semina ve phoi! lam on giup minh voi!

chào các bạn, mình đang là sinh viên năm 2 công nghệ sinh học, nhóm mình đang chuẩn bị cho một buổi thảo luận về phôi.
hiện nay, bọn mình chưa chính thức học môn này nên những đề tài có thể trình bày phải đơn giản, dễ hiểu và mang tính hấp dẫn một chút.
đề cương của bọn mình sẽ gồm phần đầu tiên là trình bày về khái niệm phôi, quá trình phát triển của phôi một loài nào đó(có lẽ sẽ là phôi người).Sau đó sẽ là những ứng dụng: nhân bản vô tính( phân tích kĩ về sự kiện cừu dolly và tuyên bố mới đây về việc tạo được phôi người ở hàn quốc), việc thụ tinh trong ống nghiệm ở bệnh viện từ dũ.
khi thi khoa này , mình thi vào bằng khối a cho nên kiến thức sinh học của mình không tốt chút nào cả. Mong mọi người góp ý cho đề cương này, gợi ý cho mình một số vấn đề trong phôi học ( cái nào hay hay mà đừng có hàn lâm quá!)
với cả ai có tài liệu về phôi nào hay hay, hoặc chỉ cho mình các trang web cũng được, nhất là những cái nào có nhiều hình ảnh minh hoạ ấy, cảm ơn các bạn thật nhiều nhé!
Tuy hiện nay, tụi mình chưa học sâu về sinh lắm nhưng tuần nào lớp cũng tự tổ chức semina về sinh để khiến cho mọi người yêu thích hơn về ngành học mà mình theo đuổi. Lớp các bạn thì sao, có sáng kiến gì để việc học trở nên thú vị hơn ko?

De tai cua ban tuy chi noi ve PHOI nhung that ra kha rong. HuyNguyen co kha nhieu tai lieu ve stem cell, human clonning den ART va ban co the tu do lua don phu hop yeu cau. Chung toi vua update (24/03/04) mot so tai lieu moi ve an de nay. Neu ban o SG co the den 201 Luong Nhu Hoc de dat hang, HuyNguyen se co gang giup ban
phongtailieu@huynguyenltd.com
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nhưng em lại ko ở thành phố hồ chí minh anh ạ!
thôi thu hẹp đề tài lại vậy, mọi người có thể cho em biết những hướng ứng dụng của công nghệ phôi trong tương lai ko?, những cái đang còn là ý tưởng hoặc đang nghiên cứu.
chỉ cần nêu tên thôi cũng được còn chi tiết em tự tìm.

Ethical Aspects of Advanced Reproductive Technologies
JOSEPH G. SCHENKER
   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EMBRYONIC STEM CELLS
NEUROLOGICAL DISORDERS
ETHICAL ASPECTS OF PREEMBRYO...
CONCLUSION
 
The progress achieved during the last 25 years in the assisted reproductive technology field has been phenomenal. Many countries currently practice genetic material donation, human embryo cryopreservation, selective embryo reduction, preimplantation genetic diagnosis, and surrogacy. While embryo research and therapeutic cloning are carried out only in a few centers, thus far human cloning has been universally condemned. Nonetheless, the rapid evolution and progress of these various techniques of assisted reproduction has opened a Pandora''s box of ethical issues that must be urgently addressed.
Key Words: ethics õ? cloning õ? embryo research õ? genetic material donation
   INTRODUCTION
 
Ethics is the enterprise of disciplined reflection on the moral intuitions and the moral choices that people make. Medical ethics is the moral obligation that governs the practice of medicine. There are several significant historical trends and events that have influenced the formation and evolution of bioethics. The medical profession has a long history of ethical codes, which dates back to at least 2000 B.C. and the Codex Laws by Hammurabi. Medical ethics in the past was based on an adherence to professional rules of conduct by practicing physicians. In the last half of the twentieth century ethics has materialized as a field of its own. Moral philosophers, theologians, lawyers, and sociologists advise physicians on what they should do and how they should act, and now dominate medical ethics, also known as bioethics. Modern bioethics is based on a pluralistic and multidisciplinary approach, deriving its sources from medicine, biology, philosophy, law, theology, social and behavioral sciences, and history. Bioethics is defined as the systematic study of human conduct in the area of the life sciences and health care, in so far as this conduct is examined in the light of moral values and principles. It includes issues related to individuals as well as to public, national, and international policy-related issues.
The moral foundation of modern biomedical ethics is based upon four prima facie principles: respect of autonomy, beneficence, nonmaleficence, and justice.1 Medical ethics has become an important part of all medical research and treatment necessarily affected by the pluralism of modem society, which is influenced by religion, culture, and historical events.
The advent of new medical technologies brought with it the dilemma of withholding or withdrawing them from patients when their use becomes inappropriate or unwanted. Issues concerning right and wrong in dealing with life and death, as well as the issues of equality, justice, and personal preferences, are evoked. The advent of organ transplantation brought about further debate, related mainly *****pply and demand. Advances in reproductive technologies created yet another series of ethical questions, such as what is important in parenting: the genes, the procreative effort, or the rearing environment? Gamete and embryo donation and in vitro fertilization (IVF) enable pregnancies outside of conventional social arrangements, for instance, surrogate or single parents. Modem genetics have created yet another new set of ethical questions, such as privacy, insurance risks, denied employment opportunities, and possible stigmatization if a person''s genetic makeup becomes known. There are fears of a resurgence of the eugenics movements or of germ line genetic engineering and biologic genetic enhancement. The availability of early diagnostic methods such as ultrasound, chorion villous sampling, and amniocentesis to diagnose abnormal fetuses, brings forth the question of whether to terminate the pregnancy. This has again and again reheated the argument surrounding abortion. The public is at present aggressively interested in how and to whom health care is delivered. Society''s concern for ethical issues of medical practice has led to a growing need of the medical profession to be fully aware of the public view, not only on the individual patient-physician relationship, but also on how the new developments in medicine affect the issues of human rights, social structure, and health policy.
