3
Animal Cloning

In this chapter, we address the following questions in our task statement:

What is the state of science on cloning of animals? How does this science apply to cloning of people?

To organize its response to those questions, the panel developed a series of subquestions, which appear as the section headings in the following text. For a general overview of the history and current status of animal cloning, see Solter (2000) [1] and Lewis et al. (2001) [2].

WHICH MAMMALIAN SPECIES HAVE BEEN CLONED, AND HOW EFFICIENT ARE THE REPRODUCTIVE CLONING PROCEDURES?

The animals that have been reproductively cloned through transfer of postembryonic nuclei are sheep [3-5], cattle [6-18], goats [19; 20], pigs [21; 22], and mice [23-29]. Similar attempts have been made in rhesus macaques, but the only success has been in experiments with nuclei from preimplantation embryos rather than postembryonic cells [30; 31]. In addition, reproductive cloning efforts in rabbits, rats, cats, dogs, and horses are ongoing [32].

The cloning efficiencies for various species are listed in Table 1 (developed by the panel) and Tables 3 and 4 (developed by Lewis et al., 2001[2]) in Appendix B. These efficiencies vary greatly—in general they



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Scientific and Medical of Aspects: Human Reproductive Cloning 3 Animal Cloning In this chapter, we address the following questions in our task statement: What is the state of science on cloning of animals? How does this science apply to cloning of people? To organize its response to those questions, the panel developed a series of subquestions, which appear as the section headings in the following text. For a general overview of the history and current status of animal cloning, see Solter (2000) [1] and Lewis et al. (2001) [2]. WHICH MAMMALIAN SPECIES HAVE BEEN CLONED, AND HOW EFFICIENT ARE THE REPRODUCTIVE CLONING PROCEDURES? The animals that have been reproductively cloned through transfer of postembryonic nuclei are sheep [3-5], cattle [6-18], goats [19; 20], pigs [21; 22], and mice [23-29]. Similar attempts have been made in rhesus macaques, but the only success has been in experiments with nuclei from preimplantation embryos rather than postembryonic cells [30; 31]. In addition, reproductive cloning efforts in rabbits, rats, cats, dogs, and horses are ongoing [32]. The cloning efficiencies for various species are listed in Table 1 (developed by the panel) and Tables 3 and 4 (developed by Lewis et al., 2001[2]) in Appendix B. These efficiencies vary greatly—in general they

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Scientific and Medical of Aspects: Human Reproductive Cloning are low, whether looked at in terms of live births per embryo produced in the laboratory or live births per embryo transferred to the uterus (see Table 1). Note that the two highest percentages are derived from one experiment and are outliers; in this experiment, the numbers are small and half the newborns (four of eight) died soon after birth [7]. In monkeys, reproductive cloning with adult nuclei has not been successful, but cloning with nuclei from the individual cells of several eight-cell embryos yielded 53 embryos for transfer; these resulted in four pregnancies, two of which gave normal offspring and two of which were lost [30; 31]. The results summarized in Table 1 and the cloning literature can be looked at from several points of view. It is clear that many healthy, apparently normal, clones have been born and have survived to fertile adulthood (for example, see [21; 27; 28; 33]). Dolly has given birth to lambs [34-36], and in the case of mice, six generations of clones have been produced serially, although the efficiency declined with succeeding generations [25]. While some cloned mice may die soon after birth [23], one detailed follow-up of five surviving cloned mice revealed no serious problems, and the weight gain seen after several weeks might have been caused by non-cloning-related genetic effects [37]. On the negative side, however, it is quite clear that across multiple species there are far more failures in the development of cloned fetuses than there are live normal births. This low efficiency of cloning reflects, among other causes, a high rate of fetal loss after embryo transfer and implantation. Spontaneous abortion is also common in natural pregnancies, but there is a major difference in the timing of fetal and neonatal loss between animal reproduction based on reproductive cloning and reproduction based on in vitro fertilization (IVF). Whereas most fetal losses in conventional zygotic pregnancies occur in the first trimester, with reproductive cloning, fetuses are lost throughout pregnancy and in the early neonatal period [6; 8; 9; 13; 23; 24; 29; 32; 38; 39]. In humans, late gestational fetal loss causes increased maternal morbidity and mortality. Cloning studies in animals have shown that a high proportion of pregnancies involving cloned fetuses have abnormalities, including abnormal placentation, pregnancy toxemia, and hydroallantois—excessive fluid accumulation in the uterus often associated with fetal abnormality [14; 33; 43; 100; 101; 115]. Those pregnancy complications can cause fetal loss and risk maternal health. For example, in the cow-cloning study by Hill et al. (1999)[8], four of the 13 pregnant mothers and their fetuses died because of complications late in pregnancy. Results of animal studies suggest that reproductive cloning of humans would similarly pose a high risk to the health of both fetus or infant and mother and lead to associated psychological risks for the mother as a consequence

