CLONING HUMAN BEINGS The Science of Animal Cloning Commissioned Paper by Janet Rossant, Ph.D. Samuel Lunenfield Research Institute-Mount Sinai Hospital CONTENTS What Is Cloning? B-3 The Scientific Question: Does Differentiation Involve Irreversible Changes in Genetic Content? B-4 The Stability of the Differentiated State: Our Understanding Today B-6 Nuclear Transfer in Mammals: The Early Experiments B-7 Reprogramming in the Oocyte Environment B-8 Nuclear Transfer in Mammals: The Current State of Play B-9 And Then Came Dolly B-10 Why Pursue Animal Cloning Research? B-12 1. Making Clones for Research Purposes B-12 2. Propagating Desirable Stocks B-13 3. Improved Generation and Propagation of Transgenic Livestock B-13 4. Generating Targeted Gene Alterations B-14 How Can We Use Information from Nuclear Transfer Experiments for Human Benefit? B-17 Ethical Concerns B-17 References B-18 B-1 WHAT IS CLONING? The word “clone” is used in many different contexts in biological research, but in simple terms it means a set of genetically identical individuals. Scientists talk about cloning DNA—the process of making and propagating a set of identical copies of a particular piece of genetic material, or about cloning cells—taking a single cell in culture and allowing it to multiply into a cell line. These techniques are some of the basic tools of the trade of modern biomedical research and are not currently a source of much public concern. But when we talk about cloning whole animals, especially mammals, the public rightly wants to know what is going on and why. Genetically identical copies of whole organisms—clones—are commonplace in the plant breeding world. Many valuable horticultural or agricultural strains are maintained solely by vegetative propagation from an original plant, and never by sexual reproduction. This reflects the ease with which it is possible to regenerate a complete plant from a small cutting. The ability to propagate a valuable animal strain in the same way would revolutionize the agricultural business. However, in the animal kingdom, development is much less flexible than in plants. Many simpler invertebrate species have the ability to regenerate a whole organism from a small piece, although this is not necessarily their usual mode of reproduction. Vertebrates have lost this ability entirely, although regeneration of missing limbs, organs, or tissues can occur to varying degrees. Although an adult vertebrate cannot make another adult, natural cloning does occur, in a limited way, with the formation of identical twins. These arise in humans and other mammals by chance separation of a single embryo into halves at an early stage of development. The resulting offspring will be genetically identical, deriving from one zygote—the result of the fusion of one egg and one sperm. A clone of two is not very remarkable, but it is a clone nonetheless. Experimental separation of cells from the early embryos of several mammalian species has shown that it is possible in some cases to get larger clones from one egg (Figure 1). In mice, only separated two-cell blastomeres are capable of generating entire mice (Rossant 1976), but in some domestic species, like sheep, it is possible to get separated eight-cell blastomeres to develop into viable offspring (Willadsen 1981). At best, efficiency of this technique is never 100 percent, so the number of clones is small. However, the experiments are scientifically important, because they demonstrate that the cells of the early embryo are totipotent; that is, they retain the full potential to form an entire animal. As development proceeds, cells begin to differentiate into specialized cell types and can no longer revert back to the beginning of development (Figure 1). If this stability of the differentiated state could be reverted in some way, then producing animals from later differentiated cells or even adult cells would become feasible. Nuclear transfer experiments, first performed in amphibians in the 1960s, demonstrated how this could be done. B-3 Figure 1: Preimplantation development in mammals THE SCIENTIFIC QUESTION: DOES DIFFERENTIATION INVOLVE IRREVERSIBLE CHANGES IN GENETIC CONTENT? The information for all the proteins produced in all the specialized cell types of the body is encoded in the DNA of the zygote and must be passed on intact into the next generation via the germ cells that form the eggs and sperm. However, there is no absolute requirement that the somatic cells—those cells not destined to give rise to the germ cells—retain the full genetic content in an unmodified form. A differentiated cell like a neuron must keep a set of neural-specific genes active and silence those genes specifically required to make muscle, liver, and other tissues. How does it do this? Is it an active process, in which genes are still present but repressed in some way, or is the DNA for the inactive genes lost or irreversibly inactivated in some way? In the early 1960s our understanding of the mechanisms of gene regulation was still rudimentary and this general question was extremely important. Elegant experiments in Xenopus