- Email: [email protected]
Ban Human Cloning
C Blackwell Wissenschafts-Verlag 2002
Differentiation (2002) 69:147–149
COMMENTARY
Marie A. Di Berardino
Ban Human Cloning
Accepted in revised form: 23 October 2001
One of the important features of basic research is the increased significance it acquires over time, as new knowledge and techniques expand its applications that, in some cases, yield unanticipated applications. This statement certainly applies to animal cloning in metazoans. As a witness to the first tadpoles produced by transferring blastula cell nuclei into enucleated frog eggs (Briggs and King, 1952), I can attest that animal cloning was not developed for the ultimate purpose of cloning humans, but rather to study nuclear potential of cells during progressive stages of amphibian embryogenesis (reviewed by Di Berardino, 1997). In fact, for several years, the pioneers terminated their experiments when the nuclear-transplant-tadpoles were 11 days old, the time when Rana pipiens tadpoles begin to feed. Only in later years were nuclear-transplant-tadpoles raised to adulthood to examine their fertility. Early speculations on human cloning by a scientist were made in 1966 by the geneticist Joshua Lederberg in the American Naturalist where he hypothesized the eugenic advantages of human cloning as well as other forms of genetic engineering (see Kass, 2001, p. 44). Soon science-fiction writers and journalists jumped into the act, but their speculations were considered mere fiction by most persons. However, in the 1980s with success in mammalian cloning from embryonic animal cells, the possibility of cloning human embryos became credible. Finally, with the birth of the sheep Dolly, the first animal
M. A. Di Berardino Department of Biochemistry, MCP-Hahnemann University School of Medicine, 2900 Queen Lane, Philadelphia, PA 19129, USA e-mail: mad26/drexel.edu Fax: π1 215 843 8849 U. S. Copyright Clearance Center Code Statement:
cloned from an adult cell (Wilmut et al., 1997), the probability of cloning adult humans became realistic. Animal cloning, as other basic research, was initiated to seek fundamental knowledge for the benefit of mankind. Now that human cloning is possible, though with miniscule success, we must decide whether it should be attempted. This dilemma is not new in science. Discoveries such as recombinant DNA, nuclear energy, anesthesia, electricity, airplanes, etc. can be used either for the benefit or destruction of mankind. So, too, the applications of animal cloning must be responsibly analyzed now and for future generations. Obviously, in a society heterogeneous in its cultural, ethical, and religious values, there is no consensus regarding human cloning. Our responsibility is to debate this issue so that a reasonable agreement can be achieved. The concerns of human cloning center mainly on two proposals: reproductive and therapeutic cloning. In this article I shall argue that human cloning should not be attempted for scientific and ethical reasons. The science and technique of somatic cell nuclear transfer (cloning) is still in its infancy and not ready for clinical trials in humans. I base this statement on the fact that only 0.1 – 5 % of the attempts to transfer somatic nuclei from mammalian animal cells into enucleated oocytes result in live births (reviewed in Di Berardino, 2001). Furthermore, on average only about 50 % of newborns survive and many suffer severe abnormalities (Hill et al., 1999, 2000). The percentage of mammalian animal clones born is usually calculated on the number of blastocyst clones transferred to surrogate mothers, but the actual success should be based on the number of attempts to transfer a somatic nucleus to an oocyte. The latter method of calculation reveals the real challenge to human cloning. For example, Dolly resulted from 434 attempts to fuse a cell from a mammary gland to an oocyte (Wilmut et al., 1997). If these data were extrapolated for proposed human trials, one would need
0301–4681/2002/6904–147 $ 15.00/0
148
oocytes donated from at least 40 women. It is true that success in achieving newborn animal clones has improved, but the problem still exists: obtaining sufficient women as oocyte donors for human trials. The gravest problem is that 95.0 – 99.9 % of the attempts to produce mammalian animal clones resulted in death during gestation and, among newborns, about half died after birth while many survivors suffered severe abnormalities. Although there is a high rate of failure during normal human sexual reproduction, the failures are not intentionally caused by the parents, whereas if human cloning were permitted, the participants would be directly responsible for the deaths. Based on current data in mammalian animal studies, attempts at human cloning would result in a vast amount of human wastage. Committees in responsible USA hospitals, judging other kinds of clinical trials in humans, would not approve studies yielding such high death rates. At