Nuclear transfer technologies: between successes and doubts

ELSEVIER

NUCLEAR TRANSFER TECHNOLOGIES:

BETWEEN SUCCESSES AND DOUBTS

J.P. Renardla, Qi Zhou*, D. LeBourhis3, P. Chavatte-Palmer’, Vignon’

I. Hue’, Y. Heyman’, and X.

‘Biologie du Dtveloppement et Biotechnologies, INRA, 92 170, Jouy en Josas, France; *Institute of Developmental Biology, Chinese Academy of Sciences (CAS), Beijing, 100080, China; %NCEIA Services techniques, BP65,94703 Maisons-Alfort, France

ABSTRACT Cloning of mammals by nuclear transfer can lead to the birth of healthy adult animals but more often compromises the development of the reconstructed embryos. A high incidence of fetal and postnatal losses has been observed in several species, revealing the existence of longlasting effects induced by the nuclear transfer procedures. Remodeling of donor chromatin by the recipient cytoplasm after nuclear transfer is frequently associated with the deregulation of specific genes, and recent observations point to the potential importance of time-dependent DNA methylation events in the occurrence of these alterations. Screening strategies to design nuclear transfer procedures that would mimic the epigenetic remodeling occurring in normal embryos are being designed, and improvement in the efficiency of procedures could imply a pre-conditioning of donor cells. Early mammalian development appears to be rather tolerant to epigenetic abnormalities, raising the possibility that even a fully functional reprogrammed genome may have been subjected to some epigenetic alterations. Bringing nuclear transfer to routine practice requires greater knowledge and understanding of the basic biological processes underlying epigenetic controls of nuclear activities. An important issue at present is to limit the production of those aberrant phenotypes that may result in significant insult to the nature and welfare of animals. 0 2001 by Elsevier Science Inc. INTRODUCTION Nuclear transfer technologies are now widely used by numerous laboratories to produce animals from embryonic and adult cells. The effects of these technologies on the development of reconstructed embryos remain, however, largely uncontrolled in all species so far successfully cloned. The sources of variation that likely affect the outcome of nuclear transplantation are numerous and include the techniques used for embryo reconstruction, the treatment of the donor cells before nuclear transfer, the type of donor cell, and the source of oocytes. Significant technical improvements over the past four years account for an increase in the pre-implantation

Acknowledgment: We are grateful to Alice Jouneau and to Senan Baqir for their suggestions and comments on the manuscript.

aCorrespondence and reprints requests; e-mail: [email protected] Theriogenology 57:203-222,2002 0 2001 Elsevier Science Inc.

0093-691X/02/$-see front matter PII: SOO93-691X(01)00667-7

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development of reconstructed embryos to the blastocyst stage. But fetal and perinatal development remains low and highly variable between experimental series This is well exemplified with adult somatic cell cloning in cattle, where the rates of live offspring per nuclear transfer embryo reported by different groups can vary between 0 and 83% and that of postnatal mortality between 0 and 100% (12, 31, 33, 34, 48, 84, 94). Thus, nuclear transfer appears, at least today, as a stochastic process that only occasionally leads to normal development. However, morphologically and physiologically normal somatic cell clones have now come to adulthood in several laboratories. These animals demonstrate that the network of interacting molecules that carry out the development of mammals can remain fully functional when produced from nuclei of differentiated cells. This argues for the possibility of significant progress in the engineering of embryonic development. The aim of this paper is to provide an insight into the biological events that today place nuclear transfer technologies between successes and doubts. LONG LASTING EFFECTS ASSOCIATED WITH NUCLEAR TRANSFER Whatever cell type tested or species utilized, less than 4% of embryos reconstructed by nuclear transfer develop to live offspring. A majority of losses occur during the first third of gestation in mice, cattle, and sheep. However, in cattle and sheep, a high rate of abortion occurs during the last third of gestation as well as a significant amount of perinatal mortality. Moreover, offspring obtained after cloning often present lethal syndromes. Although those syndromes can be observed with in vitro-produced embryos, their prevalence is much higher with clones. Early Fetal Losses Early pregnancy losses established from somatic nuclear transfer are commonly above 50% in sheep (93,95), cattle (12, 94), and goats (3). Upon implantation, those fetuses that fail to develop often show retarded development, although such a feature had previously been observed with concepti derived from embryonic nuclei (78). When compared with in vitro-produced embryos, the frequency of early fetal mortality is about two times higher with somatic nuclei obtained from either fetuses or adult cultured fibroblasts than with embryonic nuclei (3 1). Most of the losses observed with somatic nuclei occur during the second month of pregnancy in cattle (3 1, 94). They are most frequently associated with functional deficiencies that occur at the onset of placentation as evidenced by poor development of placentomes and abnormal vascularization of extra embryonic tissues (cattle: 34; 94; sheep: 21). Placental growth defects and failure to initiate adequate blood supply have also been reported in ruminants following transfer of in vitro-produced embryos (90, 99). Lack of some placentomes and abnormal vascularization of extra embryonic tissues could lead to fetal malnutrition that, in turn, would influence development long after birth (4). Variations in the degree of placental alterations do exist, as animals derived from somatic cloning or IVF and culture have placental abnormalities during early or late gestation (32,87,99). Late Fetal Losses High rates of abortion during late gestation are routinely observed in cloning experiments performed in the ovine (74, 95), bovine (12, 84) and murine (86) species. These late miscarriages are also frequently associated with an abnormal development of the placenta (31, 32, 61, 63, 87). Using the same technical protocol for embryo reconstruction and/or culture, we have recently shown, in cattle, that, while gestation losses over 70 days are almost inexistent

