CHAPTER 2 Nuclear Transplantation and New Frontiers in Genetic Molecular Medicine D. JOSEPH JERRY, PH.D. and JAMES M. ROBL, PH.D. with Ethics Note by LEONARD M. FLECK, PH.D. BACKGROUND Nuclear transplantation made its debut as a novel tool for defining the genetic basis for differentiation and probing the extent to which these mechanisms may be reversible. From these beginnings, a mature technology has emerged with applica- tions ranging from animal agriculture to clinical medicine. Thus, nuclear transplan- tation can be used to generate identical animals and transgenic livestock. Cloned livestock can be used to intensify genetic selection for improved productivity and have also been proposed as a reliable source of tissues and cells for xenotrans- plantation in humans. At a more fundamental level, these cloning experiments demonstrate that somatic cells retain developmental plasticity such that the nucleus of a single cell, when placed within an oocyte, can direct development of a complete organism. INTRODUCTION Nuclear transplantation is the process by which the nucleus of a donor cell is used to replace the nucleus of a recipient cell (Fig. 2.1). Somatic cells are most often used as the nuclear donors and are transferred, using micromanipulation, to enucleated oocytes. The factors contained within the cytoplasm of oocytes appear responsi- ble for reprogramming somatic cell nuclei and are essential for the success of nuclear transplantation. Genetic reprogramming may be harnessed to alter the developmental potential of cells to allow regeneration of tissues or provide cellular therapies. Conversely, it is possible that illegitimate activation of these factors/ mechanisms may lead to deleterious genetic reprogramming resulting in the devel- opment of cancer. Reprogramming mechanisms may also provide novel targets for cancer therapy and other diseases that involve genetically programmed differenti- 25 An Introduction to Molecular Medicine and Gene Therapy. Edited by Thomas F. Kresina, PhD Copyright © 2001 by Wiley-Liss, Inc. ISBNs: 0-471-39188-3 (Hardback); 0-471-22387-5 (Electronic) ation or developmental changes in cells. Along with these possibilities, great ethical questions surround the potential use of this technology for creating cloned humans. In an effort to clarify these difficult questions, a historical perspective of the use of nuclear transplantation to define the molecular and cellular basis for differenti- ation is presented. The technical challenges and variations among organisms are considered in an effort to explore how these advances may be applied both in the laboratory and in the clinic. However, these accomplishments must also be consid- ered within the framework of the limitations imposed by ethical concerns and tech- nical challenges that remain. NUCLEAR TRANSPLANTATION: A TOOL IN DEVELOPMENTAL BIOLOGY In 1938, the door to human cloning was opened when it was proposed by Speman (1938) that the potency of a cell could be tested by transfer of nuclei from differ- 26 NUCLEAR TRANSPLANTATION AND NEW FRONTIERS IN GENETIC MOLECULAR MEDICINE (a) (b) FIGURE 2.1 Comparison of embryos resulting from normal fertilization and nuclear trans- plantation. (a) Metaphase II oocytes (2N) come in contact with sperm (1N) causing the extrusion of the second polar body. This leaves a haploid (1N) complement of maternal chro- mosomes. The resulting pronuclear embryo is diploid containing equal genetic material from both parents. (b) In nuclear transplantation, the first polar body and metaphase chromosomes are removed leaving a cytoplast. The diploid donor cell is then introduced. The interphase chromatin undergoes premature chromatin condensation followed by reentry into S phase of the mitotic cell cycle resulting in a diploid embryo following division. entiated cells to unfertilized eggs. However, this experiment had to await the devel- opment of early nuclear transplantation techniques. By 1952, it could be shown that nuclei from blastula-stage Rana pipiens embryos could be transferred to enucleated frog oocytes and that these embryos could develop to blastocyst-stage embryos. Blastomeres as Nuclear Donors Early successes spawned a flurry of experiments demonstrating that blastomeres from early cleavage embryos could direct embryonic development when transferred to enucleated oocytes, and therefore, retained pleuripotency. Efforts using blas- tomeres as donor nuclei were soon followed by experiments using cells in more extreme states of differentiation. Somatic Cells as Nuclear Donors Initially, intestinal cells from Xenopus laevis feeding tadpoles were used as donor