the present time, the cloning procedure is a roulette game and will continue to be a game of chance until we learn how to synchronize the cell cycle of the donor cell with that of the recipient oocyte and to reprogram the genomic expression of donor nuclei, including genetic imprinting and DNA methylation, to conform with those patterns in normal pronuclei (Kang et al., 2001; Reik et al., 2001; Rideout et al., 2001). In addition to severe limitations in the cloning procedure, I have other scientific concerns. Most metazoan animal species produce their offspring not asexually but through sexual reproduction. Why did sexual reproduction evolve and persist in complex metazoan animals? Obviously, sexual reproduction provides for genetic diversity, stemming from genetic recombination and random distribution of chromosomes during meiosis. Also, during meiosis there is a DNA repair process, one that is additional to that occurring in somatic cells. Furthermore, the processes of oogenesis and spermatogenesis select against some abnormal germ cells. Although this selection process is not full proof, it is non-existent in somatic cells. Even if cloning from adult cells becomes efficient, serious hazards would still exist. Somatic donor cells during their lifetime could acquire mutations from radiation, chemicals, aging and/or errors in DNA replication that would be transferred to the clone. Also, mutations arise frequently in cells during culture, and these could be transmitted to the clone. Prospects for identifying all mutations in an apparently normal embryo prior to transfer to the uterus are probably impossible. Although in time it might be possible to examine the epigenetic properties of many imprinted genes from a few blastomeres of the embryo clone, knowledge of the DNA sequence of all the clone’s genes appears impossible. Furthermore, the sampling of blastomeres may be misleading. In my experience in frog cloning, many normal appearing blastula clones were composed of a mosaic of cells, some with apparently normal chromosomes
and others with abnormal ones (reviewed in Di Berardino, 1997). Thus, sampling of blastomeres from a clone could result in false negatives. No studies can guarantee that adult animal clones are completely normal. A few studies have examined the rate of aging and certain behavioral traits. For example, Wakayama et al. (2000) cloned mice from cumulus cells through six generations, and found no evidence for accelerated aging, telomere shortening, or abnormal behavior. They evaluated learning ability in the Morris water maze and Krushinsky tests, as well as strength and agility, and also tested for premature aging, such as a decline in activity and loss of coordination. I applaud this report as a scientific study, but such analyses in mice do not prove to me that a cloned human would be mentally normal, nor did the authors make such a claim. My main point is how are we going to look into the minds of sheep cow, pig, or goat clones and diagnose them as emotionally and intellectually normal? Furthermore, if we could, would it be relevant to the human mind? There are still other scientific concerns. Will telomere shortening in the donor cell limit the life span of the clone? Some clones have shortened telomeres, but others do not (reviewed in Di Berardino, 2001). Can the disparity in results be accounted for by differences in nuclear reprogramming or donor cell types? Obviously, this question requires more study. Another question, scarcely explored in mammalian animal cloning, concerns maternal RNAs and proteins in oocytes. Will maternal gene products from foreign donors always be compatible with the donor nucleus? One well-known case of nuclear-cytoplasmic incompatibility was reported for the DDK strain of mice in which matings between DDK females and non-DDK males are virtually (95 % embryo lethality) infertile, but matings between DDK males and non-DDK females are fully fertile (reviewed by Latham, 1999). We should also reflect on certain nucleocytoplasmic hybrids produced long ago in amphibians. Rana pipiens and Rana palustris, when crossed in the laboratory, produced normal hybrid embryos that develop in normal metamorphic frogs. However, enucleated Rana pipiens eggs injected with blastula nuclei from Rana palustris and the reciprocal combination developed abnormally (Hennen, 1972). Why the hybrid genome could function in either cytoplasm but a non-hybrid genome could not was never explained. However, we can speculate that a critical molecular interaction had to occur directly between a gene product in the oocyte and its homologous haploid genome or indirectly with one of its gene products, otherwise normal development failed. These examples illustrate how little we know about possible deleterious outcomes from the cloning process. Finally, very little concern has focused on female animals bearing the clones through gestation. Surrogate mothers suffer a high rate of spontaneous abortions.
