved. This science is continuing to make progress worldwide, even in developing counties. For example, in Brazil, Embrapa-Cenargen recently pioneered the cloning of the first bovine calf from somatic cells, born in March 2001 (see Figure 6-2).
Most people and the scientific media were surprised with the publication of the article “The First Human Cloned Embryo” in the magazine Scientific American in November 2001. Even the most optimistic followers believed that experiences with human cloning would not produce results so early. ACT, a small biotechnology company in Massachusetts, was the first to accomplish the cloning of human cells for therapeutic use. Dr. Michael West, the company's chief executive officer, emphatically stated that his company's objective was research in cloning for exclusively therapeutic treatments and not for reproductive or human cloning purposes. Nevertheless, the public had a strong reaction to this news.
Those who favor the use of embryonic stem cells tend to see the potential to cure genetic problems, and they emphasize the hope of a cure and improved lives for patients with fatal genetic diseases. Opponents recognize that human life begins at conception, and they believe that the price of a cure should not result in the taking of another life, as harvesting embryonic stem cells for this research results in the destruction of embryos. Therefore, embryonic stem cells can be seen as a matter of life by those who can benefit from this technology, or as a matter of death by those who do not agree with the sacrifice of embryos for the production of stem cells.
This is not an easy debate. Imagine a case in which the only hope of cure for a young mother with two small children is the use of embryonic stem cell therapy. Even if this mother's dramatic situation might suggest that it would be ethical to sacrifice a mass of frozen cells stored in liquid nitrogen to obtain the needed stem cells for the therapy, the point that deserves to be addressed is this: Who would have the right to sacrifice a defenseless life (embryo) to save another (adult individual)?
The use of stem cells from bone marrow, umbilical cord, and other parts of the adult human body has not generated as much controversy. The potential benefits from stem cell therapy have been widely discussed. However, the use of embryonic stem cells has raised heated debates in public and scientific arenas. These cells are usually harvested from spare embryos generated through in vitro fertilization that have not been implanted in prospective mothers. Even if the scientist that uses stem cells were not responsible for producing them, he or she would be aiding in this process by creating a demand that results in the destruction of embryos, being an accomplice in the process. This is the same rationale used by the governments that burn ivory confiscated from smugglers, as well as the refusal of the scientific community to use the knowledge generated by the Nazis in the horrific human experiments conducted at the concentration camps during World War II.
This and many other recent discoveries in biotechnology have been occupying the world media. Although the scientific bases for cloning are easy to understand, the greater challenge for society is to address its ethical issues.
The lack of ethical references and the speed of development of new knowledge have exposed the society's lack of readiness to address current ethical issues. Sometimes society fears a technology with great potential benefits; other times it is apathetic about technology with proven negative impacts. Individualism and relativist morale, ideals in fashion in this postmodern society, are fertile ground for justifiable mistakes. These ideologies emphasize that nobody should deny anything to himself or herself that is good unless it is especially harmful to his or her neighbor. The ethical boundaries of society reflect the moral principles that it possesses. Society is dynamic and so are its ethical values. This doesn't mean, however, that the principles within society should develop in a liberal way.
Humans were created with intelligence and this allows them to develop new technologies and expand science. Along with this intelligence they have the freedom to choose between good and bad.
Why should one not be in favor of the evolution of the human race? What are the limits of what is morally acceptable? Any answer that deserves consideration should address the dilemmas of society in light of its principles, morals, and religious beliefs. These are some of the challenges society must deal with.
For more information on cloning refer to the following Web sites:
National Academy of Sciences: http://www4.nationalacademies.org/nas/nashome.nsf
New Scientist Magazine: http://www.newscientist.com/hottopics/cloning
After the cloning experience of Dolly the sheep, human cloning is theoretically and technically possible. The procedure would consist of taking an egg, removing its chromosomes, and then fusing it with a somatic cell from the individual to be cloned. Some believe that it is inevitable that some scientist will try to clone humans, if it is not already occurring. There seems to be a consensus that within a few years the news of the birth of the first human clone will be the major headline in the media. Scientists in South Korea reported success in creating a cloned human embryo, but it was destroyed instead of being implanted in a surrogate mother. Even if the first human clone is decades from birth, the idea that scientists are secretly trying to do it is a real possibility.
Scientists with an economic interest in this science have been expressing their viewpoint that it would be ethically acceptable to clone human beings. They argue that an embryo up to 10 days after fertilization cannot be considered a life because development of the brain begins at about 14 days after fertilization. It would be interesting to know how those scientists define the ethical limits in relation to their objectives.
It has been assumed by some that human cloning serves only the interests of the narcissists or neo-Nazis, those who would like to create the perfect race. In fact, several scenarios have been created that justify cloning of the Homo sapiens “animal.” Some of those scenarios can seem extremely appealing, but an ethical analysis of the dilemmas that clones, their relatives, and society would face during their life indicates that cloning of the most intelligent and rational of the animals is not politically, socially, or religiously acceptable.
Some of the following scenarios show the complexity of the subject:
Consider the situation of a homosexual man who feels frustrated with his incapacity to bear children and wants to be cloned.