Developments in reproductive medicine over the last 20 years have created unexpected unprecedented public interest in certain aspects of human reproduction. The range of ethical questions that have been raised with the introduction of assisted reproduction have been debated by governments and medical groups around the world. It is very difficult to find solutions to the ethical problems in reproductive technologies that are acceptable in a pluralistic society, and it is even more problematic to reach concensus on universal policy. Many believe that nowadays the physician should not be left alone to make decisions of an ethical nature. This has prompted the development of ethics committees in institutions, and by governments and international bodies.
The cloning of several animal species, the isolation of human embryonic stem cells, and the discovery that adults may be reprogrammed, taken together give substance to hopes for novel principles of treatment, but it raises new ad***ional ethical dilemmas.
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EMBRYONIC STEM CELLS
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Stem cells are cells that have the ability to divide for indefinite periods in culture and to give rise to specialized cells. In the very early stages of embryonic development, stem cells have the capability of dividing indefinitely and then differentiating into any type of cell in the body. Recent studies have revealed that much of this remarkable developmental potential of embryonic stem cells is retained by small populations of cells within most tissues in the adult. The development of stem-cell lines that can produce many tissues of the human body is an important scientific breakthrough. Transplanted stem cells will allow them to replace lost or dysfunctional cell populations in degenerative disorders. This research has the potential to revolutionize the practice of medicine and improve the quality and length of life. At present, human stem-cell lines have been developed from two sources with methods previously developed in work with animal models:
Human embryos 5- to 9-weeks postfertilization, obtained as a result of therapeutic termination of pregnancy. The cells were obtained from gonadal ridges and mesenteries of human embryos.2
Stem cells were isolated directly from the inner cell mass of human embryos at the blastocyst stage, received from IVF clinics. These embryos were in excess of the clinical need for infertility treatment, and embryos were made for purposes of reproduction.3
Trials have been carried out to obtain human stem cells by somatic-cell nuclear transfer (SCNT). Therapeutic cloning involves the transfer of the nucleus from one of the patient''s cells into an enucleated donor oocyte for the purpose of making medically useful and immunologically compatible cells and tissues. A method successful in animal models.4
There are several important indications why human stem-cell lines formation is important to advances in health care. Research on stem cells could help us to understand the complex events that occur during human development. A primary goal of this work would be the identification of the factors involved in the cellular decision-making process that results in cell specialization. For this, stem cells need to be grown on a large scale, genetic modifications introduced into them, and their differentiation directed. To guide different stem cells into the desired lineage requires the identification of factors that direct their differentiation There are several potential applications of stem-cell technology in human medicine: basic embryological research, functional genomics, growth factor and drug discovery, toxicology, and cell transplantation. Some of serious medical con***ions, such as cancer and birth defects, are due to abnormal cell specialization and cell division.
Most important clinical pathological con***ions result from disruption of cellular function or destruction of tissues of the body. Stem cells, stimulated to develop into specialized cells, offer the possibility of a renewable source of replacement cells and tissue to treat diseases, con***ions, and disabilities common in aging patients.
ÂÂ NEUROLOGICAL DISORDERS
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Demyelination is the major pathology in diseases of the nervous system. Animal experiments showed that transplanted myelinogenic cells can remyelinate the damaged axons and restore function. Cells can be obtained from multipotential stem cells that have been expanded and committed to oligodendrocyte lineage before transplantation. Embryonic stem (ES) cell-derived oligodendrocyte precursors were transplanted into a rat model of myelin disease. The cells efficiently myelinated axons in both the brain and spinal cord, but did not improve neurological function in these animals.5
The derivation of neural progenitor cells from human ES cells is of value both in the study of early human neurogenesis and in the creation of an unlimited source of donor cells for neural transplantation therapy. In our Institution Hadassah Medical Center in Jerusalem, the generation of enriched and expandable preparations of proliferating neural progenitors from human ES cells was obtained. The neural progenitors could differentiate in vitro into the three neural lineages: astrocytes, oligodendrocytes, and mature neurons. When human neural progenitors were transplanted into the ventricles of newborn mouse brains, they incorporated in large numbers into the host brain parenchyma, demonstrated widespread distribution, and differentiated into progeny of the three neural lineages. The transplanted cells migrated along established brain migratory tracks in the host brain and differentiated in a region-specific manner, indicating that they could respond to local cues and participate in the processes of host brain development.6
Spinal Cord Damage
Spinal cord injury is a major source of morbi***y following trauma. Degenerative diseases of the spinal cord often lead to premature death. Mouse ES cells have been grafted into the injured spinal cord of immunosuppressed rats. Surviving cells developed into neurons, oligodendrocytes, and astrocytes, and supported partial recovery of motor function in the hind limbs that were affected by the spinal damage. However, no clinical trial has demonstrated significant functional effects of grafting primary embryonic neural tissue to the spinal cord in humans.