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Scientific and Medical of Aspects: Human Reproductive Cloning of late spontaneous abortions or the birth of a stillborn child or a child with severe health problems. WHAT DEFECTS HAVE BEEN OBSERVED IN CLONED ANIMALS? A wide array of abnormalities and defects have been observed in reproductively cloned animals, both before and after birth [4; 6; 8-10; 13; 16; 20; 23; 24; 29; 32; 38-45]. However, these abnormalities have not always been studied in detail, possibly because most reproductive animal cloning has been done for commercial purposes and there is less interest in the failures than in the successes. The panel was told that funding for studies to catalog and understand the basis of the abnormalities is sorely needed [39]. The reported defects in cloned animals are summarized in Table 1 and detailed in Table 2. The most notable defects are increased birth size, placental defects, and lung, kidney, and cardiovascular problems [39; 46]. Other problems have included liver, joint, and brain defects, immune dysfunction, and postnatal weight gain. Thus, a wide variety of tissues and organs can fail to develop properly in cloned animals, and some of the reported defects (such as aberrant growth and development of lung tissue and the immune system) cannot be diagnosed or prevented with current technology, such as prenatal screening with ultrasonography. Many of the defects seen in cloned cattle and sheep (for example, high birth weight, abnormal placentation, fluid accumulation associated with maternal and fetal distress, and cardiovascular abnormalities) are the same as those described for “large offspring syndrome” (LOS). This is frequently seen in uncloned offspring produced after in vitro fertilization and embryo manipulation in these species (but not in others, including humans [47]) and is attributed to, among other things, the exposure of eggs and embryos to suboptimal culture conditions in the laboratory [41; 47-49]. In spite of much work to identify the causative factors (given the economic benefits that could come from efficient embryo manipulation in cattle), the etiology and species specificity of LOS are not understood. All that can be said is that it probably results from abnormal gene expression in the early embryo, including the misexpression of imprinted genes (see later) [41; 47]. As will be discussed again below, this highlights the fact that perturbations in gene expression during the preimplantation period can have serious consequences for later development. For the purposes of this report, it is important to stress two other things: some of the postnatal defects described in cloned cattle have not so far been associated with LOS (for example, [13]); and species that do not show LOS after normal

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Scientific and Medical of Aspects: Human Reproductive Cloning embryo manipulation or IVF (for example, mouse, goat and pig) still have a very low reproductive cloning efficiency, with prenatal and early postnatal losses [19-23; 29; 50]. Moreover, until the molecular basis of LOS is known, it is not possible to say that the syndrome would not occur in human reproductive cloning attempts. Animal cloning can also result in danger to the mother of any cloned offspring. Increased maternal morbidity and mortality can result from late gestational fetal loss, increased size of the fetus, abnormal placentation, pregnancy toxemia, and, most notably, hydroallantois and/or hydramnios (excessive fluid accumulation in the uterus often associated with fetal abnormality and maternal distress) [6; 8-11; 16]. These effects have been seen most prominently in studies with cattle and sheep. For example, in the cattle cloning study by Hill et al. (1999) [8], four of the 13 pregnant cows and their fetuses died because of complications late in pregnancy. Tim King and Ian Wilmut (pers. comm.) have noted that hydroallantois can affect up to 5% of established sheep pregnancies involving cloned offspring, although this condition is “extremely rare” in normal pregnancies. Documentation of these and related maternal problems appears to be relatively sparse in the literature, possibly because the focus of research has been on the cloned offspring rather than the pregnant cows. In conclusion, if results from animal reproductive cloning studies are extrapolated to humans, they suggest that reproductive cloning of humans could carry a very high risk to the health of both fetus or infant and mother and lead to associated psychological risks for the parents as a consequence of late spontaneous abortions or the birth of a stillborn child or a child with severe health problems. Moreover, if the cloned human fetus or placenta grew abnormally large, this could cause problems before a cesarean section would be an option, particularly if multiple embryos are placed in the uterus, which is the procedure in most IVF clinics in the United States. There is no reason, at this time, to expect the efficiency of implantation to be better for reproductive cloning than IVF. WHAT ARE SOME POSSIBLE REASONS FOR THE DEFECTS? Failures in several aspects of mammalian development are likely to contribute to the defects observed in cloned animals, and probably no one cause is responsible for all the problems. Some of the processes that are likely to be suboptimal have been enumerated [1; 2] and are outlined in the final sections of this chapter. Two processes, reprogramming and imprinting, are thought to be especially problematic [32; 38; 51].