nuclei. A small fraction of the nuclear transplantation embryos developed to the swimming tadpole stage. In these experiments, seven embryos completed meta- morphosis to produce normal adult males and females.The adult clones were fertile, demonstrating the completeness of the nuclear reprogramming. Other studies used renal adenocarcinoma cells from R. pipiens as donor nuclei to produce normal swim- ming tadpoles. Therefore, not only could differentiated somatic cell nuclei undergo reprogramming, but tumor cells could also be recruited to participate in normal embryonic development following nuclear transplantation. Nonetheless, rates of development of embryos were reduced greatly when “dif- ferentiated” cells were used as nuclear donors compared to blastula or gastrula endodermal cells. Restrictions in the extent of development was most apparent in the Mexican axolotl. Only 0.6% of nuclear transplantation embryos from neurula- stage notochord cells formed swimming tadpoles, whereas 33% of nuclear trans- plantation embryos from blastulae cells reached this stage. Therefore, the vast majority of notochord nuclei were severely restricted in their developmental capac- ity. Failure of development following nuclear transplantation was associated with the presence of chromosomal abnormalities, which included ring chromosomes, anaphase bridges, chromosome fragments, and variable numbers of chromosomes. From these results, Briggs and co-workers (1964) concluded: “The central question therefore concerns the origin of these chromosomal abnormalities. Are they to be regarded as artifacts, or do they indicate a genuine restriction in the capacity of the somatic nuclei to function normally following transfer into egg cytoplasm?” Alter- natively, others suggested that these differences may reflect the relative proportions actively dividing cells within the tissues.These questions remain to be settled despite the passage of three decades. In subsequent experiments, primary cultures were used as a source of nuclei in an effort to provide more uniform populations. Also, “serial nuclear transplanta- tion” gained favor to improve rates of development beyond the blastocyst stage. Serial nuclear transplantation involved a first round of nuclear transfer to produce partially cleaved blastocysts. Although the vast majority (<0.1%) of first-transfer embryos failed to develop beyond the blastocyst stage, they apparently contained a higher proportion of cells with nuclei that were capable of undergoing NUCLEAR TRANSPLANTATION: A TOOL IN DEVELOPMENTAL BIOLOGY 27 nuclear reprogramming following a secondary nuclear transplantation, also referred to as “recloning.” Selection of the most well-developed embryos from initial nuclear transfers allowed enrichment for embryos that contained minimal genetic damage resulting from the manipulations. The positive effects of serial nuclear transplantation were not improved by additional rounds of nuclear transplantation, suggesting that sequential nuclear reprogramming was not taking place. Using serial nuclear transplantation, partial or complete blastulae were obtained at rates of 22 to 31% using cultures of kidney, lung, heart, testis, and skin from adult frogs as donor nuclei for serial nuclear transplantation. Swimming tadpoles devel- oped when nuclei for the initial transfers were from adult kidney, lung, and skin but not heart. Based on these results, it would appear that <10% of cells from the primary nuclear transplant embryos were able to undergo successful genetic repro- gramming and direct successful development of tadpoles. It is also important to note that some developmental abnormalities were evident in tadpoles derived from nuclear transplantation. The descriptions were not extensive, but anal and cardiac edema were reported and resulted in subsequent death. Since a relatively small proportion of donor nuclei were able to form even blastocysts following nuclear transplantation, it remained possible that embryos resulted only from a subpopulation of cells that retained stem cell-like characteris- tics. To rule out this possibility, primary cell cultures were established from foot- web explants and were shown to be differentiated by the expression of keratin in >99.9% of cells. Although no first-transfer embryos developed beyond early cleavage embryos, serial transplantation resulted in swimming tadpoles with well- differentiated organs. Attempts to confirm these results in Drosophila yielded development of larvae but no adults.This result was extended by Schubiger and Schneiderman (1971) when it was shown that preblastoderm nuclei could be transplanted