Parkinson''s Disease
Parkinson''s disease is due to degenerative changes in the ventral midbrain, causing a striatal dopamine deficit and a severe movement disorder. Parkinson''s disease so far is the only disorder that has been treated successfully with transplantation of embryonic brain tissue. A limitation for embryonic-brain-tissue neural-transplantation trials in Parkinson''s disease is the need to use multiple donors for each patient. In future, to use a stem cell that could be proliferated in an unlimited fashion and then differentiated into a dopamine-producing cell with a full repertoire of neuronal features may be a curative approach. Recently, undifferentiated mouse ES cells were found to develop into fully differentiated dopamine neurons after transplantation into a experimental rat model.7
Diabetes
Diabetes, which affects millions of people in the world, results from abnormal function of pancreatic islets. Current diabetes drug therapies do not provide sufficiently tight control of blood glucose to avoid late-stage complications. Transplantation approaches using the whole donor pancreas or isolated islet-cell transplantations are limited by a shortage of donors, major surgery, and long-term immunosuppression. Lumelsky and coworkers succeeded in generating cells that produce insulin and other pancreatic endocrine hormones from mouse ES cells. When injected into diabetic mice, the insulin-producing cells undergo rapid vascularization and maintain a clustered, isletlike organization.8
Cardiovascular Disorders
Cardiovascular disorders with their effects upon the brain, heart, kidneys, other vital organs, and extremities, are the leading causes of morbi***y and mortality in Western countries.
Transplant of healthy heart muscle cells could provide new hope for patients with chronic heart disease whose hearts can no longer pump adequately. The goal is to develop heart muscle cells from human pluripotent stem cells and transplant them into the failing heart muscle in order to augment the function of the failing heart. Preliminary work in mice and other animals has demonstrated that healthy heart-muscle cells transplanted into the heart successfully repopulate the heart tissue and work together with the host cells.9 Experiments carried out in Israel show that human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. The human ES cell-derived cardiomyocytes displayed structural and functional properties of early-stage cardiomyocytes. Establishment of this unique differentiation system may have a significant impact on the study of early human cardiac differentiation, functional genomics, pharmacological testing, cell therapy, and tissue engineering.10
Cancer Research and Therapy
The major treatment strategy for cancer patients has been to kill the tumor cells by radiation or chemotherapy, an approach that is effective for some types of cancer, but not for others. Human stem-cell experimentation may be important to cancer research. Stem cells may be used to treat the tissue toxicity brought on by cancer therapy. The isolation and characterization of stem cells and in-depth study of their molecular and cellular biology may help us to understand why cancer cells survive despite very aggressive treatments. Once the cancer cell''s ability to renew itself is understood, new therapeutic strategy can be introduced for prevention and treatment of this common and lethal con***ion.
Ad***ional degenerative con***ions, and disabilities like osteoarthritis and rheumatoid arthritis, can be treated in the future by applying this new therapeutic approach.
Future Research
The use of stem cells for therapeutic purposes has been proposed for many different diseases. However, basic research is still needed before translation of the present experimental evidence of basic research to clinical trials. It is important to determine the best con***ions for growing the cells and directing their differentiation into specialized cells, such as neurons, muscle cells, and insulin-producing cells. Investigators will also need to learn about some of the key genes that control the capability of an embryonic stem cell to proliferate in an undifferentiated state. Research is needed on some of the existing cell lines regarding safety testing in culture and in animal model systems. Before stem cells (ES) for transplantation can be clinically applied, the well-known problem of immune rejection must be overcome. The human stem cells derived from embryos created by IVF would be genetically different from the recipient. Research using human embryonic stem cells will also facilitate the development of approaches to avoid immune rejection of transplanted cells, as well as their integration into and ability *****rvive in target tissue. Trials have been carried out to obtain human stem cells by SCNT. Therapeutic cloning involves the transfer of the nucleus from one of the patient''s cells into an enucleated donor oocyte for the purpose of making medically useful and immunologically compatible cells and tissues. A method that is successful in animal models.
Israeli scientists have been at the forefront of ES cell research. They were key players in the landmark isolation of stem cells from human embryos in 1998, and of the first 12 publications on human ES cells, 10 included Israeli authors.11
The use of human stem cells raises many ethical issues, for example, the moral status of the embryo, the consents required to use embryonic stem cells and other genetic material, genetic privacy, and ownership of genetic information.
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   ETHICAL ASPECTS OF PREEMBRYO RESEARCH
 
Human embryonic stem research and its potential clinical application is a hope for new modalities of treatment, especially for elderly generation suffering of degenerative medical con***ions. Since at the present time the promising source of stem cells is early blastocyst-stage embryos or tissues derived from the gonadal ridge of aborted fetuses-embryonic germ cells, the research involves the use of early embryos obtained as a result of therapeutic termination of pregnancy and/or human embryos received from IVF clinics. These embryos were in excess of the clinical need for infertility treatment and were made available for purposes of reproduction.
The potential use SCNT technology requires creation and disaggregation of human preembryos. Therapeutic cloning of stem cells may finally lead to reproductive cloning.
The use of early human embryos that are destroyed, the research is inextricably linked with the abortion debate in most countries.
Modern medical ethics is based on a multidisciplinary and a pluralistic approach. The pluralistic nature of modern ethics results in the impossibility of achieving a consensus on almost any moral statement. When considering an ethical argument concerning human subjects, some generally universal principles are accepted, such as respect for human dignity, doing no harm, the "slippery slope" argument, the autonomy rights of the patients, and beneficence.
Moral Status of the Human Preembryo
The central question regarding therapeutic approaches of research on the preembryo is its moral status. There are three options with which to consider the moral status of a human preembryo:12
The preembryo has no moral status. It is merely a collection of undifferentiated cells lacking individuality. Its status is no different from that of any other human tissue or cluster of cells. Furthermore, because the preembryo is not an integral part of the mother''s body, she has the full right to permit preembryo research, if she wishes to do so.
The preembryo has the full status of a human being. A new genotype is established during fertilization and some of the preembryos have the potential to become full-term fetuses, children, and adults. The preembryo has its own rights, and the gamete donors are only its guardians. The interests of the mother are irrelevant to the future of the preembryo. Society is obligated to apply therapeutic measures to the preembryo as an individual patient.