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Scientific and Medical of Aspects: Human Reproductive Cloning FAILURES IN REPROGRAMMING What is reprogramming, and why is it necessary? Reprogramming is the process by which DNA and associated proteins in the nucleus transplanted from the somatic cell are reset so that the genes are ready to coordinate early developmental processes and make products required for growth of the early embryo [1; 52]. When researchers place animal somatic cell nuclei into enucleated eggs, they expect to “coerce” the adult cell nucleus into responding to egg cytoplasm as though it were the nucleus of a zygote. The nucleus should switch off many of the genes that were active in the adult cells and “restart” the genes needed to support the growth of embryonic tissues. Reprogramming must be completed in a relatively short time—within a few days in most mammals—so that the gene products that are normally supplied by the zygote nucleus can be delivered to the developing embryo [1]. In sexual reproduction, the process of reprogramming is not necessary, because the chromosomes come from germ cells, not somatic cells. The DNA in the egg and sperm are preprogrammed during the long processes of egg and sperm development and continue to be programmed through early development [53]. Does reprogramming fail during cloning? After nuclear transplantation, there is probably insufficient time to accomplish reprogramming before the embryo begins to develop into a blastocyst. Incomplete or incorrect reprogramming is likely to result in the embryos making products in an inappropriate and uncoordinated manner. However, gene expression in embryos after nuclear transplantation has not been surveyed extensively or systematically except in one case, when errors were found [54]. Other studies are under way with mice, and it will be possible to compare the resulting data with the extensive available information about gene expression in normal early embryos of this species [1]. Abnormalities in the methylation of a DNA region were seen in cloned bovine blastocysts compared with embryos derived by IVF [55]. Additional investigations into the molecular events of reprogramming (such as the identification of proteins that enter or leave the transferred nucleus) have also just begun [52; 56]. It is important to note that reprogramming errors could involve any genes. Those who wish to assess the safety of human reproductive cloning would have to survey a large fraction of or perhaps all genes at various times to check the integrity of a cloned embryo. Moreover, they would

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Scientific and Medical of Aspects: Human Reproductive Cloning have to examine the quality and quantity of gene activity and whether it is appropriate for the particular cell type. Furthermore, some errors can be manifest only in particular tissues and only later in development. FAILURES IN GENOMIC IMPRINTING What is imprinting? Imprinted genes usually have a “mark” imposed on or near them in the egg or the sperm, so the copy of a gene inherited from the mother behaves differently from the copy inherited from the father [57-59]. In the embryo and resulting offspring, the mark controls whether the gene is expressed. The best characterized of these marks is a methyl chemical group, which is added to some segments of the DNA in regions near the imprinted genes that are termed imprint control regions. Methylation is a mark that can be measured; other marks will probably be found in the future, but for now they are unknown. For normal development to occur, an embryo needs one set of chromosomes with the imprints imposed by the father and another set with imprints imposed by the mother. In experimental studies with mice, embryos that inherit both copies of their chromosomes from the mother’s germ cells can be generated; they inherit two versions of the mother’s imprint. (Similarly, mouse embryos that inherit two copies of the father’s chromosomes can be made.) Such genome-wide imprinting errors in mice result in fetal abnormalities and death [60-64]. Moreover, the size of the fetus and placenta may be abnormal. In humans, mutations that perturb or inactivate one copy of an imprinted region can result in the development of tumors in children or adults [65] or several well-known genetic disorders in children [66-68]. Three such disorders are Prader-Willi syndrome, Angelman syndrome, and Beckwith-Weidemann syndrome; these are characterized by various combinations of mental retardation and congenital abnormalities [67]. When are imprinting patterns established? Imprints are first erased and then re-established in a purely maternal or paternal pattern during the early development of the germ cells in the ovary or testis [58]. Further modifications occur in some genes during or after fertilization [69-74]. Maternal and paternal imprints are retained in somatic cells, although changes occur later in life in some tissues. Methylated regions are usually faithfully replicated in cell division, but errors occur [38], and some marks can be erased as cells multiply and develop

