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"...contemplative and humorous essays by writers and scholars from a variety of disciplines and backgrounds."
divisions and progressive differentiation, first into the early embryo and subsequently into all of the cell types that make up the adult animal. Transfer of a single nucleus at a specific stage of development, to an enucleated unfertilized egg, provided an opportunity to investigate whether cellular differentiation to that stage involved irreversible genetic modification. The first offspring to develop from a differentiated cell were born after nuclear transfer from an embryo-derived cell line that had been induced to become quiescent. Using the same procedure, we now report the birth of live lambs from three new cell populations established from adult mammary gland, fetus and embryo. The fact that a lamb was derived from an adult cell confirms that differentiation of that cell did not involve the irreversible modification of genetic material required for development to term. The birth of lambs from differentiated fetal and adult cells also reinforces previous speculation that by inducing donor cells to become quiescent it will be possible to obtain normal development from a wide variety of differentiated cells.
from adult keratinocytes established in culture support development to the juvenile, tadpole stage. Although this involves differentiation into complex tissues and organs, no development to the adult stage was reported, leaving open the question of whether a differnetiated adult nucleus can be fully reprogrammed. Previously we reported the birth of live lambs after nuclear transfer from cultured embryonic cells that had been induced into quiescence. We suggested that inducing the donor cell to exit the growth phase causes changes in chromatin structure that facilitate reprogramming of gene expression and that development would be normal if nuclei are used from a variety of differentiated donor cells in similar regimes. Here we investigate whether normal development to term is possible when donor cells derived from fetal or adult tissue are induced to exit the growth cycle and enter the G0 phase of the cell cycle before nuclear transfer.
embryo, (2) a day-26 fetus and (3) mammary gland of a 6-year-old ewe in the last trimester of pregnancy. Morphology of the embryo-derived cells is unlike both mouse embryonic stem (ES) cells and the embryo-derived cells used in our previous study. Nuclear transfer was carried out according to one of our established protocols and reconstructed embryos transferred into recipient ewes. Ultrasound scanning detected 21 single fetuses on day 50-60 after oestrus. On subsequent scanning at ~14-day intervals, fewer fetuses were observed, suggesting either misdiagnosis or fetal loss. In total, 62% of fetuses were lost, a significantly greater proportion than the estimate of 6% after natural mating. Increased prenatal loss has been reported after embryo manipulation or culture of unreconstructed embryos. At about day 110 of pregnancy, four fetuses were dead, all from embryo-derived cells, and post-mortem analysis was possible after killing the ewes. Two fetuses had abnormal liver development, but no other abnormalities were detected and there was no evidence of infection.
were represented. One weak lamb, derived from the fetal fibroblasts, weighed 3.1 kg and died within a few minutes of birth, although post-mortem analysis failed to find any abnormality or infection. At 12.5%, perinatal loss was not dissimilar to that occurring in a large study of commercial sheep, when 8% of lambs died within 24 h of birth. In all cases the lambs displayed the morphological characteristics of the breed used to derive the nucleus donors and not that of the oocyte donor. This alone indicates that the lambs could not have been born after inadvertent mating of either the oocyte donor or recipient ewes. In addition, DNA microsatellite analysis of the cell populations and the lambs at four polymorphic loci confirmed that each lamb was derived from the cell population used as nuclear donor. Duration of gestation is determined by fetal genotype, and in all cases gestation was longer than the breed mean. By contrast, birth weight is influenced by both maternal and fetal genotype. The birth weight of all lambs was within the range for single lambs born to Blackface ewes on our farm (up to 6.6 kg) and in most cases was within the range for the breed of the nuclear donor. There are no strict control observations for birth weight after embryo transfer between breeds, but the range in weight of lambs born to their own breed on our farms is 1.2-5.0 kg, 2-4.9 kg and 3-9 kg for the Finn Dorset, Welsh Mountain and Poll Dorset genotypes, respectively. The attainment of sexual maturity in the lambs is being monitored.
upon the maintenance of normal ploidy and creating the conditions for developmental regulation of gene expression. These responses are both influenced by the cell-cycle stage of donor and recipient cells and the interaction between them (reviewed in ref. 9). A comparison of development of mouse and cattle embryos produced by nuclear transfer to oocytes or enucleated zygotes suggests that a greater proportion develop if the recipient is an oocyte. This may be because factors that bring about reprogramming of gene expression in a transferred nucleus are required for early development and are taken up by the pronuclei during development of the zygote.
oocyte at metaphase II, it is only possible to avoid chromosomal damage and maintain normal ploidy by transfer of diploid nuclei, but further experiments are required to define the optimum cell-cycle stage. Our studies with cultured cells suggest that there is an advantage if cells are quiescent (ref. 1, and this work). In earlier studies, donor cells were embryonic blastomeres that had not been induced into quiescence. Comparisons of the phases of the growth cycle showed that development was greater if donor cells were in mitosis or in the G1 (ref. 10) phase of the cycle, rather than in S or G2 phases. Increased development using donor cells in G0, G1 or mitosis may reflect greater access for reprogramming factors present in the oocyte cycoplasm, but a direct comparison of these phases in the same cell population is required for a clearer understanding of the underlying mechanisms.
cell types should prove to be totipotent after enhancing opportunities for reprogramming by using appropriate combinations of these cell-cycle stages. In turn, the dissemination of the genetic improvement obtained within elite selection herds will be enhanced by limited replication of animals with proven performance by nuclear transfer from cells derived from adult animals. In addition, gene targeting in livestock should now be feasible by nuclear transfer from modified cell populations and will offer new opportunities in biotechnology. The techniques described also offer an opportunity to study the possible persistence and impact of epigenetic changes, such as imprinting and telomere shortening, which are known to occur in somatic cells during development and senescence, respectively.
is, to our knowledge, the first mammal to develop from a cell derived from an adult tissue. The phenotype of the donor cell is unknown. The primary culture contains mainly mammary epithelial (over 90%) as well as other differentiated cell types, including myoepithelial cells and fibroblasts. We cannot exclude the possibility that there is a small proportion of relatively undifferentiated stem cells able to support regeneration of the mammary gland during pregnancy. Birth of the lamb shows that during the development of that mammary cell there was no irreversible modification of genetic information required for development to term. This is consistent with the generally accepted view that mammalian differentiation is almost all achieved by systematic, sequential changes in gene expression brought about by interactions between the nucleus and the changing cytoplasmic environment.
