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ScienceWeek
SCIENCE POLICY: HUMAN CLONING AND NUCLEAR TRANSPLANTATION
The following points are made by Rudolf Jaenisch (New Engl. J. Med. 2004 351:2787):
1) In addition to the moral argument against the use of somatic-cell nuclear transfer for the creation of a child ("reproductive cloning"), there are overwhelming scientific reasons to oppose this practice. In contrast, many believe that the practice of somatic-cell nuclear transfer with the goal of generating an embryonic stem-cell line (sometimes referred to as "therapeutic cloning") is justified, because it holds the promise of yielding new ways of studying and treating a number of diseases. Once isolated from a patient, an embryonic stem cell thus derived would be "customized" to the needs of the patient who had served as the nuclear donor and thus would obviate the need for immunosuppressive treatment as part of a therapeutic application. In addition, because embryonic stem cells can generate most, if not all, types of cells in vitro, a stem cell isolated from a patient with a complex genetic disease could be used to study the pathogenesis of the disease in culture.
2) In contrast to an embryo derived by in vitro fertilization, a cloned embryo has little, if any, potential to develop into a normal human being. By circumventing the normal processes of gametogenesis and fertilization, nuclear cloning prevents the proper reprogramming of the clone's genome that is a prerequisite for the development of an embryo into a normal organism. It is unlikely that these biologic barriers to normal development can be overcome in the foreseeable future. However, embryonic stem cells derived from a cloned embryo are functionally indistinguishable from those that have been generated from embryos derived through in vitro fertilization. Both have an identical potential to serve as a source for cells that may prove useful for research or therapy.[1]
3) Most cloned mammals derived by nuclear transfer die during gestation, and those that survive to birth frequently have the large offspring syndrome, a neonatal phenotype characterized by respiratory and metabolic abnormalities and an enlarged, dysfunctional placenta. In order for a donor nucleus to support development into a clone, it must be "reprogrammed" to a state compatible with embryonic development. Inadequate reprogramming of the donor nucleus is most likely the principal reason for the developmental failure of clones. The transferred nucleus must properly activate genes that are important for early embryonic development and must also suppress genes associated with differentiation that have been transcribed in the original donor cell.
4) However, gene-expression analyses indicate that 4 to 5 percent of the overall genome and 30 to 50 percent of imprinted genes are not correctly expressed in tissues of newborn cloned mice.[2] These data represent strong molecular evidence that cloned animals, even if they survive to birth, have serious gene-expression abnormalities. Moreover, as cloned mice age, severe pathological alterations in multiple organs and major metabolic disturbances that were not apparent at younger ages become manifest. A case in point is Dolly the sheep, the first mammal cloned from a somatic cell, which appeared healthy at a young age but died prematurely with numerous pathological abnormalities. These findings suggest that clones that survive to birth merely represent the least abnormal animals: subtle abnormalities that originate in faulty reprogramming may simply not be severe enough to interfere with their survival. Indeed, given the available evidence, it may be exceedingly difficult, if not impossible, to generate healthy cloned animals or humans.[3]
References (abridged):
1. Jaenisch R. The biology of nuclear cloning and the potential of embryonic stem cells for transplantation therapy. Washington, D.C.: President's Council on Bioethics, January 2004.
2. Hochedlinger K, Jaenisch R. Nuclear transplantation, embryonic stem cells, and the potential for cell therapy. N Engl J Med 2003;349:275-286
3. McHugh PR. Zygote and "clonote" -- the ethical use of embryonic stem cells. N Engl J Med 2004;351:209-211
New Engl. J. Med. http://www.nejm.org
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Related Material:
NUCLEAR CLONING AND EPIGENETIC REPROGRAMMING
The following points are made by W.M. Rideout et al (Science 2001 293:1093):
1) Epigenetic modification of the genome ensures proper gene activation during development and involves (i) genomic methylation changes, (ii) the assembly of histones and histone variants into nucleosomes, and (iii) remodeling of other chromatin-associated proteins such as linker histones, polycomb group, nuclear scaffold proteins, and transcription factors (1).
2) The two parental genomes are formatted during gametogenesis to respond to the oocyte environment and proceed through development. The zygote biochemically remodels the paternal genome shortly after fertilization and before embryonic genome activation (EGA) occurs. To successfully recapitulate these processes, the somatic nuclei transferred into an oocyte must be quickly reprogrammed to express genes required for early development.
3) Epigenetic reprogramming after fertilization and nuclear transfer has been studied in Xenopus and mammals (1).The programming of the genome that occurs as primordial germ cells (PGCs) differentiate into mature gametes establishes the markedly different chromatin configurations of sperm and oocyte. As demonstrated by normal preimplantation development of uniparental embryos, both parental genomes share the ability to independently direct cleavage (early development to the blastocyst stage) despite profound differences in their epigenetic organization (2,3).
4) In spermatogenesis, chromatin is sequentially remodeled, silenced, and ultimately compacted with protamines (4), processes crucial for normal fertilization (5). However, completion of these events is not strictly required for development as normal pregnancies can result from intracytoplasmic sperm injection with round spermatids or secondary spermatocytes. In contrast, the genome of the oocyte is organized in a structure more like that of a somatic cell, with chromatin whose nucleosomes contain an oocyte-specific linker histone. In comparison with the male pronucleus, the female pronucleus is more transcriptionally repressive, contains relatively deacetylated histone, and is deficient in generalized transcription factors. This repressive chromatin structure may protect the oocyte genome against the extensive epigenetic modifications imposed on the paternal genome after fertilization.