149
Among the fetuses surviving to birth, many are significantly overweight and have enlarged placentas (Hill et al., 1999, 2000). In fact, many overweight fetal clones have been delivered by Caesarian section to preserve the lives of the clone and the mother. What would be the physiological and biochemical risks of a human female carrying a clone to birth? Why would anyone attempt human cloning when such questions have been barely examined in animal studies? In my previous comments I delineated a number of scientific reasons why I oppose the extension of cloning to humans. The physical dangers to the child and mother alone support my contention that human cloning would be unethical. I have other ethical reasons why human cloning should not be attempted. Asexual reproduction is unnatural to higher organisms. Why should we introduce retrograde evolution and mimic the ameba? The production of clones is a manufacturing business. This technology could destroy the normal relations within members of a family and lead to further destruction of the family unit. Even animals, especially primates, have a family structure to nurture their own. In my research career, I experimented with frog gametes and embryos. It is repugnant to me to equate human oocytes with mass amounts of frog oocytes that can be poked, cut up and modified, then discarded after they have served their purpose. Even though fertilization of a human oocyte by a spermatozoan is a chemical reaction, there still is a significant difference between human and non-human fertilized oocytes – the fertilized human oocyte has the potential to be a human child. This fact alone should prevent us from experimenting with human embryos. Unless we maintain respect for human life, we will be inferior to animals, our evolutionary ancestors, for even animals have concern for their offspring. For the above scientific and ethical reasons I support an international law to ban human cloning and one enacted immediately in the United States of America. Though such laws may not prevent all attempts at human cloning, they will certainly severely curtail such attempts. Although this article was requested to focus on human cloning, I would like to end by illustrating some of the many beneficial applications of animal cloning to medicine, agriculture, and the basic sciences (reviewed by Di Berardino, 2001). The production of transgenic clones producing human proteins is indeed a breakthrough. This feat was first reported by Schnieke et al., (1997) in sheep and followed by others in goats pigs, and additional sheep. After clinical testing, the various human proteins from transgenic clones can be used to treat diverse human diseases. Animal cloning can also be used to produce large domestic animals as models of human diseases, to test pharmaceuticals on animals with a uniform genetic background and, in the future, to aid
in xenotransplantation of organs to humans. In agriculture, cloning can be applied to producing animals with outstanding traits and resistance to diseases, as well as saving endangered species. In the basic sciences, animal cloning has already yielded important information e.g., in nuclear reprogramming and genetic imprinting, and some insight into cellular aging and cancer. Furthermore, we expect these and various other fundamental questions in biology and medicine to be assisted by animal cloning.