Embryo-derived cells were obtained from embryonic disc of a day-9 embryo from a Poll Dorset ewe cultured as described, with the following modifications. Stem-cell medium was supplemented with bovine DIA/LIF. After 8 days, the explanted disc was disaggregated by enzymatic digestion and cells replated onto fresh feeders. After a further 7 days, a single colony of large flattened cells was isolated and grown further in the absence of feeder cells. At passage 8, the modal chromosome number was 54. These cells were used as nuclear donors at passages 7-9. Fetal-derived cells were obtained from an eviscerated Black Welsh Mountain Fetus recovered at autopsy on day 26 of pregnancy. The head was removed before tissues were cut into small pieces and the cells dispersed by exposure to trypsin. Culture was in BHK 21 (Glasgow MEM; Gibco Life Sciences) supplemented with L-glutamine (2 mM), sodium pyruvate (1 mM) and 10% fetal calf serum. At 90% confluency, the cells were passaged with a 1:2 division. At passage 4, these fibroblast-like cells had modal chromosome number of 54. Fetal cells were used as nuclear donors at passages 4-6. Cells from mammary gland were obtained from a 6-year-old Finn Dorset ewe in the last trimester of pregnancy. At passages 3 and 6, the modal chromosome number was 54 and these cells were used as nuclear donors at passage numbers 3-6.
Oocytes were recovered from Scottish Blackface ewes between 28 and 33 h after injection of gonadotropin-releasing hormone (GnRH), and enucleated as soon as possible. They were recovered in calcium- and magnesium-free PBS containing 1% FCS and transferred to calcium-free M2 medium containing 10% FCS at 37 [degrees] C. Quiescent, diploid donor cells were produced by reducing the concentration of serum in the medium from 10 to 0.5% for 5 days, causing the cells to exit the growth cycle and arrest in G0. Confirmation that cells had left the cycle was obtained by staining with antiPCNA/cyclin antibody (Immuno Concepts), revealed by a second antibody conjugated with rhodamine (Dakopatts).
of the oocyte were induced by the same electrical pulses, between 34 and 36 h after GnRH injection to donor ewes. The majority of reconstructed embryos were cultured in ligated oviducts of sheep as before, but some embryos produced by transfer from embryo-derived cells or fetal fibroblasts were cultured in a chemically defined medium? Most embryos that developed to morula or blastocyst after 6 days of culture were transferred to recipients and allowed to develop to term. One, two or three embryos were transferred to each ewe depending upon the availability of embryos. The effect of cell type upon fusion and development to morula or blastocyst was analysed using the marginal model of Breslow and Clayton. No comparison was possible of development to term as it was not practicable to transfer all embryos developing to a suitable stage for transfer. When too many embryos were available, those having better morphology were selected.
60 after oestrus and to monitor fetal development thereafter at 2-week intervals. Pregnant recipient ewes were monitored for nutritional status, body condition and signs of EAE, Q fever, border disease, louping ill and toxoplasmosis. As lambing approached, they were under constant observation and a veterinary surgeon called at the onset of parturition. Microsatellite analysis was carried out on DNA from the lambs and recipient ewes using four polymorphic ovine markers.
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.
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.
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.
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.
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.
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.
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. 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.
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.
Several important questions remain unanswered about the feasibility in mammals of nuclear transfer cloning using adult cells as the source of nuclei:
in other cases? Only one animal has been produced to date. Thus, it is not clear that this technique is reproducible even in sheep.
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.
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 tell 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 it may lead to a genetic disease or cancer. The risks of such events occurring following nuclear transfer are difficult to estimate.
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* to rapidly propagate desirable animal stocks* to improve the efficiency of generating and propagating* to produce targeted genetic alterations in domestic animals* to pursue basic knowledge about cell differentiation
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.
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.
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
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.
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.
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 genes (Colman, 1996).
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.
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.
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).
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.
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.
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.
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.
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.
could provide a potential source of organs or tissues of a pre-determined 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.
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.
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 nonfunctional sperm, one might envision using cloning of one member of the couple's nuclei to produce a child.
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.
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.
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.
|Viable Offspring Derived from Fetal and Adult Mammalian Cells||21|
|The Science and Application of Cloning||29|
|Dolly's Fashion and Louis's Passion||41|
|What's Wrong with Cloning?||54|
|Sheep, Joking, Cloning and the Uncanny||78|
|Sameness Is All||88|
|Sex and the Mythological Clone||114|
|Pt. III||Ethics and Religion|
|Cloning Human Beings: An Assessment of the Ethical Issues Pro and Con||141|
|To Clone or Not to Clone||181|
|Human Cloning and the Public Realm: A Defense of Intuitions of the Good||190|
|Pt. IV||Law and Public Policy|
|The Constitution and the Clone||207|
|On Not Banning Cloning for the Wrong Reasons||221|
|The Demand for Human Cloning||233|
|A Rush to Caution: Cloning Human Beings||262|
|Recommendations of the Commission||289|
|Pt. V||Fiction and Fantasy|
|World of Strangers||297|
|Flourish Your Heart in This World||310|