laevis by John Gurdon, following earlier experiments in Rana temporaria by Briggs and King (1952), provided strong evidence that the genetic content of differentiated somatic cells is essentially unchanged from that of the early embryo. Nuclei from donor differentiated cells were injected into recipient eggs from which the nucleus, containing the DNA, had been inactivated (Figure 2). If the donor nucleus could direct normal development of the recipient egg, this would strongly suggest that differentiation cannot involve permanent B-4 changes in the genetic material. The first series of experiments used intestinal epithelial cells from swimming tadpoles (Gurdon 1962), and adult frogs were produced, albeit at a very low efficiency. The intestinal cells used were highly specialized brush-border gut cells, but were not derived from the final adult frog and so might not be terminally differentiated. Gurdon and colleagues (1975) performed another carefully controlled series of experiments in which they used nuclei from adult skin cells for transfer. Over 99 percent of the cultured cells expressed keratin, a differentiated marker of skin cells, and 4 percent of the nuclei transferred eventually gave rise to fully developed tadpoles. These experiments provided the strongest evidence to date that the nuclei of terminal differentiated cells could be reactivated by the cytoplasm of the egg and redirect normal development. Figure 2: Nuclear transfer in amphibians However, no viable adult frog was ever produced from an adult differentiated nucleus and there was a strong decline in the rate of recovery of feeding tadpoles with progressive age of nuclei transferred. This left open the theoretical possibility that complete reactivation of the adult B-5 nucleus was prevented by some kind of irreversible change in the genetic material, and that there was, indeed, a progressive decline in nuclear potential with age. However, careful analysis at the time suggested that the major reason for developmental failure of transplant embryos was chromosomal abnormalities acquired as a consequence of the process of nuclear transplantation itself. The cell cycle of adult cells is much slower than the rapid pace of cell division in the early frog embryo. Expecting a transplanted nucleus to reprogram its gene expression, replicate its DNA, and enter normal embryonic cell cycles within an hour of nuclear transfer is unrealistic. The remarkable thing is that some nuclei manage to do so, rather than that so many do not. The general conclusion from the amphibian nuclear transfer experiments of Gurdon and others was that the differentiated state did not involve major irreversible changes in the DNA. This conclusion was reached in the 1960s and early 1970s and has not been challenged in the intervening years (Gurdon 1974). THE STABILITY OF THE DIFFERENTIATED STATE: OUR UNDERSTANDING TODAY As our understanding of the regulation of gene expression has grown, we have learned that most patterns of differentiated gene expression are maintained by active control mechanisms (Blau 1992), in which combinations of regulatory proteins bind to control sequences adjacent to genes and turn them on or off. The particular differentiated state of a cell depends on its particular combination of regulatory proteins. This is not the only mechanism of gene control. There are some cases in which actual rearrangements and deletions of DNA occur, as in the expression of the immunoglobulin and T-cell receptor genes in lymphocytes. Heritable modification of the DNA by methylation can also affect gene expression. However, the overwhelming evidence suggests that given the right environment, it should be possible to activate or inactivate almost any gene in a cell. This environment need not be the cytoplasm of the egg. Cell fusion experiments, in which different cell types are fused into one multinucleate cell called a heterokaryon, have demonstrated that extensive reprogramming of differentiated nuclei can occur. For example, when muscle cells are fused with non-muscle cells of various sorts, muscle-specific genes are activated in the non-muscle cells (Blau et al. 1985). Similarly, globin genes can be activated in many cell types after fusion with erythroid cells (Baron and Maniati 1986). These and other kinds of experiments have led to the isolation of specific protein factors that regulate cell differentiation, such as the myogenic factors that regulate the formation of the muscle cell lineages (Weintraub 1993). All of this information has shown that the stability of the differentiated state is not absolute and, therefore, it should be theoretically possible to reprogram adult cells to reinitiate earlier programs of differentiation. Nuclear transfer experiments pointed the way and molecular biology is continuing to define the components of the regulation of cellular differentiation. NUCLEAR TRANSFER IN MAMMALS: THE EARLY EXPERIMENTS B-6 Following success in nuclear transfer experiments in frogs, there were some attempts in the 1970s to repeat the experiments in mice, the mammal of choice for experimental manipulation.