References Briggs, R and King, T.J. (1952) Transplantation of living nuclei from blastula cells into enucleated frogs’ eggs. Proc Natl Acad Sci USA 38:455–463. Di Berardino, M.A. (1997) Genomic potential of differentiated cells. Columbia University Press, New York. Di Berardino, M.A. (2001) Animal cloning – the route to new genomics in agriculture and medicine. Differentiation 68:67–83. Hennen, S. (1972) Morphological and cytological features of gene activity in an amphibian hybrid system. Develop Biol 29:241– 249. Hill, J.R., Burghardt, R.C., Jones, K., Long, C.R., Looney, C.R., Shin, T., Spencer, T.E., Thompson, J.A., Winger, Q.A. and Westhusin, M.E. (2000) Evidence for placental abnormality as the major cause of mortality in first-trimester somatic cell cloned bovine fetuses. Biol Reprod 63:1787–1794. Hill, J.R., Roussel, A.J., Cibelli, J.B., Edwards, J.F., Hooper, N.L., Miller, M.W., Thompson, J.A., Looney, C.R., Wethusin, M.E., Robl, J.M. and Stice, S.L. (1999) Clinical and pathologic features of cloned transgenic calves and fetuses (13 case studies). Theriogeneology 51:1451–1465. Kang, Y-K., Koo, D-B., Park, J.-S., Choi, Y-H., Chung, A-S., Lee, K-K. and Han, Y-M. (2001) Aberrant methylation of donor genome in cloned bovine embryos. Nature Genetics 28:173–177. Kass, L.R. (2001) The wisdom of repugnance. In: Brannigan, M.C. (ed.) Ethical issues in human cloning Seven Bridges Press, LLC, New York and London, pp 43–66. Latham, K.E. (1999) Epigenetic modification and imprinting of the mammalian genome during development. Current Topics in Develop Biol 43:1–47. Reik, W., Dean, W. and Walter, J. (2001) Epigenetic reprogramming in mammalian development. Science 293:1089–1093. Rideout, W.M. III, Eggan, K. and Jaenisch, R. (2001) Nuclear cloning and epigenetic reprogramming of the genome. Science 293:1093–1098. Schnieke, A.E., Kind, A.J., Ritchie, W.A., Mycock, K., Scott, A.R., Ritchie, M., Wilmut, I., Coleman, A. and Campbell, K.H.S. (1997) Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science 278:2130– 2133. Wakayama, T., Shinkai, Y., Tamashiro, K.L.K., Niida, H., Blanchard, D.C., Blanchard, R.J., Ogura, A., Tanemura, K., Tachibana, M., Perry, A.C.F., Colgan, D.F., Mombaerts, P. and Yanagimachi, R. (2000) Cloning of mice to six generations. Nature 407:318–319. Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J. and Campbell, K.H.S. (1997) Viable offspring derived from fetal and adult mammalian cells. Nature 385:810–813.
Differentiation (2002) 69:147–149
COMMENTARY
Marie A. Di Berardino
Ban Human Cloning
Accepted in revised form: 23 October 2001
One of the important features of basic research is the increased significance it acquires over time, as new knowledge and techniques expand its applications that, in some cases, yield unanticipated applications. This statement certainly applies to animal cloning in metazoans. As a witness to the first tadpoles produced by transferring blastula cell nuclei into enucleated frog eggs (Briggs and King, 1952), I can attest that animal cloning was not developed for the ultimate purpose of cloning humans, but rather to study nuclear potential of cells during progressive stages of amphibian embryogenesis (reviewed by Di Berardino, 1997). In fact, for several years, the pioneers terminated their experiments when the nuclear-transplant-tadpoles were 11 days old, the time when Rana pipiens tadpoles begin to feed. Only in later years were nuclear-transplant-tadpoles raised to adulthood to examine their fertility. Early speculations on human cloning by a scientist were made in 1966 by the geneticist Joshua Lederberg in the American Naturalist where he hypothesized the eugenic advantages of human cloning as well as other forms of genetic engineering (see Kass, 2001, p. 44). Soon science-fiction writers and journalists jumped into the act, but their speculations were considered mere fiction by most persons. However, in the 1980s with success in mammalian cloning from embryonic animal cells, the possibility of cloning human embryos became credible. Finally, with the birth of the sheep Dolly, the first animal
M. A. Di Berardino Department of Biochemistry, MCP-Hahnemann University School of Medicine, 2900 Queen Lane, Philadelphia, PA 19129, USA e-mail: mad26/drexel.edu Fax: π1 215 843 8849 U. S. Copyright Clearance Center Code Statement:
cloned from an adult cell (Wilmut et al., 1997), the probability of cloning adult humans became realistic. Animal cloning, as other basic research, was initiated to seek fundamental knowledge for the benefit of mankind. Now that human cloning is possible, though with miniscule success, we must decide whether it should be attempted. This dilemma is not new in science. Discoveries such as recombinant DNA, nuclear energy, anesthesia, electricity, airplanes, etc. can be used either for the benefit or destruction of mankind. So, too, the applications of animal cloning must be responsibly analyzed now and for future generations. Obviously, in a society heterogeneous in its cultural, ethical, and religious values, there is no consensus regarding human cloning. Our responsibility is to debate this issue so that a reasonable agreement can be achieved. The concerns of human cloning center mainly on two proposals: reproductive and therapeutic cloning. In this article I shall argue that human cloning should not be attempted for scientific and ethical reasons. The science and technique of somatic cell nuclear transfer (cloning) is still in its infancy and not ready for clinical trials in humans. I base this statement on the fact that only 0.1 – 5 % of the attempts to transfer somatic nuclei from mammalian animal cells into enucleated oocytes result in live births (reviewed in Di Berardino, 2001). Furthermore, on average only about 50 % of newborns survive and many suffer severe abnormalities (Hill et al., 1999, 2000). The percentage of mammalian animal clones born is usually calculated on the number of blastocyst clones transferred to surrogate mothers, but the actual success should be based on the number of attempts to transfer a somatic nucleus to an oocyte. The latter method of calculation reveals the real challenge to human cloning. For example, Dolly resulted from 434 attempts to fuse a cell from a mammary gland to an oocyte (Wilmut et al., 1997). If these data were extrapolated for proposed human trials, one would need
0301–4681/2002/6904–147 $ 15.00/0
148
oocytes donated from at least 40 women. It is true that success in achieving newborn animal clones has improved, but the problem still exists: obtaining sufficient women as oocyte donors for human trials. The gravest problem is that 95.0 – 99.9 % of the attempts to produce mammalian animal clones resulted in death during gestation and, among newborns, about half died after birth while many survivors suffered severe abnormalities. Although there is a high rate of failure during normal human sexual reproduction, the failures are not intentionally caused by the parents, whereas if human cloning were permitted, the participants would be directly responsible for the deaths. Based on current data in mammalian animal studies, attempts at human cloning would result in a vast amount of human wastage. Committees in responsible USA hospitals, judging other kinds of clinical trials in humans, would not approve studies yielding such high death rates. At the present time, the cloning procedure is a roulette game and will continue to be a game of chance until we learn how to synchronize the cell cycle of the donor cell with that of the recipient oocyte and to reprogram the genomic expression of donor nuclei, including genetic imprinting and DNA methylation, to conform with those patterns in normal pronuclei (Kang et al., 2001; Reik et al., 2001; Rideout et al., 2001). In addition to severe limitations in the cloning procedure, I have other scientific concerns. Most metazoan animal species produce their offspring not asexually but through sexual reproduction. Why did sexual reproduction evolve and persist in complex metazoan animals? Obviously, sexual reproduction provides for genetic diversity, stemming from genetic recombination and random distribution of chromosomes during meiosis. Also, during meiosis there is a DNA repair process, one that is additional to that occurring in somatic cells. Furthermore, the processes of oogenesis and spermatogenesis select against some abnormal germ cells. Although this selection process is not full proof, it is non-existent in somatic cells. Even if cloning from adult cells becomes efficient, serious hazards would still exist. Somatic donor cells during their lifetime could acquire mutations from radiation, chemicals, aging and/or errors in DNA replication that would be transferred to the clone. Also, mutations arise frequently in cells during culture, and these could be transmitted to the clone. Prospects for identifying all mutations in an apparently normal embryo prior to transfer to the uterus are probably impossible. Although in time it might be possible to examine the epigenetic properties of many imprinted genes from a few blastomeres of the embryo clone, knowledge of the DNA sequence of all the clone’s genes appears impossible. Furthermore, the sampling of blastomeres may be misleading. In my experience in frog cloning, many normal appearing blastula clones were composed of a mosaic of cells, some with apparently normal chromosomes
and others with abnormal ones (reviewed in Di Berardino, 1997). Thus, sampling of blastomeres from a clone could result in false negatives. No studies can guarantee that adult animal clones are completely normal. A few studies have examined the rate of aging and certain behavioral traits. For example, Wakayama et al. (2000) cloned mice from cumulus cells through six generations, and found no evidence for accelerated aging, telomere shortening, or abnormal behavior. They evaluated learning ability in the Morris water maze and Krushinsky tests, as well as strength and agility, and also tested for premature aging, such as a decline in activity and loss of coordination. I applaud this report as a scientific study, but such analyses in mice do not prove to me that a cloned human would be mentally normal, nor did the authors make such a claim. My main point is how are we going to look into the minds of sheep cow, pig, or goat clones and diagnose them as emotionally and intellectually normal? Furthermore, if we could, would it be relevant to the human mind? There are