The preembryo is a potential human being. This definition is a relatively new philosophical entity, representing a compromise between the preceding two approaches. It is this opinion that is generally accepted today by most scientists, physicians, and ethicists. Inasmuch as the preembryo is a potential human being, it should be handled with dignity and its rights should be respected as long as they do not conflict with major social, maternal, or other ethical interests. Yet, it should not be treated as a person, because it has not yet developed the features of personhood, is not yet established as developmentally individual, and may never realize its biologic potential.
The Origin of Human Life
The question arises as to when the status of a potential human being is acquired. This issue has not yet been resolved by the many sides involved in the argument. There are different religious and ethical views regarding the origin of human life. The view of the Roman Catholic Church is that life begins at conception. Another view is that life begins with the implantation of the preembryo. According to another opinion, life begins when brain activity starts.
This could be interpreted as being 8 weeks after conception at the point when the embryo is responsive to stimuli, or later in gestation when there is evidence of higher brain activity. Some consider human life to begin when the human conceptus becomes a "person," has some degree of sentience or even active volition.
According to Grobstein,13 there are six aspects of individuality that become identifiable during a human life and are important to the determination of status. These are in order of their appearance during development: genetic, developmental, functional, behavioral, psychic, and social. The emphasis of this notion of individuality is the development of sentience and self-awareness. Therefore, the individual does not come into existence until about 26 weeks'' gestation. However, the moral status of the unborn human at different stages cannot be answered by scientific facts alone.13
In the common law system, the question of the status of the human preembryo has arisen in criminal as well as civil law. It is necessary to define the person in order to determine offences against the person.
Laws pertaining *****ccession, marriage, domestic relations, and the area of negligence in recovery for prenatal injuries stand to gain from specific definitions of status. Surprisingly, most legal actions have not been able *****pply a definite answer.
Human life cannot be attributed to the preembryo in the in vitro status. There are three main views concerning when the status of a potential human being is achieved:
The status of a potential human being is achieved at conception with the fusion of human gametes, at which point the zygote acquires the entire genetic information for human development.
The status of a potential human being is acquired at implantation, when no active procedure is required to maintain growth, and the chances of the preembryo to reach the neonatal stage are increased to more than 50%.
The status of a potential human being is acquired with the appearance of the primitive streak.
Acceptance of this concept resolves most of the ethical questions that concern preembryo research.
Respect for Human Dignity
A preembryo should be treated with respect for its potential to become a person. The creation of embryos or their abortion for the purpose of contributing their cells would demean the potential or actual humanity of the embryo, and would harm the principle of respect for human dignity. According to this principl, preembryo and embryo research can be performed as long as the preembryos were not created or aborted for research purposes alone.
Doing no harm, preembryonic or embryonic tissue often becomes available as a result of pregnancy termination.
The legality of induced abortions remains controversial in the Western culture. It is possible that the subsequent use of preembryonic or embryonic tissue for research will encourage abortions that would otherwise not occur.
This would make the issue of pre embryo research unseparable from the issue of abortions, thus causing much greater controversy than exists already. Many women are ambivalent about abortion. This ambivalence may be tipped in favor of pregnancy termination if the women are informed that some good may come of it, that is, they are given some incentive. This argument dictates that the use of preembryonic or embryonic tissue should not be allowed if it stimulates abortions that would otherwise not take place. Similarly, the subsequent use of IVF preembryos might encourage their intentional production for research purposes. A clear distinction must be made between preembryos from spontaneous abortion, where the incentive argument does not apply, and preembryos from induced abortions.
The use of preembryos from spontaneous abortion would only partially solve the problem, because 50% of these preembryos are chromosomally abnormal, and thus their use may be limited. A distinction can be made as to the cases when tissue from induced abortion could be used:
Induced abortion in order to save the mother''s life is a generally acceptable procedure, except by those who believe that the preembryo acquires full human status on conception. The use of preembryonic tissue from these abortions may not be more questionable than the use of preembryos from spontaneous abortions. In order to prevent conflicst of interest from affecting the advice given to patients on the issue, it is proposed that the mother, as well as the medical personnel who perform induced abortions, should not be allowed any direct or indirect benefit from the subsequent use of tissue.
Induced abortion for maternal reasons other than life-saving procedures. A physician or medical center should not feel obligated to participate in research or therapy involving such tissue, if morally incapable of doing so.
Induced abortion for contributing embryonic tissue is unacceptable and harms the principle of respect for human dignity. However, if we accept the idea that the preembryo is an integral part of the mother''s body, she may be able to do with it as she wishes. Preembryo research could apply for the first category.
Slippery Slope Argument
The slippery slope argument claims that if a given act is allowed, it may end with unacceptable results. This is because there is a series of acts in between the original act and the unacceptable outcome that cannot be distinguished one from the other. This argument claims that it is difficult to find the cutoff point between a preembryo and a human being. Starting with preembryo research, we might end up with research on human beings that is similar to the monstrous experiments conducted by the Nazis in World War II. Creating IVF preembryos solely for research purposes might similarly lead to creating neonates for medical experiments. Only a broad consensus regarding the cutoff point can give an answer to the slippery slope argument.
Autonomy Rights of the Patients
The demand for obtaining informed consent is derived from this principle: autonomy, or the right to chose, is the actual ability to choose in a fully autonomous manner, and the moral right to act according to the chosen will. The first con***ion depends on our view of the moral status of the preembryo; the last con***ion could be naturally fulfilled. The preembryo is not able to voice its choice, so the second con***ion would not apply.
If "substitute judgment" is to be accepted, then the lack of authentic preembryonic consent is not an ethical contraindication to performing preembryo research. An agreement for the use of preembryonic tissue for essential research can be based on the assumption that the preembryo is endowed with the natural goodness and the fine qualities of humanity. If the preembryo is considered an integral part of the mother, then it is her informed consent that should be acquired.