Consider the couple that wants to have a baby, but the husband is sterile. Assuming that cloning is an alternative, the couple could decide to clone the husband, and the wife could contribute as a surrogate mother. Would the child's responses to education differ though he is genetically identical to his father? Would he have the same tastes and preferences as the husband? What if a divorce occurs? How would the mother see her son, who is a copy of the man from whom she is divorced? Would the father have the right to custody of the child because he is genetically related to his father?
In another scenario, where a woman gives birth to her own clone, would she be her child's mother or twin sister with a different age?
Obviously, society changes over time. In vitro fertilization was illegal in many countries until about 20 years ago, and the idea of heart transplants was considered immoral in the past. Public opinion on human cloning will probably change in the next few years, but cloning will likely be banned globally before the birth of the first human clone. It would be a terrible mistake to wait until the birth of a baby with genetic defects before that decision is reached. Current experience with animals shows that this technology has too many technical and ethical problems to justify experimentation in humans.
Ethicists are concerned that clones would be considered inferior to human beings, and they would be subject to the limitations and expectations of the knowledge of the copied person. These expectations could be false, as both genetic factors and the environment determine personality. For example, a clone of an extroverted person could be more introverted, depending on his or her upbringing. Clones of athletes, artists, scientists, and politicians could choose different professional careers based on opportunities and the environment in which they are raised.
Predicting the future of human cloning is not an easy task. History shows that society is dynamic, that ethical values change, and moral principles distort over time. In other words, only time will tell. The challenge for bioethicists is to keep science progressing while maintaining the sanctity of life. Additional ethical arguments related to human cloning are presented in Chapter 14, “Bioethics.”
It is a mistake to think that genetically identical means identical individuals. In the 1978 movie The Boys of Brazil, based on Ira Levin's bestseller, a scientist conspires after World War II to clone Hitler, with the objective of raising a new generation of Nazi leaders. The film shows that without intense indoctrination, the clones can be influenced to pursue other activities than becoming dictators.
There is a series of scientific reasons for not cloning human beings. Although many scientists and most of the public share this point of view, it is feared that personal ambition of unscrupulous scientists would make them blind to the scientific reasons for not cloning man. The success in animal cloning is evidence that this technology might be ready to justify its application to humans. In 2000, Dr. Panayiotis Zavos, an Israeli specialist in in vitro fertilization, and Dr. Severino Antinori, an Italian specialist in reproductive physiology, announced their intention to clone humans. In April 2002, Antinori claimed that he had two women carrying cloned babies.
After the birth of Dolly and the successful cloning of mice, cattle, monkeys, goats, and pigs, it is evident that cloning is not a completely safe procedure. Cloning of mammals is considered highly inefficient, and this is unlikely to change in the foreseeable future. Many cloning experiments have resulted in developmental flaws either during gestation or in the neonatal period. Even in the best cases, only a small percentage of cloned embryos survive to birth and, of those, many die shortly after birth. There is no reason to believe this would be any different with humans. This means that to achieve the successful generation of a human clone, many others will have been sacrificed in the developmental phases.
The few animal clones that have survived and been born show abnormal size, a phenomenon called increased offspring syndrome. It is believed that incorrect functioning of the placenta is one of the main causes of embryonic death. The suspected causes of newborn death are respiratory and circulatory problems. Some seemingly healthy survivors might possess immune system dysfunction or kidney and brain malformation. Those problems have been detected in practically all species in which cloning has been accomplished. Therefore, if an attempt to clone a human is made, the concern is not just with the embryos, but also with those that will live to be abnormal children and adults.
The abnormalities in the fetuses and in those few clones that are born alive cannot be easily traced to the nucleus of the donor. The most probable explanations are flaws in the genetic reprogramming or timing and expression of the correct developmental genes. Normal development depends on a necessary sequence of changes in the configuration of DNA and proteins coded by developmental genes. Those developmental changes control the specific genetic expression in the specialized tissues.
Genetic reprogramming of the entire genome is a natural process that happens during spermatogenesis and oogenesis, which can span over months and years in humans. During cloning, reprogramming of the donor's DNA must be done within minutes or, at the most, in a few short hours, during the period of time that nuclear transfer is completed and cell division begins to form the zygote.
Prenatal mortality in clones can occur due to inadequate reprogramming that results in improper gene expression. Some surviving clones have subtle genetic defects that, over time, result in life-threatening conditions. There is no information on genetic regulation in clones, but some evidence seems to indicate errors in gene expression in cloned animals. The expression of marked genes is significantly altered when embryos are cultivated in vitro before they are implanted in the uterus, indicating that even a minimal disturbance of the embryo's environment can have profound effects on gene regulation during development.
All the current evidence now suggests that the experiments on human cloning announced by Zavos and Antinori will have the same failure rates and occurrences of abnormalities that have been detected in animal cloning. Zavos tried to calm the public, informing them their research would use genetically perfect embryos to be implanted as a quality control. However, the public perception of reproductive biotechnology will be seriously damaged if the research fails and defective babies are born from human cloning experimentation. This would likely negatively affect other areas of research, such as the advancements being made with stem cells.
The National Bioethical Advisory Commission in the United States reached the following conclusion six years ago: “At the present, the use of cloning to generate a child would be a premature experiment, and would expose the fetus and the child in development to unacceptable risks.” All the data gathered since seems to reinforce this point of view. In many countries, it is unlawful to perform research with human reproductive cells, thereby forbidding embryonic cloning.
Other ethical considerations of human reproductive biotechnology are discussed in Chapter 14.