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Scientific and Medical of Aspects: Human Reproductive Cloning into various cell types. In this case the missing marks cannot be added back again if the cell divides and replicates. Do imprinting errors happen in reproductive cloning? Many of the imprinting errors that have been studied through genetic manipulation of mice result in too much or too little fetal or placental growth. Similar effects seen in some animal reproductive cloning experiments lead scientists to suspect a common cause. Although a direct link has not yet been demonstrated in most cases, mice cloned using ES cells as nucleus donors show widespread, unpredictable and aberrant regulation of their imprinted genes, as well as developmental abnormalities [75]. ES cells are essentially embryonic cells, and it is not yet known whether the same imprinting errors will be seen in the genes of animals cloned with adult nuclei [76]. However, mouse reproductive cloning experiments with adult nuclei have revealed errors in methylation in about 0.5% of some 1000 normally methylated DNA segments studied (but not necessarily associated with imprinted genes) [77; 78]. In addition, studies on bovine blastocysts obtained by cloning from fetal fibroblasts showed abnormal DNA methylation compared with blastocysts obtained by IVF [55]. Understanding the relationship between imprinting and increased offspring size in animal reproductive cloning experiments is complicated by the fact that, as discussed and referenced earlier, overgrowth, or LOS, can occur in cattle [79] and sheep [80] simply as a result of the culturing of normal cleavage-stage embryos before implantation, as is done in IVF procedures. Although the mechanisms underlying LOS are not known, changes in the expression of genes that are imprinted in other species may occur during in vitro culture of sheep and cattle embryos [41; 47; 81]. In addition, aberrant regulation of imprinted genes has been reported after culturing mouse ES cells [82] and preimplantation mouse embryos [83], although, in the latter case, the embryos apparently develop normally [84]. Thus, abnormal development of cloned animals may result in part from the culturing of embryos in the laboratory in association with the SCNT technique. However, the presence of cloning-specific defects and a study in mice [50] suggest that at least some of the errors arise as a result of the nuclear transplantation procedure itself. Further work is needed to understand how external conditions can perturb the expression of imprinted and non-imprinted genes in the preimplantation embryos of different species, and to understand the relationship between these changes and those shown to be specifically associated with the technique of transplantation of somatic cell nuclei.

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Scientific and Medical of Aspects: Human Reproductive Cloning How widespread are imprinting effects? In addition to the growth effects mentioned above, imprinting errors are known to affect brain development and mental function [85-88] and placental function [89]. One hundred or more genes might be imprinted in humans [58; 90]. They seem to be mostly genes that are important and turned on differentially early in development. The expression of each gene varies according to the time, the tissue, the species, and the parent of origin. How might imprinting go awry in reproductively cloned animals? There are several ways in which reproductive cloning might result in the abnormal expression of imprinted genes: Imprints and methylation marks may not be maintained in all cells during adult life, and random errors may occur. If nuclei from these cells are used for reproductive cloning, the errors cannot be repaired in the embryo. It is therefore important in the future to examine the possibility that the rare cases when reproductive cloning is successful involve a small subpopulation of cells that have kept their imprints unaltered. The pattern of imprints from the nucleus donor’s parents might not be maintained or copied correctly as the chromosomes from the donor nucleus replicate in the preimplantation embryo. This problem might be exacerbated by the culture of embryos before implantation. Even if the imprinting marks are copied correctly, incorrect reprogramming might result in the imprinted genes not being read correctly in the embryonic tissues. There is evidence that imprinting of some genes is modified in the preimplantation embryo [69]. This process might work properly only if the cellular machinery is faced with two distinct sets of DNA from a sperm and an egg [70]. Nuclear transplantation, however, presents the egg cytoplasm with two sets of DNA from a single somatic cell. Could imprinting errors cause problems for the human mother? Incorrectly imprinted cells could cause problems for the mother, as well as the child. Three lines of evidence support that possibility: Some imprinting problems—for example, after sheep preimplantation embryo culture [41]—are associated with excessive growth of the fetus or placenta [49]. If LOS occurs in humans, it could be serious because humans have an extended growing time in the mother and are