Beneficence: The many benefits of preembryo research have been outlined. There can be little dispute as to the worthiness as research subjects, because they confer great benefits for the entire human race. Thus, the principle of beneficence is in favor of preembryo research.
Control of Preembryo Research
Licensing embryo research does not imply moral indifference. However, licensing without regulating does. It is obvious that cloning, placing of human embryos in other species, and altering the genetic structure must be explicitly forbidden. A high respect for the nature of their research is the fundamental characteristic of scientists. Within the ethical restraint imposed by statute and regulation, there is no convincing reason why embryo research should not be performed for sufficiently serious ends. In communities that, while putting a very high value on human lifê?"a value that impels them to take so much trouble to foster it?"do not attribute value to undifferentiated embryonic cells, embryo research may be performed. Embryo research must be subject to all the ethical restraints that are applied to any medical relationship. Informed consent must be obtained. The physician must not yield to a patient''s demand to perform research or supply treatment if the physician considers it medically inadvisable.
Considering the use of embryos acquired from pregnancy termination links us with the heated issue of abortion. Those who argue that induced abortion is completely wrong in all circumstances, argue that the use of the products of abortion, even for good purposes, compound the wrong, and should be prohibited. The only reasonable way around this, if seeking to use that material, must be by imposing a principle of separation. Discussion of the use of the embryo for research should not be opened with the pregnant woman until after she has made the decision to abort. Obtaining consent must be entirely separate for each procedure. Also, medical teams involved in abortions and in research should be separated.
The research must be approved by a Local Research Ethics Committee,14 and the passage of tissue should go through an intermediate tissue bank. In order to enhance the probability that an adequate number of normally developing embryos will result from IVF, more oocytes are fertilized than will be transferred to the woman.
Sometimes the number of resulting embryos exceeds expectations. They become "spare" embryos and may be cryopreserved for the couple''s future use, donated to a recipient, donated for research, or disposed of. The persons who should decide the disposition of the embryos are those who provide the gametes, through the process of informed consent. Their choices should be made devoid of financial or other coercion. "Spare" embryos are preferable to embryos generated specifically for research for the following reasons: the concern that women will be placed at unnecessary risk during the required ovulation induction process, and the preference for a process that is less vulnerable to the commercialization of gametes.
Research that is not intended to benefit the subject on whom it is performed requires more justification, especially if it involves the risk of harm to the subject. Neither medical practice nor biomedical research is static. Over time, procedures move from being experimental to innovative treatments to becoming accepted as part of orthodox medical practice.
The judgment of whether a research project is ethically acceptable must be made on the basis of a proposal preceding the project, and not after its completion in light of its success or failure to provide the anticipated results. Validation must be found in the results of previous research or clinical application, which provide a basis for reasonable expectation, and must be seen as a matter of professional opinion. Embryo research is argued to be justified as therapeutic research, because it is necessary to improve the success rate of IVF for the benefit of the women undergoing the treatment.
Human embryo research is subject to ethical controls that are generally not imposed on. The reason behind this is that human life is sacred and possesses unique dignity, a principle that must not be eroded under any circumstances. Research must be performed with the integrity proper to all scientific endeavors.
Research must be performed with the constant reminder of ethical limitations, which must not be forsaken. To prevent misuse, strict guidelines for regulation must be applied.
Therapeutically orientated research on embryos was banned by some individual couples, some societies, and religious authorities. For many people, embryos have intrinsic value from the moment of conception, whatever their stage of development, wherever they are, and whatever their likely future. It does not matter that the embryo might be one of several hundred thousand left over after IVF and waiting almost inevitable discard. Roman Catholic doctrine holds that embryos have intrinsic value,15 and the has Pope publicly expressed his disapproval of human stem-cell research.
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ÂÂ CONCLUSION
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It is undeniable that the human con***ion has been generally improved through systematic biologic and medical research. The new reproductive era introduces new considerations, involving simultaneously formidable risks as well as substantial benefits. Human preembryos deserve special respect because some possess the potential to become human beings.
There are dilemmas in setting and monitoring guidelines to help clarify acceptable versus unacceptable experimentationon humam preembryos. Preembryo research should be conducted under restrictions and supervision by the society.
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The future of cloning
It is now possible to make clones, or exact genetic copies, of sheep, cows, goats, mice and, probably, humans. This opens the way towards the production of replacement body parts from adult cells.
Cloning techniques have been in use for centuries. The practice of taking cuttings is universal among gardeners, and large companies now propagate desirable plant strains in their millions. Lower invertebrates can also be cloned â?" cut an earthworm or flatworm in half, for example, and the missing halves will regenerate to create two genetically identical individuals. Although vertebrates cannot be cloned by these routes, identical twins are naturally occurring genetic clones. Moreover, the method of nuclear transplantation, first developed about 40 years ago in frogs, has been successfully used to make clones of sheep, mice, cows and goats, and it could probably be applied to people too. By taking a few non-reproductive cells from adult mammals, identical replicas can be created without damage (or even inconvenience) to the donors.
But what is the way forward for cloning techniques in the next century? Combined with other cell-biological procedures, they could open the way towards a type of tissue-replacement therapy that avoids the problems of immune rejection. What can we achieve using this methodology, and what are the potential benefits for humans?
How is it done?
In vertebrates, fertilization begins with the union of the sperm and the egg. The unfertilized egg is stopped at a certain stage of the cell-division cycle, and the sperm provides an activation stimulus that triggers the resumption and completion of cell division. The egg and sperm ''pronuclei'' then swell, their chromosomes unravel from the tightly packed, ''condensed'' state in which they are stored, and DNA replication can proceed. The chromosomes then recondense, the nuclear membrane dissolves, and the fertilized egg divides into two identical daughter cells.