into oocytes, then develop 8 to 10 days when placed in a mature female.These implants were retrieved, then dissociated, and the nuclei were again used for serial nuclear transplantation. The serial nuclear transplant embryos were transferred into developing larvae where they underwent metamorphosis along with their hosts to form adult tissues. Therefore, extensive genetic reprogramming of donor nuclei was possible but required serial nuclear transplantation similar to that used in amphibia. Nonetheless, reprogramming was not sufficient to allow development of normal flies. Conclusions The work with amphibia clearly demonstrated that nuclear transplantation could be used to efficiently generate multiple cloned individuals using blastomeres from early cleavage embryos. Although rates of development were diminished when more highly differentiated cell types were used as donors for nuclear transplanta- tion, it was possible to generate live offspring. Therefore, differentiation was reversible and developmental fates were subject to reprogramming under appro- priate conditions.The extent of development following nuclear transplantation also varied considerably among tissues. Gurdon (1970) voiced caution that “nuclear transplantation experiments can only provide a minimum estimate of developmen- tal capacity of a nucleus or a population of nuclei.” It was a concern that the vari- 28 NUCLEAR TRANSPLANTATION AND NEW FRONTIERS IN GENETIC MOLECULAR MEDICINE able rates of success using cells from various tissues reflected technical challenges due to isolation or culture of specific cell types. Mechanical damage to cells during isolation may vary among tissues. The proportion of nuclear transfer embryos that result in live births may reflect the relative infrequency of specific stem cells that may be more amenable to nuclear reprogramming. The more limited reprogram- ming observed with Mexican axolotl and Drosophila may indicate that some changes are irreversible. If true, then some organisms or cell types may have bio- logical barriers preventing nuclear reprogramming. At this point, the molecular basis for nuclear reprogramming was left to conjecture. TECHNICAL DEVELOPMENTS IN NUCLEAR TRANSPLANTATION The ability to create cloned frogs fueled hopes that mammalian nuclei might also be subject to nuclear reprogramming by the oocyte cytoplasm. The value of being able to make multiple clones of genetically superior livestock for the purpose of intensifying genetic selection was not lost on agricultural scientists. As a result, efforts to apply nuclear transplantation to create cloned livestock were under- taken by several groups. This required modifications of nuclear transplantation procedures. Overview of the Procedures The nuclear transplantation procedures were pioneered in 1952 in R. pipiens where it was possible to physically enucleate oocytes. However, the membranes sur- rounding the oocyte in X. laevis precluded this. Therefore, ultraviolet (UV) irradi- ation was used to destroy the nucleus. The donor cells were most conveniently handled in suspension following trypsinization. The donor cells were drawn into a glass micropipet, then inserted into the enucleated egg between the center and the animal pole. The intact donor cell, with its nucleus, cytoplasm, and membranes, was expelled into the recipient egg. The membranes surrounding the recipient cell should heal spontaneously as the pipet is withdrawn.The eggs were then transferred to buffered media and cleavage proceeded as manipulation of the oocyte was suf- ficient activation stimulus in amphibians. Nuclear transplantation procedures in mammals involve four specific steps: (1) enucleation, (2) transfer of a donor nucleus along with its associated cytoplasm, (3) fusion of the donor nucleus and recipient cytoplasm, and (4) activation of cleavage (Fig. 2.2). Oocytes arrested in metaphase II of meiosis are most often used to prepare recipient cytoplasts because they are large cells that can be easily enucleated. Enucleation is accomplished by inserting a glass micropipet through the zona pelucida and withdrawing the polar body and metaphase chromosomes. Rather than direct injection, the intact donor cell (nucleus, cytoplasm, and mem- branes) is expelled into the perivitelline space adjacent to the enucleated oocyte with the aid of a micropipet. The enucleated oocyte and intact donor cell are then fused and treated to initiate the cell cycle,which is referred to as activation. Embryos resulting from this process would be genetically identical to the donor at the level of their genomic deoxyribonucleic acid (DNA) but are chimeric with respect to organelles. Therefore, animals prepared by nuclear transplantation are not true clones. TECHNICAL DEVELOPMENTS IN NUCLEAR TRANSPLANTATION 29 Fusion The first challenge was to develop more versatile methods for fusion of the donor and enucleated recipient cells.The use of Sendai virus to mediate fusion of the recip- ient oocyte and donor cells was ineffective in a number of species. The advent of electrical fusion of cell membranes provided a flexible and efficient method to stimulate fusion of the donor and recipient cells in a broad range of species. 