still other scientific concerns. Will telomere shortening in the donor cell limit the life span of the clone? Some clones have shortened telomeres, but others do not (reviewed in Di Berardino, 2001). Can the disparity in results be accounted for by differences in nuclear reprogramming or donor cell types? Obviously, this question requires more study. Another question, scarcely explored in mammalian animal cloning, concerns maternal RNAs and proteins in oocytes. Will maternal gene products from foreign donors always be compatible with the donor nucleus? One well-known case of nuclear-cytoplasmic incompatibility was reported for the DDK strain of mice in which matings between DDK females and non-DDK males are virtually (95 % embryo lethality) infertile, but matings between DDK males and non-DDK females are fully fertile (reviewed by Latham, 1999). We should also reflect on certain nucleocytoplasmic hybrids produced long ago in amphibians. Rana pipiens and Rana palustris, when crossed in the laboratory, produced normal hybrid embryos that develop in normal metamorphic frogs. However, enucleated Rana pipiens eggs injected with blastula nuclei from Rana palustris and the reciprocal combination developed abnormally (Hennen, 1972). Why the hybrid genome could function in either cytoplasm but a non-hybrid genome could not was never explained. However, we can speculate that a critical molecular interaction had to occur directly between a gene product in the oocyte and its homologous haploid genome or indirectly with one of its gene products, otherwise normal development failed. These examples illustrate how little we know about possible deleterious outcomes from the cloning process. Finally, very little concern has focused on female animals bearing the clones through gestation. Surrogate mothers suffer a high rate of spontaneous abortions.
149
Among the fetuses surviving to birth, many are significantly overweight and have enlarged placentas (Hill et al., 1999, 2000). In fact, many overweight fetal clones have been delivered by Caesarian section to preserve the lives of the clone and the mother. What would be the physiological and biochemical risks of a human female carrying a clone to birth? Why would anyone attempt human cloning when such questions have been barely examined in animal studies? In my previous comments I delineated a number of scientific reasons why I oppose the extension of cloning to humans. The physical dangers to the child and mother alone support my contention that human cloning would be unethical. I have other ethical reasons why human cloning should not be attempted. Asexual reproduction is unnatural to higher organisms. Why should we introduce retrograde evolution and mimic the ameba? The production of clones is a manufacturing business. This technology could destroy the normal relations within members of a family and lead to further destruction of the family unit. Even animals, especially primates, have a family structure to nurture their own. In my research career, I experimented with frog gametes and embryos. It is repugnant to me to equate human oocytes with mass amounts of frog oocytes that can be poked, cut up and modified, then discarded after they have served their purpose. Even though fertilization of a human oocyte by a spermatozoan is a chemical reaction, there still is a significant difference between human and non-human fertilized oocytes – the fertilized human oocyte has the potential to be a human child. This fact alone should prevent us from experimenting with human embryos. Unless we maintain respect for human life, we will be inferior to animals, our evolutionary ancestors, for even animals have concern for their offspring. For the above scientific and ethical reasons I support an international law to ban human cloning and one enacted immediately in the United States of America. Though such laws may not prevent all attempts at human cloning, they will certainly severely curtail such attempts. Although this article was requested to focus on human cloning, I would like to end by illustrating some of the many beneficial applications of animal cloning to medicine, agriculture, and the basic sciences (reviewed by Di Berardino, 2001). The production of transgenic clones producing human proteins is indeed a breakthrough. This feat was first reported by Schnieke et al., (1997) in sheep and followed by others in goats pigs, and additional sheep. After clinical testing, the various human proteins from transgenic clones can be used to treat diverse human diseases. Animal cloning can also be used to produce large domestic animals as models of human diseases, to test pharmaceuticals on animals with a uniform genetic background and, in the future, to aid
in xenotransplantation of organs to humans. In agriculture, cloning can be applied to producing animals with outstanding traits and resistance to diseases, as well as saving endangered species. In the basic sciences, animal cloning has already yielded important information e.g., in nuclear reprogramming and genetic imprinting, and some insight into cellular aging and cancer. Furthermore, we expect these and various other fundamental questions in biology and medicine to be assisted by animal cloning.