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Scientific and Medical of Aspects: Human Reproductive Cloning already close to the maximal size that will allow for safe birth. In addition, if multiple embryos are implanted, as in nearly all IVF procedures in the United States, the risk to the mother would be higher. Incorrectly imprinted cells can be malignant [91]. An example is seen in complete hydatidiform moles. These form when an egg that lacks a nucleus is fertilized by a sperm, so that all the DNA is contributed by the sperm. An embryo does not develop, but, possibly as a result of imprinting problems [92], a potentially malignant growth (mole) forms inside the uterus. A few human fetal cells normally circulate in the mother’s blood during and after pregnancy [93], and it has been speculated that they might be implicated in the development of some skin, autoimmune, and muscle diseases [94-98]. If incorrectly imprinted fetal cells have a growth advantage, it is theoretically possible that they could lodge in the mother’s tissues and grow into a tumor. Errors in processes other than reprogramming and imprinting are also possibilities. Some of these possibilities are listed below. MITOCHONDRIAL HETEROPLASMY AND CONFLICT What is mitochondrial heteroplasmy? Normally, mitochondria are inherited from the mother. In mitochondrial heteroplasmy, a mix of mitochondria is present in a single cell. That can happen naturally [99; 100] and has been induced in humans with ooplasmic transfer ([101]; see Chapter 4). When the SCNT procedure involves fusion of a somatic cell and an egg from two different individuals, mitochondrial heteroplasmy can result [102]. However, the relatively small number of incoming mitochondria will probably be swamped by the vast excess of egg mitochondria [103; 104] and might in any case be subject to elimination by the egg [105-107]. Could a transferred nucleus conflict with egg-derived mitochondria? When the SCNT procedure is used, the incoming nuclear DNA will encounter a foreign set of egg-derived mitochondrial DNA. That has the potential to cause problems because, for example, there are natural variants of both nuclear and mitochondrial genes, and some pair combinations work less efficiently than others [108-110]. Mitochondria are inherited almost exclusively from the mother [106]. In the mother, previous natural selection might have eliminated potentially deleterious conflicts between nuclear and mitochondrial genomes

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Scientific and Medical of Aspects: Human Reproductive Cloning [111] particularly by eliminating unfit oocytes [112; 113]. In sexual reproduction, the father’s nuclear DNA therefore encounters a “foreign” set of mitochondrial genes from the mother, but in this case products encoded by the mother’s nuclear DNA may compensate for any potential conflict between products encoded by the father’s nuclear DNA and the mother’s mitochondria. But when SCNT is performed, such compensation might no longer be present. In mice, a conflict between transplanted nuclei and foreign egg cytoplasm (which includes mitochondria) has been shown to cause growth deficiency and misregulation of some genes [114]. TELOMERE SHORTENING Could shortened telomeres result in prematurely “old” clones? Telomeres, the caps on the ends of chromosomes, shorten during aging in somatic cells. In germ cells, the caps are rebuilt by an enzyme called telomerase. Thus, there is a potential for cloned embryos, with their chromosomes from somatic cells, to have shortened telomeres. That could result in prematurely “old” cells in a clone and the misproduction of proteins from genes near the telomeres [115]. The possibility does not seem to be a major concern. Any shortening of telomeres in cloned sheep appears to be minor and can be minimized by judicious choice of the cell type used as a nucleus donor [116]. No sign of telomere shortening or aging was seen in mice cloned serially for six generations [25], and telomeres in cattle are rebuilt in cloned embryos [117-119] and can eventually be longer than [18; 120] or the same size as [117] those in age-matched control animals. Human blastocysts have high levels of telomerase activity [121]; this suggests that they might be able to rebuild telomeres after reproductive cloning. MUTATIONS Could adult-donor nuclei carry more mutations than do gamete nuclei? The source of a nucleus for reproductive cloning would have to be chosen very carefully. Sun-exposed skin cells, for example, might be a bad source of nuclei, because their DNA could have many mutations induced by the sun’s ultraviolet radiation. Cells that have been grown in culture dishes for a considerable time might also make poor nucleus donors, because growth in culture favors the accumulation of growth-promoting mutations that are often associated with cancer development. However, it should be noted that normal calves were born from cloning