30 NUCLEAR TRANSPLANTATION AND NEW FRONTIERS IN GENETIC MOLECULAR MEDICINE FIGURE 2.2 Summary of nuclear transplantaion in mammals.With the aid of a micropipet, the metaphase plate and first polar body are removed from an oocyte arrested in metaphase II to generate a recipient cytoplast. A donor cell (nucleus and cytoplasm) is transferred to the perivitelline space using a micropipet. Electrical pulses are used to stimulate fusion of the plasma membranes of the donor cell and recipient cytoplast causing the donor nucleus to enter recipient cytoplasm and initiation of cell division. During the first cell cycle, cyto- plasm of the oocyte causes condensation of the chromatin followed by replication of the DNA. If successful, the embryo will continue to undergo cleavage to form a normal blastocyst. Enucleation Complete removal of chromosomes was also more challenging in mammalian oocytes. Treatment with cytoskeletal inhibitors, cytochalasin B, and colcemid stabi- lized the plasma membrane and prevented rupturing. This allowed a large pipet to be inserted through the zona pellucida and adjacent to the pronuclei without pen- etrating the membrane.The pronucleus can then be removed in a membrane-bound cytoplast along with the polar body as shown in Figure 2.2. Fluorescent vital dyes are now used to visualize chromatin to ensure complete removal of the metaphase II chromosomes. Activation Resumption of the cell cycle in metaphase II oocytes is referred to as activation and results in cleavage of the cell. Activation following nuclear transplantation also proved to be a formidable problem and variable among species. This may belie the lower efficiencies associated with nuclear transplantation in rodents. In cattle, fer- tilization of oocytes by sperm was shown to initiate changes in calcium concen- trations in the oocyte cytoplasm. The electrical pulses used to induce fusion were also shown to cause calcium increases but were minimally effective in activating the oocyte following nuclear transplantation. Procedures to elevate calcium fol- lowed by the extended inhibition of MPF activity, using the kinase inhibitor 6- dimethylaminopurine, have been shown to support rates of development to the blastocyst stage that are equivalent to that of in vitro fertilized oocytes. Cell Cycle Synchronization Between Nuclear Donor and Recipient Oocyte Synchrony of the cell cycle between recipient oocyte and donor nucleus was also subject to refinements. Nuclear transplantation between metaphase donors and metaphase II recipient oocytes would appear to be the ideal match. Although modest success has been achieved, this approach remains technically challenging. The difficulty in using G2 or M-phase donor cells is that the cells are tetraploid at this stage of the cell cycle. Therefore, cell division must occur following nuclear transfer to produce a diploid two-cell embryo. The difficulty lies with the fact that premature chromatin condensation (PCC) occurs following nuclear transplantation followed by reentry into S phase leading to tetraploid embryos. Nuclei from cells that are in G1 also undergo PCC following nuclear transfer and proceed to S phase resulting in diploid embryos. To successfully utilize recipient oocytes in metaphase II with donor nuclei that are most likely in the G1 or S phases of the cell cycle, it is necessary that the oocyte be given an activation stimulus following fusion with the donor cell. The metaphase II oocyte cytoplasm has been shown to initiate immediate breakdown of the nuclear envelope of the donor cell, condensation of the chromosomes followed by reformation of the nuclear envelope and dramatic swelling of the nucleus as activation progresses. This sequence of events may be crucial for nuclear proteins of the donor cell to be lost and replaced by the oocyte nuclear proteins with nuclear reformation allowing reprogramming of the chromatin. TECHNICAL DEVELOPMENTS IN NUCLEAR TRANSPLANTATION 31 DEFINING THE LIMITS OF NUCLEAR REPROGRAMMING IN MAMMALS With technical hurdles addressed, further investigations undertook