References Briggs, R and King, T.J. (1952) Transplantation of living nuclei from blastula cells into enucleated frogs’ eggs. Proc Natl Acad Sci USA 38:455–463. Di Berardino, M.A. (1997) Genomic potential of differentiated cells. Columbia University Press, New York. Di Berardino, M.A. (2001) Animal cloning – the route to new genomics in agriculture and medicine. Differentiation 68:67–83. Hennen, S. (1972) Morphological and cytological features of gene activity in an amphibian hybrid system. Develop Biol 29:241– 249. Hill, J.R., Burghardt, R.C., Jones, K., Long, C.R., Looney, C.R., Shin, T., Spencer, T.E., Thompson, J.A., Winger, Q.A. and Westhusin, M.E. (2000) Evidence for placental abnormality as the major cause of mortality in first-trimester somatic cell cloned bovine fetuses. Biol Reprod 63:1787–1794. Hill, J.R., Roussel, A.J., Cibelli, J.B., Edwards, J.F., Hooper, N.L., Miller, M.W., Thompson, J.A., Looney, C.R., Wethusin, M.E., Robl, J.M. and Stice, S.L. (1999) Clinical and pathologic features of cloned transgenic calves and fetuses (13 case studies). Theriogeneology 51:1451–1465. Kang, Y-K., Koo, D-B., Park, J.-S., Choi, Y-H., Chung, A-S., Lee, K-K. and Han, Y-M. (2001) Aberrant methylation of donor genome in cloned bovine embryos. Nature Genetics 28:173–177. Kass, L.R. (2001) The wisdom of repugnance. In: Brannigan, M.C. (ed.) Ethical issues in human cloning Seven Bridges Press, LLC, New York and London, pp 43–66. Latham, K.E. (1999) Epigenetic modification and imprinting of the mammalian genome during development. Current Topics in Develop Biol 43:1–47. Reik, W., Dean, W. and Walter, J. (2001) Epigenetic reprogramming in mammalian development. Science 293:1089–1093. Rideout, W.M. III, Eggan, K. and Jaenisch, R. (2001) Nuclear cloning and epigenetic reprogramming of the genome. Science 293:1093–1098. Schnieke, A.E., Kind, A.J., Ritchie, W.A., Mycock, K., Scott, A.R., Ritchie, M., Wilmut, I., Coleman, A. and Campbell, K.H.S. (1997) Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science 278:2130– 2133. Wakayama, T., Shinkai, Y., Tamashiro, K.L.K., Niida, H., Blanchard, D.C., Blanchard, R.J., Ogura, A., Tanemura, K., Tachibana, M., Perry, A.C.F., Colgan, D.F., Mombaerts, P. and Yanagimachi, R. (2000) Cloning of mice to six generations. Nature 407:318–319. Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J. and Campbell, K.H.S. (1997) Viable offspring derived from fetal and adult mammalian cells. Nature 385:810–813.