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Scientific and Medical of Aspects: Human Reproductive Cloning experiments in which nuclei were derived from cells obtained from a 17-year-old bull and then cultured for 3 months in the laboratory [17]. In this study, six healthy calves were born from a total of 15 pregnancies involving nine abortions and 54 embryos transferred. The overall efficiency of live births (11% of embryos transferred) was thus not significantly lower than with nuclei from younger animals and less extensive cell culture (see Table 1 in Appendix B). X-CHROMOSOME INACTIVATION Can cloned female animals shut off one of their X chromosomes? Females and males differ in their complement of sex chromosomes: females have two X chromosomes, and males have one X chromosome and one Y chromosome. Females reduce the production from X-chromosome genes to the level seen in males by shutting down almost an entire X chromosome. Experiments in mice [122] suggest that cloned embryos can successfully recapitulate that process, so failure of X inactivation is unlikely to be a source of defects in cloned animals. Can reprogramming and imprinting errors be understood and controlled, and can cloning efficiencies be improved? Our survey of the literature on animal cloning, as well as presentations at the workshop, revealed great variability in its efficiency (Table 1). Moreover, it is clear that although healthy clones can in some cases be produced, success is not a reproducible phenomenon, and the precise molecular mechanisms responsible for the high failure rate are almost entirely unknown. The optimal method for animal reproductive cloning cannot be determined from current studies, because the number of variables makes direct comparisons between multiple studies difficult or impossible. Studies often differ in species used, method of nuclear transplantation (fusion or injection, and single transfer or serial transfer), method of egg activation, expertise of the investigators, and condition of cells used as nucleus donor (for example, different cell type, cell cycle stage, and time of growth in culture before nuclear transplantation). In the sections above, several of the most likely problems, including defects in genetic reprogramming and defects in imprinting, have been outlined and discussed. Several other potential sources of error have been summarized elsewhere with pertinent references [1]. For example, one problem may lie in the methods now used to activate the egg after nuclear transplantation. Immediately after normal fertilization, waves of increased calcium concentration pass through the egg in an orderly way, and this

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Scientific and Medical of Aspects: Human Reproductive Cloning may impart some organization on the egg cytoplasm or membrane important for gene activation and later development [123]. When the egg is activated by an electric shock or by chemicals, as is the case in animal cloning, these calcium waves do not occur in an orderly way. Another example is that problems may arise if the donor cell is replicating its DNA at the time the nucleus is taken for nuclear transplantation [6]. A number of different strategies have been used by different groups to try to overcome those and other problems and so increase the efficiency of cloning. In some cases, progress has been made, but no clear picture has emerged, particularly when nuclear transplantation from adult rather than embryonic cells is used. Studies were undertaken to determine whether inbreeding may be important in the poor efficiency of cloning in mice, since many mouse strains commonly used in the laboratory are inbred. Inbred mice are generally less fertile than hybrid or outbred mice and their embryos may be more difficult to culture in the laboratory [124]. In two cloning studies [26; 50], researchers did find that inbred animals showed much poorer cloning success than outbred animals, but even in outbred strains, cloning efficiency was low 0.36-1.8% of the hybrid cloned embryos produced from nuclei of hybrid cells resulted in live births). That suggests that inbreeding, although it plays a role, cannot by itself account for the poor efficiency of cloning in mice. One of the first approaches to overcoming reprogramming problems involved culturing the donor cells in the laboratory under conditions in which the cells become quiescent and shut down the activity of their genes [3]. However, this strategy was not the solution to low cloning efficiency (see, for example, [6]). Nevertheless, it is possible that in the future some particularly quiescent cells in an adult tissue (for example, stem cells) will be found to be better nucleus donors than others. Another early approach to improve reproductive cloning efficiency involved delaying the activation of the egg after nuclear transplantation; theoretically, this should allow more time for the regulatory proteins to be stripped off the incoming DNA and for cytoplasmic proteins to bind to the DNA [23]. Again, this strategy has not led to a solution to low cloning efficiencies. In the future, new and improved methods of activation might allow the process to be controlled more precisely [1]. Finally, several groups have tried the technique of serial nuclear transfer or recloning in an attempt to overcome both reprogramming and egg-activation problems. The strategy here is to carry out nuclear transplantation in the usual way, by transferring a nucleus into an enucleated egg, then activating the egg and allowing the embryo to develop to the two-cell stage. Nuclei are then taken from this embryo and transferred into the