5) In summary: Cloning of mammals by nuclear transfer (NT) results in gestational or neonatal failure with at most a few percent of manipulated embryos resulting in live births. Many of those that survive to term succumb to a variety of abnormalities that are likely due to inappropriate epigenetic reprogramming. Cloned embryos derived from donors, such as embryonic stem cells, that may require little or no reprogramming of early developmental genes develop substantially better beyond implantation than NT clones derived from somatic cells. Although recent experiments have demonstrated normal reprogramming of telomere length and X chromosome inactivation, epigenetic information established during gametogenesis, such as gametic imprints, cannot be restored after nuclear transfer. Survival of cloned animals to birth and beyond, despite substantial transcriptional dysregulation, is consistent with mammalian development being rather tolerant to epigenetic abnormalities, with lethality resulting only beyond a threshold of faulty gene reprogramming encompassing multiple loci.
References (abridged):
1. K. E. Latham, Int. Rev. Cytol. 193, 71 (1999)
2. M. H. Kaufman, S. C. Barton, M. A. Surani, Nature 265, 53 (1977)
3. J. McGrath and D. Solter, Cell 37, 179 (1984)
4. K. Steger, Anat. Embryol. 199, 471 (1999)
5. C. Cho, et al., Nature Genet. 28, 82 (2001)
Science http://www.sciencemag.org
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Related Material:
SOMATIC CELL NUCLEAR TRANSFER
The following points are made by I. Wilmut et al (Nature 2002 419:583):
1) Cloning by present methods is very inefficient owing to the extraordinary demands placed on the oocyte cytoplasm in reprogramming a somatic nucleus rather than a sperm nucleus. The cumulative loss observed throughout development is assumed to reflect inappropriate expression of many genes whose harmful effect is exerted at different stages of development. These fundamental limitations to cloning are being addressed by analyses of the underlying cellular mechanisms. In time, this information may be used in the development of treatments to cause cells of one phenotype to "transdifferentiate" to another.
2) Several laboratories have used a variety of somatic cell types to create cloned sheep, cattle, mice, pigs, goats, rabbits and cats (1). However, those laboratories have failed so far to obtain offspring in other species, including the rat, rhesus monkey and dog. Consistent among all published research is that only a small proportion of embryos reconstructed using adult or fetal somatic cells developed to become live young, typically between 0 and 4%. The low overall success rate is the cumulative result of inefficiencies at each stage of the process.
3) In addition to embryonic loss, somatic cell nuclear transfer is also associated with very high rates of fetal, perinatal and neonatal loss, and production of abnormal offspring. Not all of these effects are due solely to nuclear transfer, since similar problems are reported after embryo culture(2). Typically, at least one-third of the cattle and sheep confirmed pregnant with cloned embryos lose their fetuses during gestation(1). Abnormal development of the placenta, including vascular reduction, is a principal contributor to loss particularly during early pregnancy in sheep and cattle(3). It may also contribute to some of the defects reported in neonates(4). In cattle the rate of loss is also increased in the second and third trimesters of pregnancy after nuclear transfer (compared with in vitro fertilization (IVF)), with greater losses when adult rather than fetal or embryonic nuclei are used(5). The overaccumulation of placental fluid in hydroallantois occurs rarely in natural cattle pregnancies, but can affect up to 2% and 40% of pregnancies established with IVF and cloned embryos, respectively. In cloned mice, the placentae are often 2 3-fold heavier than from natural mating, although a lack of vascularization has not been reported.
4) As the fate of cloned embryos is determined by molecular events within hours of nuclear transfer, it is disappointing that so little is known about these events during the early development of cloned embryos. During somatic cell nuclear transfer a great deal is asked of the molecular mechanisms that have evolved to regulate fertilization and pregnancy. Viewed in this light, it is still surprising that somatic cell cloning ever produces viable offspring. Although some improvements in efficiency are to be expected from optimization of present procedures, greater benefits might be expected from intervention to assist reprogramming of the transferred nucleus. At present the means to enhance the success of nuclear transfer are not known, but may involve the use of remodeling complexes and factors that remove somatic epigenetic modifications before transfer. In addition to application of this information in nuclear transfer, new understanding of mechanisms that regulate developmental plasticity will lead to methods to change cells of one phenotype to another as a means of providing histocompatible cells for treatment of degenerative diseases. A new era of developmental biology and regenerative medicine awaits.
5) In summary: Despite present experimental difficulties, cloning by nuclear transfer from adult somatic cells is a remarkable demonstration of developmental plasticity. When a nucleus is placed in oocyte cytoplasm, the changes in chromatin structure that govern differentiation can be reversed, and the nucleus can be made to control development to term.
References (abridged):
1. Wilmut, I. & Peterson, L. A. Somatic cell nuclear transfer (cloning) efficiency [online] http://www.roslin.ac.uk/public/webtablesGR.pdf (2002).
2. Young, L. E. & Fairburn, H. R. Improving the safety of embryo technologies: possible role of genomic imprinting. Theriogenology 53, 627-648 (2000)
3. Hill, J. R. et al. Evidence for placental abnormality as the major cause of mortality in first-trimester somatic cell cloned bovine fetuses. Biol. Reprod. 63, 1787-1794 (2000)
4. Hill, J. R. et al. Clinical and pathologic features of cloned transgenic calves and fetuses (13 case studies). Theriogenology 51, 1451-1465 (1999)
5. Heyman, Y. et al. Frequency and occurrence of late-gestation losses from cattle cloned embryos. Biol. Reprod. 66, 6-13 (2002)
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