Nuclear transfer subverts fertilization by replacing the female genetic material of an unfertilized egg with the nucleus from a different cell. This was first done successfully on frogs in the 1950s, in the United States and Britain. A non-reproductive (somatic) cell, such as an intestinal epithelial cell, was ruptured by suction into a glass microneedle. Its nucleus, surrounded by a layer of cytoplasm, was then injected into an unfertilized egg from which the female genetic material had been removed or destroyed by ultraviolet irradiation. Some of these nuclear-transplant embryos developed normally to swimming tadpole or adult stages, and genetic markers were used to show that only the transplanted nucleus â?" and not the egg pronucleus â?" had contributed the genetic material of the resulting embryo1.
Mechanical disruption of donor cells was also tried in mammals. Until recently, however, it was an unsuccessful or unreproducible method of nuclear transfer. Consistent success in producing live, full-term animals was achieved in livestock by fusing a donor cell from an early embryo with either unfertilized or mock-fertilized eggs as recipients2 (Fig. 1). The female genetic material is sucked out, then the recipient cell is fused with the donor nucleus using electrical or chemical methods. In most experiments, the donor nucleus comes from a dividing cell, and it should be able to divide in harmony with the recipient. Nuclear transfer in livestock, using somatic cells as donors and unfertilized eggs as recipients, has been successful (the most famous example being Dolly the sheep), but only in a small percentage of cases3. Live mouse births were initially achieved only by using donor nuclei from very early embryos, although a piezo-electrically controlled microinjection device has facilitated success with the nuclei of adult cells4.
Figure 1 Nuclear-transplantation techniques in mammals. ÂÂFullÂlegend
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How can we tell that mammals obtained by nuclear transfer are genetically identical to the donor cell? Confirmation has come from microsatellite5 and DNA fingerprinting6 analyses. However, only genetic material in the nucleus will be identical to the nuclear donor. A cell''s energy-producing factories, the mitochondria, also contain their own genome. Because mitochondria are located in cytoplasm, they are inherited through the cytoplasm-packed recipient (the egg) rather than through the donor7. The mitochondrial DNA in mammalian clones is therefore entirely maternal in origin.
Conservation of the genome
For successful cloning, it is probably essential for donor cells to contain a full complement of genes. Germline cells (the eggs and sperm) have a complete set of genes; that is, those needed for all cell types such as skin cells, intestine and so on. But for nuclear transfer to work, an adult cell that has already been programmed as, say, a skin cell, needs to be somehow reprogrammed so that it regains genetic totipotency â?" the ability to guide the formation of all the different cell types that make up an animal.
Early experiments with the frog Rana pipiens showed that nuclear totipotency is lost very early in development, at a stage known as gastrulation8. However, in another species of frog, Xenopus laevis, a transplanted nucleus has been shown to retain genetic totipotency. Indeed, the first clones of adult males and females were obtained in this species from embryonic nuclei (Fig. 2). Although the proportion of successful nuclear transfers decreased as increasingly differentiated donor cells were used (that is, cells more specialized for a particular function), at least a few normal tadpoles (1.5% of the total nuclei transplanted) were obtained from the most advanced donor cells9. These experiments culminated in the derivation of genetically marked, fertile male and female adult frogs by transplanting nuclei from intestinal epithelial cells of feeding tadpoles10. Later experiments used other kinds of differentiated cells as donors, including striated muscle cells, adult skin cells11 and blood cells12. Thus the principle of totipotency or multipotency was established.
Figure 2 Cloned frogs. ÂÂFullÂlegend
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For many years, attempts to transplant nuclei in mammals were unsuccessful, probably because the recipients were fertilized eggs. Nuclear transfer to unfertilized eggs, as in amphibians, has been remarkably successful. Dolly the sheep3 was obtained using cultured mammary-gland cells as donors, and similar experiments with adult mice and cows have yielded fertile adults4, 13. Therefore, totipotency of some adult somatic cells has been demonstrated in mammals.
Nevertheless, the yield of normal animals from nuclear transplantation is low. Starting with cells from adult animals, or with fully differentiated cells from tadpoles, only 0.1â?"1.0% of all eggs receiving transplanted nuclei are born alive (mammals) or reach the swimming stage (tadpoles). Most nuclear transplants either fail to divide or they develop abnormally, possibly due to problems in reprogramming the transplanted nucleus. In amphibians there is the ad***ional problem that incompletely replicated chromosomes are forced to divide prematurely, becoming broken or lost. In mammals, the importance of synchronizing division of the donor nucleus and recipient cell has only recently been appreciated14.
An important conclusion to come from nuclear-transfer experiments is that the processes of cell differentiation and ageing do not lead to genetic changes in somatic cells. However, there are a few special exceptions. For example, immunoglobulin genes undergo rearrangements to generate antibody diversity. Moreover, mutations can occur in non-reproductive cells, although it is unlikely that changes affecting both copies of an essential gene occur in more than one in 104 cells. Finally, telomeres, the specialized structures at the ends of chromosomes, shorten progressively with age in somatic cells. In mice unable to generate telomeres, however, it takes several generations before this has any noticeable effect15. Moreover, sheep made through somatic nuclear transfer have shorter telomeres, but this has had no detectable effects so far7.