the task of determining the point during development when cells lost their pluripotency and, therefore, had become differentiated. An initial report of successful nuclear transplantation in mice offered promise but was unable to be confirmed by other investigators. Blastomeres as Nuclear Donors In sheep, blastomeres from 8-cell and 16-cell embryos were shown to develop to blastocysts following nuclear transplantation and form viable embryos after trans- fer to the oviduct of recipient ewes. This was the first reproducible evidence that mammals could be cloned by nuclear transplantation as reported in Nature in 1986. Cattle (1987) and rabbits (1988) were soon added to the growing list of mammals that had been cloned with the assistance of nuclear transplantation. Full-term devel- opment of mice from nuclear transfer of blastomeres was eventually demonstrated in 1987. However, the rates were low compared to sheep and cattle, possibly due to differences in the requirements for activation following nuclear transfer. Cloning in pigs was also reported in 1989, but was limited to one live pig. These results emphasize the considerable variation in the success in cloning mammals using blas- tomeres as donor cells. Unlike earlier results using nonmammalian species, serial nuclear transplantation did not offer any substantial improvement in developmen- tal potential. Inner Cell Mass as Nuclear Donors Efforts to obtain cloned animals using cells derived from the inner cell mass (ICM) were initially unsuccessful in mice. However, live births were reported in cattle using nuclear donors from the ICM. These data supported the concept that the ICM cells retained their primitive state and remain able to be reprogrammed by nuclear transplantation. Nonetheless, results from mice, rabbits, and cattle all suggest that reprogramming of cellular fates is dramatically restricted in eight-cell embryos and beyond. Embryonic Stem Cells as Nuclear Donors The more limited ability of ICM cells to participate in embryonic development fol- lowing nuclear transplantation appeared to contradict results emerging from exper- iments with embryonic stem (ES) cells. ES cells had been derived from the ICM and maintained in vitro under conditions to prevent differentiation and were shown to contribute to many different tissues in aggregation chimeras. The most stringent verification of the totipotency of the ES cells was that they contributed to the germline, but this has been accomplished only in mice. Therefore, it appeared that ES cells retained totipotency. An obvious extension of these experiments was to use ES cells as donors for nuclear transplantation. However, establishment of ES cell lines from species other than mice proved to be more difficult. Even in mice, success in establishing and 32 NUCLEAR TRANSPLANTATION AND NEW FRONTIERS IN GENETIC MOLECULAR MEDICINE maintaining totipotent ES cell lines has been largely limited to the 129 strain. Selec- tion methods to eliminate differentiated cells have been developed recently to prepare ES cells from nonpermissive strains of mice. Use of the epiblast for deriv- ing ES cell lines also appears promising. In spite of the challenges, ES-like cells have been produced from cattle, rabbits, pigs, and sheep. Initial work using “short-term” cultures of bovine ES-like cells for nuclear trans- plantation resulted in live births. However, when bovine ES cell lines that had been in culture for extended periods were used as nuclear donors, the results were less promising. Normal fetal development was achieved following nuclear transplanta- tion of bovine ES-like cells, but pregnancies failed due to improper development of the extraembryonic membranes of the fetal placenta. This occurred in spite of the fact that similarly derived ES cells were shown to contribute to a variety of tissues in aggregation chimeras. Rabbit ES cells were also used for nuclear transplantation. Fetal development of nuclear transplantation embryos derived from rabbit ES cells appeared to be normal, but no live births were reported. These data suggest that the ability of ES cells to form chimeras and their success in nuclear transplantation may be distinct features. Somatic Cells as Nuclear Donors Although nuclear transplantation was shown to be successful using blastomeres in a variety of species, the dramatic decreases in rates of success using ICM and ES cells had diminished the enthusiasm among developmental biologists for cloning mammals from somatic cells. The prevailing wisdom was thoroughly shaken by the reports of Dolly—a normal sheep that developed to term following nuclear transplantation