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Scientific and Medical of Aspects: Human Reproductive Cloning cytoplasm of an unfertilized egg from which the chromosomes have been removed, or a normally fertilized egg from which both the male and female nuclei have been removed (for diagram, see [1]). The embryo then continues to develop into a blastocyst for transfer. This serial transfer does two things: it allows the nucleus more time to be exposed to egg cytoplasm for possible reprogramming, and in some cases it uses an egg that has been activated normally by fertilization. The first use of this technique in mice gave high cloning efficiency [125], but the original nuclei came from embryos, not adults; when it was used with adult nuclei, there was no improvement in cloning efficiency [23], or only a very low efficiency was obtained with fetal losses [29]. In experiments with pigs, a relatively high cloning efficiency was also achieved [21], but the effect was not repeated in cattle [10]. It should be noted that if this procedure were applied to human cloning, it would involve not only donation of large numbers of unfertilized eggs, but also large numbers of fertilized eggs, or zygotes. In conclusion, research into the science of genetic reprogramming and animal cloning is in its infancy, and much more information is needed. It is unlikely that the poor outcomes of cloning are the result of only one defect arising from the nuclear transplantation procedure. More likely, they arise from the accumulated effects of sometimes unpredictable and stochastic (random) errors in several complex and interdependent biological processes. HOW DOES THE SCIENCE OF ANIMAL REPRODUCTIVE CLONING APPLY TO THE CLONING OF HUMANS? Theoretically, it should be possible to use animal-cloning techniques for reproductive cloning of humans. Reproductive cloning with nuclear transplantation from adult cells has not yet been performed successfully in nonhuman primates, so no data on the efficiency or safety of the procedure in primates are available. Such data might be helpful in assessing the possible results of a human reproductive cloning attempt, given the close evolutionary relationship and reproductive similarities of humans and nonhuman primates. It cannot be ruled out that the abnormalities observed in cloned animals would occur in humans produced with reproductive cloning [51], especially given the widespread conservation of basic developmental mechanisms between different mammalian species and the impressive level of conservation—for example, between mice and humans—of placental anatomy and the genes controlling placental function [126]. Nevertheless, differences do exist in the developmental programs of various

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Scientific and Medical of Aspects: Human Reproductive Cloning mammals, including humans, and at the present time, we do not know whether attempts at human cloning would reveal fewer, more, or different abnormalities. FINDINGS 3-1. In general, the efficiency of reproductive cloning in animals remains extremely low despite several years of experimentation. 3-2. Animal cloning results in a wide variety of abnormalities, including greater than normal size (both during gestation and after birth), greater early- and late-gestation fetal morbidity and mortality, greater postnatal mortality, and various developmental defects in the immune, cardiovascular, and possibly nervous systems. (Subtle behavioral and mental defects might be undetectable in animal models.) In addition to the risks inherent in the overproduction of oocytes from egg donors, increased maternal morbidity and mortality are to be expected. 3-3. The most likely reasons for the abnormalities are failures in reprogramming in the adult nucleus used for reproductive cloning, so that it fails to turn on all the appropriate embryo-specific genes at the right times, and errors in imprinting. 3-4. Before human reproductive cloning is feasible, a great deal more research is necessary, including studies of cloning in nonhuman primates. Research focused on gaining an understanding of all aspects of reprogramming and imprinting, determining which steps in the reproductive cloning technique contribute to the overall low efficiency, and determining how these problems can be overcome would be most useful. REFERENCES 1. SOLTER D. Mammalian cloning: advances and limitations. Nat Rev Genet 2000 Dec, 1(3):199-207. 2. LEWIS IM, MUNSIE MJ, FRENCH AJ, DANIELS R, TROUNSON AO. The cloning cycle: From amphibia to mammals and back. Reprod Med Rev 2001, 9(1):3-33. 3. WILMUT I, SCHNIEKE AE, MCWHIR J, KIND AJ, CAMPBELL KH. Viable offspring derived from fetal and adult mammalian cells. Nature 1997 Feb 27, 385(6619):810-3. 4. SCHNIEKE AE, KIND AJ, RITCHIE WA, MYCOCK K, SCOTT AR, RITCHIE M, WILMUT I, COLMAN A, CAMPBELL KH. Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science 1997 Dec 19, 278(5346):2130-3. 5. MCCREATH KJ, HOWCROFT J, CAMPBELL KH, COLMAN A, SCHNIEKE AE, KIND AJ. Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature 2000 Jun 29, 405(6790):1066-9.

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