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Gene reprogramming
The pattern of gene expression in adult cells is very different from that in embryonic cells. In amphibians, for example, a number of genes expressed in embryos five hours after fertilization are not expressed in differentiated (specialized) larval or adult cells16. Conversely, some genes are expressed in adult cells but not in early embryos. When embryos are analysed a few hours after the transfer of adult cell nuclei, gene expression cannot be distinguished from that in embryos grown from fertilized eggs17. This means that the exchange of cytoplasm around a nucleus, from that of an adult cell to that of an egg, causes a dramatic switch in gene expression in only a few hours. A nucleus that was once part of an intestine, skin or muscle cell is therefore transformed into that of an embryonic cell.
The situation is probably different for imprinted genes in mammals. Imprinting, which is required for normal development, is a process by which about 30 genes are marked during sperm and egg formation such that they are switched off in the embryo18. Imprinted genes are unlikely to be reprogrammed by nuclear transfer, because embryos would not survive if they were. Moreover, imprinting is normally reversed during egg and sperm formation, rather than after fertilization.
Another focus of reprogramming attention is the inactive X chromosome. During early development of female mammals, one of the two X chromosomes is randomly inactivated in those tissues contributing to the fetus19. However, in the embryonic tissues that contribute to the placenta, the paternal X chromosome is always the one that is inactivated19. These observations prompt a question, so far unanswered, concerning the use of female cells in cloning. Will the inactive X chromosome become reactivated in the tissues of the newly created animal? If not, will embryos reconstructed from a donor containing an inactive maternal X chromosome be viable, given that the maternal X chromosome is usually inactivated in the paternal tissue (which will therefore have no active X-chromosomal genes)?
In amphibians, reprogramming of gene expression is accompanied by massive enlargement of the nucleus and exchange of proteins with those in the cytoplasm. When a nucleus is transplanted to a frog egg, it undergoes 12 rounds of division before new gene activity commences. Does gene reprogramming require the formation of new DNA (DNA replication)? To test this, several nuclei can be injected at once into growing egg cells in which replication does not take place. The transplanted nuclei nevertheless change their pattern of gene expression to conform with that of a growing egg cell20. These experiments show that gene reprogramming can occur on the same actual genes as were present before nuclear transfer, and does not require the formation of new DNA. Key molecules found in eggs that may bring about reprogramming include nucleoplasmin and certain embryo-specific histones (proteins around which the DNA is wrapped).
New tissues for old
Damaged or diseased tissues often cannot be repaired by drugs or other medication, and most organs and tissues regenerate very poorly in mammals. In a few cases, artificial materials such as replacement joints, or mechanical devices such as renal dialysis machines, work remarkably well. But ultimately, the most satisfactory remedy is to transplant organs or tissues from other people.
Transplantation works reasonably well for kidneys, hearts and so on, but there are three major disadvantages. First, the supply of such organs is extremely limited, depending largely on donations from accident victims. Second, treatment is very expensive (about Ê100,000, or US$160,000, for a replacement heart). Third, recipients need to be given immunosuppressive drugs to avoid rejection of the transplanted organ owing to the genetic differences between donor and recipient. Although the use of animal organs has been considered for transplantation, the genetic incompatibility is even greater with these, and even animal organs that have been engineered to contain human immune regulator genes are still targets for rapid rejection.
An alternative strategy involves stem cells; these are cells that can renew themselves and also give rise to a variety of differentiated cell types. Mammalian bone marrow, for example, contains a range of haematopoietic (blood-forming) stem cells. Some of these can be isolated and encouraged to proliferate using natural signalling molecules such as members of the interleukin family. These stem cells can then be made to progress to form more restricted stem cells (which can form a more limited range of cell types) and, eventually, to form fully differentiated cells such as erythrocytes or granulocytes. This type of blood stem-cell therapy has been practised for many years in humans, but the quantity and quality of available stem-cell types is very limited. The prospects also seem good for stem cells obtained from olfactory placodes. These neural stem cells can be proliferated in culture, and they have been shown to restore function in the mouse central nervous system.
It is also possible for stem cells of one type to generate occasional cells of a different type, although the con***ions that achieve this are not known. For example, neural stem cells can generate haematopoietic stem cells when transplanted to mice that have been irradiated to eliminate their own blood stem cells21. Stem cells from human bone marrow have been reported to generate functional neural cells22. An ideal stem cell is that exemplified by mouse embryonic stem or germ cells, which are derived experimentally from early mouse embryos or germline cells respectively. These cells can be proliferated in culture and, when transplanted to hosts, can differentiate into all types of adult cell. Human cells with several properties of mouse embryonic stem cells have also been described23, 24. However, all embryonic stem and germ cells are currently obtained by killing normally generated early embryos, raising ethical concerns for human material.
Despite their advantages, transplantation and stem-cell strategies still suffer from the problem of immunological incompatibility, a problem that could be avoided if material derived from a patient''s own tissues could be transplanted into them. This is already done with skin for burns patients, but the amount of material is very limited and, for most tissues, it is not yet possible to obtain enough stem cells, if they can be obtained at all. One approach to the problem is to freeze, at birth, samples of cells from the umbilical cord. This tissue is rich in stem cells which, after proliferation and differentiation, might be of use later in life.
Therapeutic cloning
Given the problems of rejection, the lack of identified stem cells for most tissues, and the difficulties of using normal human embryos as a source of embryonic stem cells, an altogether different route is to combine therapeutic cloning with the use of differentiation factors (Fig. 3). Although not yet practicable, this scheme offers a realistic possibility for the future.
Figure 3 Steps involved in therapeutic cloning.   Full legend
 
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The first stage is to use nuclear transplantation to reprogramme the nucleus of a human adult cell and obtain a blastocyst (a very early embryo). This step, in effect, transforms an adult cell into an embryonic cell of the same genetic constitution. By doing a number of such transplants in series, the yield of embryos will be increased. The next stage is to expand this population of embryo cells in culture by promoting proliferation but not differentiation. This has not yet been done with embryos obtained by nuclear transplantation, but it works well for mouse and human embryonic stem-cell-like cells, showing that it can be done in principle.