of a donor nucleus from a single mammary epithelial cell. Not only was Dolly cloned from somatic cells but it was from adult cells providing a dra- matic confirmation of the earlier work of Gurdon (1970). This was followed by nuclear transplantation of embryonic fibroblasts to clone cattle, sheep, and goats. Cumulus cells from adult animals have also been used as donor cells to clone mice and cattle. The results from animals cloned using somatic cells from mammals substan- tiate much of the work performed in amphibians; however, the data are far from complete (summarized in Table 2.1). It is clear that a variety of somatic cell types are capable of undergoing nuclear reprogramming following nuclear trans- plantation and yield live offspring. However, efficiency of nuclear reprogram- ming is very dependent on the donor cells. Cumulus cells and fetal fibroblasts have proven to be competent donors in two species, whereas trophectodermal cells were consistently negative in two studies. Under different conditions, trophecto- dermal cells were used to produce cloned mice. These differences arise from differences in the techniques used, suggesting that procedures may be optimized further. The differences among cell types may also reflect incompatibilities in the cell cycle between donor and recipient cells. Some cell types may contain irreversible genetic blocks due to differentiation. Irreversible gene silencing can result from multiple G :C to A: T transition mutations, termed “repeat-induced point mutations,” induced by methylation.The proportions of stem cells, which may be more amenable to undergoing nuclear reprogramming, are also likely to vary among tissues as well. DEFINING THE LIMITS OF NUCLEAR REPROGRAMMING IN MAMMALS 33 34 NUCLEAR TRANSPLANTATION AND NEW FRONTIERS IN GENETIC MOLECULAR MEDICINE TABLE 2.1 Relative Success of Nuclear Transplantation Using Different Donor Nuclei Species Blastocysts Live Births References (%) (% of transfers) Tissue Embryonic or Sheep (fibroblasts) 37.9 7.5 Wilmut et al., 1997 fetal cells Sheep (fibroblasts) 6–20 5–20 Schnieke et al., 1997 Cattle (fibroblasts) 12 14 Cibelli et al., 1998 Goats (fibroblasts) 34–49 3.5 Baguisi et al., 1999 Adult somatic cells Fibroblasts Sheep 11 3.4 Wilmut et al., 1997 Cumulus cells Mice — 2.3 Wakayama et al., 1998 Cattle 49 83 Kato et al., 1998 Neuronal cells Mice 22 — Wakayama et al., 1998 Sertoli cells Mice 40 — Wakayama et al., 1998 Oviductal cells Cattle 23 75 Kato et al., 1998 Granulosa cells Cattle 69 10 Wells et al., 1999 Trophectoderm Mice — 0 Tsunoda et al., 1998 — 0 Collas and Barnes, 1994 32–64 8 Rabbit 0 — TOWARD AN UNDERSTANDING OF THE MECHANISMS OF GENETIC REPROGRAMMING Cloning animals has been the focus of the efforts in nuclear transplantation to date because this provides the most stringent test of the underlying phenomenon of genetic reprogramming. Cloning has been, in some ways, an unfortunate endpoint because of the ethical dilemmas that arise from the potential application of this tech- nology to humans.The prospect of human cloning and its moral and ethical implica- tions has diverted both public and political attention away from the fundamental goal of identifying the molecular basis for reprogramming the DNA to allow cells to regain developmental plasticity. Once these mechanisms are understood, they may be harnessed to interconvert cell types. The implications and medical therapeutic applications of cellular interconversion are staggering (summarized in Table 2.2). For example,skin cells from a leukemia patient could be converted to hematopoietic stem cells for reconstituting the hematopoietic system following chemotherapy without risk of “residual disease” from the transplanted cells, a major reason for failure of autologous bone marrow transfers. Alternatively, new approaches toward disease etiology may be explored. Cancer could be viewed as the converse situation where a cell acquires new phenotypes as the result of inappropriate genetic repro- gramming. Cancer cells harbor many genetic changes (see Chapter 11), but the phe- notype is, in part, reversible. Thus the question arises: How to reverse the cancer phenotype through genetic reprogramming? The most dramatic example of such “reprogramming” of cancer cells is the ability of embryonal carcinoma cells to par- ticipate in normal development to produce chimeric mice. Adenocarcinoma cells have also been shown to produce normal offspring after nuclear transplantation. Additionally, the cellular microenvironment has been shown to “reprogram” globin [...]