Having obtained a certain number of cells, the next step is to differentiate them in a desired direction. In amphibians this works well when blastula cells are exposed to signalling molecules that act early in development, including bone-morphogenetic proteins, the fibroblast growth factor, Cerberus and noggin. A molecule called activin, which belongs to the transforming growth factor- family, is particularly useful. Activin is very stable, easy to make in a biologically active form, effective at extremely low concentrations, and it can direct blastula cells towards a wide variety of cell types depending on the concentration at which it is used25. Combinations of other secreted factors generate an even greater variety of differentiated cell types. A mass of differentiated cells could then be transplanted back to the original nuclear donor.
Might such extensive manipulations cause some of the resulting cells to become cancerous? Possibly, but this could, in principle, be avoided, at least for those tissues that are not continuously replaced in the body. If a gene (or genes) were introduced to limit the number of divisions that the cells may undergo before their terminal differentiation (a state in which they can no longer divide), cells would be unable to proliferate out of control. Another way to achieve this objective could be to reduce the length of telomeres during in vitro culture.
Of course, several steps will need further development before this procedure is of practical use. These include:
An increased efficiency of nuclear transfer.
The ability to derive a population of embryonic stem-cell-like cells from nuclear-transplant blastocysts.
An increased ability to differentiate such embryonic stem cells into functional tissues.
Methods to screen for any cell lines that may have incurred genetic damage, and that would not therefore be suitable for human in vivo use.
This scheme has several advantages. It requires unfertilized eggs rather than embryos, and the nuclear-transplant blastocysts do not need to be able to develop normally (they only need to form the required cell types). Moreover, populations of cells or simple tissues may be useful, avoiding the need to create complex organs. Above all, the newly generated cells would be almost totally compatible with the recipient. We anticipate that any incompatibilities, owing to minor histocompatibility antigens in the transplanted cells, which will differ from those of the patient, should not be significant. This supposition could easily be tested by skin transplantation between a group of cloned animals13.
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Legality and ethics
The publication of Dolly the sheep''s existence had an immediate and generally negative effect on public opinion worldwide, and sparked off a flurry of restrictive legislative activity. Most invective was aimed at the prospect of human reproductive cloning; that is, the production of a person using nuclear transfer. Both safety and ethical reasons were cited against such cloning. Belated sober reflection revealed that few of these moral arguments were sound. For example, the ethical objection about the welfare of cloned children has been shown to apply equally well to the case of natural procreation26: if the anticipated psychological damage to clones from societal prejudice is enough to ban the practice, then the psychological damage to a mixed-race child from racism should justify a like ban on procreation from mixed-race couples. Clearly the latter would be absurd.
Therapeutic cloning is ethically less contentious because a new person is not produced. However, as for abortion, the issue of the deliberate destruction of a potential person is raised27. Therapeutic cloning is illegal in many countries (Fig. 4), where legislation prevents the use of human eggs for therapeutic research. This prohibits therapeutic cloning using unfertilized eggs, even though a nuclear-transplant embryo would be used to make embryonic stem-cell-like cells, and so would have no potential for survival because it would not be implanted.
Figure 4 Countries in which anti-cloning legislation has been, or will shortly be, passed. ÂÂFullÂlegend
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No such legislation has yet been enacted in the United States, except that federal funds cannot be used for reproductive cloning, nor can they be used in generating embryos for therapeutic cloning. They can, however, be used for research on cell lines derived from human embryos. In the United Kingdom it is legal to use human embryos up to 14 days old for research connected with fertility or contraception. It is illegal to use human eggs for any purpose where the intention is to create an embryo, even if only for cell replacement. Permission to do this (and, hence, to allow therapeutic cloning) could be granted by the Secretary of State for Science under an ad***ion to the present Human Embryology Act.
Perhaps a way round these issues is to use cells or eggs from other species. However, as yet, normal development has not been obtained with nuclear transfers between species. All but one of the human-nucleus to cow-egg transfers attempted died before the embryo had divided five times, and other mammalian combinations behaved similarly28, 29. It is therefore unlikely that animal eggs could be used as an alternative to human eggs as recipients for human nuclei.
The future of cloning
Live births have been achieved using somatic nuclear transfer in mice, sheep, cows and goats, and the technique would probably also be successful in humans. However, the low efficiency of the procedure may restrict its immediate application to the production of small herds of identical animals. There will be special applications, though, where the production of single animals will be useful. For example, the technique could be used to inactivate prion genes in ruminants30 (scrapie/BSE research), to disrupt the 1â?"3 galactosyl transferase gene in pigs31 (for xenotransplantation), and to knock out the cystic-fibrosis transmembrane-conductance-regulator gene in sheep32 (creating an animal ''model'' in which this disease could be studied). Nuclear transfer is currently the only option for gene targeting in livestock, and one of us (A.C.) has shown that sheep can be generated with targeted changes in particular genes.
Our belief, however, is that the greatest eventual benefit of the new technology will be in therapeutic cloning; the use of somatic-cell nuclear transfer to generate replacement tissues or organs. This would avoid the risks of tissue rejection by supplying a person with new tissue of exactly their own genetic type. All of the main steps in the therapeutic cloning procedure have been achieved individually, albeit at a low efficiency. We now need to improve the success rate for generating nuclear-transplant embryos from adult tissues, to find ways of generating embryonic stem-cell cultures from nuclear-transplant embryos, and to control more accurately the pathways of stem-cell differentiation and the formation of whole organs in vitro. As soon as any new scientific technique works at all, it is almost always improved in both efficiency and ease of operation. This seems likely to be the case for cloning technology too.
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