... cell cloned transgenic bovine neurons for transplantation in parkinsonian rats Nat Med 4:569–574, 1998 44 NUCLEAR TRANSPLANTATION AND NEW FRONTIERS IN GENETIC MOLECULAR MEDICINE Developmental Biology, Nuclear Remodeling, Tissue Engineering and Xenotransplantation Blasco MA, Lee H-W, Hande MP, Samper E, Lansdorp PM, DePinho RA, Greider CW Telomere shortening and tumor formation by mouse cells lacking... 42 NUCLEAR TRANSPLANTATION AND NEW FRONTIERS IN GENETIC MOLECULAR MEDICINE • • • within the cytoplasm of oocytes, which allows reprogramming of somatic cell nuclei Nuclear transplantation can be used to generate identical animals and transgenic livestock Cloned livestock can be used to intensify genetic selection for improved productivity and have also been proposed as a reliable source of tissues and. .. stem cells, and somatic cells A variety of somatic cell types are capable of undergoing nuclear reprogramming following nuclear transplantation to yield live offspring However, efficiency of nuclear reprogramming is very dependent on the donor cells A by-product of nuclear transplantation technology may be the ability to interconvert cell types for use in cell therapies In addition, nuclear transplantation. .. NUCLEAR TRANSPLANTATION AND NEW FRONTIERS IN GENETIC MOLECULAR MEDICINE TOWARD AN UNDERSTANDING OF THE MECHANISMS OF GENETIC REPROGRAMMING 37 gene expression in chimeric mice Thus, one could envision new fields of investigation detailing cellular reprogramming mechanisms determining cell type and function based on the local tissue or organ microenvironment Methylation and Acetylation Methylation and acetylation... therapies and organs for xenotransplantation Cells for allotransplantation Cells for autologous transplantation Precise genetic modifications can be introduced by homologous recombination Preparation of transgenic livestock for cell-based therapies and organs for xenotransplantation Cells for allotransplantation Precise genetic modifications can be introduced by homologous recombination Applications 36 NUCLEAR. .. Failure to maintain proper genetic imprint- 38 NUCLEAR TRANSPLANTATION AND NEW FRONTIERS IN GENETIC MOLECULAR MEDICINE ing may be responsible for the embryonic death that occurs beyond the blastocyst stage Cellular Senescence Many questions as to the “age” of cells following nuclear transplantation persist Erosion of telomeric repeats has been associated with aging and cellular senescence in vitro It is clear... of these cells), then it seems we have a minor ethical problem at best On the other hand, if future research requires tens of thousands of embryos to be created and destroyed in order to meet highly specialized future research needs, then we have a more serious ethical problem At 40 NUCLEAR TRANSPLANTATION AND NEW FRONTIERS IN GENETIC MOLECULAR MEDICINE present we have no way of knowing which state... genetic modifications Benefits Generally result in higher rates of development following nuclear transplantation Benefits/Drawbacks Comparison of Nuclear Transplantation Donors and Applications to Therapies Nuclear Donor TABLE 2.2 Limited to experimental uses Limited to experimental uses Applications TOWARD AN UNDERSTANDING OF THE MECHANISMS OF GENETIC REPROGRAMMING 35 10–70% Adult somatic cells Benefits/Drawbacks... al 1998) Successful early embryonic development following nuclear transplantation has been reported in primate, for rhesus monkeys As an extension of these studies, fibroblasts could be obtained from a patient and used as donor cells for nuclear transplantation to yield blastocysts ES cells could then be prepared from the ICM of the nuclear transplantation blastocysts This approach, precluded from federally... cytoplasmic activity on the development in vivo of sheep embryos after nuclear transplantation Biol Reprod 40:1027–1035, 1989 Speman H Embryonic development and induction Hafner, New York, 1938 Stice SL, Robl JM Nuclear reprogramming in nuclear transplant rabbit embryos Biol Reprod 39:657–664, 1988 Thompson EM Chromatin structure and gene expression in the preimplantation mammalian embryo Reprod Nutr . CHAPTER 2 Nuclear Transplantation and New Frontiers in Genetic Molecular Medicine D. JOSEPH JERRY, PH.D. and JAMES M. ROBL, PH.D. with Ethics Note by LEONARD M. FLECK, PH.D. BACKGROUND Nuclear transplantation. differentiate into adipose (fat), chondrocytes (cartilage), and osteocytes (bone). Nuclear transplantation may 38 NUCLEAR TRANSPLANTATION AND NEW FRONTIERS IN GENETIC MOLECULAR MEDICINE provide a means. species. 30 NUCLEAR TRANSPLANTATION AND NEW FRONTIERS IN GENETIC MOLECULAR MEDICINE FIGURE 2.2 Summary of nuclear transplantaion in mammals.With the aid of a micropipet, the metaphase plate and first