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with in vitro-produced embryos, they represent about 4% of the pregnancies initiated with donor embryonic nuclei, but 33 to 44% of those resulting from the transfer of, respectively, fetal and adult somatic nuclei (31). The most striking feature of these developmental abnomalities is the excessive accumulation of amniotic fluid and significantly increased birth weight. These growth defects are also observed with in vitro-fertilized embryos (26, 32, 90). Because abnormal placentation may be the cause of those fetal anomalies, we designed an in vivo means of monitoring placental development. For that, we measured the level of pregnancy serum protein 60 (PSP60) in maternal blood. This PSP60 is secreted by placental cells in cattle (73), and levels of this protein increase as placental development advances. We observed that PSP60 levels significantly increase 1 month earlier in cows carrying somatic cell nuclear transfer embryos, whose development failed before term, as compared with cloned embryos undergoing normal development (our unpublished observations). Concomitantly, we confirmed abnormal placentome sizes and development of hydroallantois in the post-mortem examination of fetuses at late developmental stages. Pregnancy specific protein B (BSPb) has also been reported to be transiently higher at Day 35 for pregnancies that stopped before Day 90 (34). These approaches can be used as diagnostic methods to predict the occurrence of abnormal pregnancies established with cloned embryos. Postnatal Development Postnatal mortality has been reported for all species cloned until now. These losses are frequently associated with prolonged pregnancies and dystocia (40, 68, 74, 95). Body weight of these animals is generally higher than normal (11, 12, 95, 94) and may occasionally be twice the mean value of the corresponding breed (74, 80, 94). A syndrome called Large Offspring Syndrome (LOS) may also arise after embryo culture (6, 75, 100) or pronuclear injection (89). This syndrome results from epigenetic modifications of imprinted genes (100). Such epigenetic changes occurring in the early embryo could be propagated through subsequent cell cycles and could affect gene expression during fetal or postnatal development (18; see subsequently). Interestingly, in the caprine species, cloning experiments revealed no increase in prenatal losses after Day 40 of gestation, and birth weights and the numbers of cotyledons were within the normal range (3, 43, 104). In the mouse, a high variability in the expression of imprinted genes without correlation to fetal overgrowth or neonatal mortality has recently been reported with EScell nuclear transfer (36). Morphologically normal mammalian clones have been routinely produced by various laboratories over the world. In several instances, however, those clones are physiologically normal at birth except for one given function. For instance, we have produced several cloned calves over the past 2 yrs that unexpectedly died 6 to 12 wks after birth despite being carefully monitored. In one case, we precisely diagnosed the occurrence of a thymic aplasia that was directly related to the cloning process (68). An atrophied kidney was also observed at autopsy for two cloned calves that died 1 and 2 days, respectively, after normal delivery (our unpublished observations). In several other cases, we noticed elevated concentrations of leptin or fluctuations in body temperature characterized by increases in spikes lasting 12 to 48 hrs over 3 to 4 wks after birth (our unpublished observations). Liver or heart hypertrophy, with no other apparent abnormalities, has been reported for cattle and sheep obtained from somatic cell cloning or in vitro-produced embryos (26,47).

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These observations clearly indicate that apparently normal clones may, even transiently, exhibit physiological peculiarities that are not typical of those syndromes associated with deregulation of imprinted genes. More clinical observations, together with cellular and molecular analysis of phenotypes, will contribute to our understanding of the pleiotropic effects generated by nuclear remodeling. NUCLEAR REMODELING UPON EMBRYO RECONSTRUCTION For successful development of embryos resulting from nuclear transfer, the donor nucleus has to obtain an embryonic pattern of DNA replication and transcription under the control of the recipient oocyte. It is generally agreed that synchrony between the donor nucleus and recipient cytoplasm enhances the successful development of the reconstituted embryo. Functional Consequences of Cycle Synchronization

Between the Nucleus and Cytoplast

The recipient cytoplasm is generally obtained by enucleation of a matured oocyte at the Metaphase-II (M-II) stage. A nucleus introduced after enucleation will be submitted to the activities of cytoplasmic cell cycle regulators such as p34cdc2/cyclin B kinase, the maturation promoting factor (MPF), which induces the remodeling of nuclear structure (27). A high MPF activity, as is present in the M-II oocyte, induces nuclear envelope breakdown, premature chromosome condensation (PCC), and subsequently the DNA replication of the foreign nucleus (14, 15). Alternatively, if oocytes are aged for a period of time after the end of maturation, the MPF activity progressively decreases until reaching an interphase-like stage (28) in which the nucleus will continue its natural cell cycle progression (13). A prerequisite for the reconstruction of a karyotypically normal embryo is the correct cell cycle synchronization between the cytoplast and the karyoplast at the time when the transplanted nuclei are exposed to MPF (10). Chromosomal abnormalities are observed when PCC occurs in the S or G2 phase of the nuclear cell cycle because of a potential reduplication of the genome (14). On the basis of these data, it is considered that the donor cells should be in the M phase or Gl phase of the cell cycle when fused with or injected into M-II oocytes. When M stages are used, the chromosome constitution of the reconstituted embryos will depend on the second polar body extrusion after the activation. Efforts to coordinate the cell cycle stage of donor nuclei and recipient oocytes significantly improved the rate of in vitro development of reconstituted embryos. This improvement, however, is variable from one species to another (Table 1) and is not reflected in later stages of development. The ES-cell nuclei, synchronized in metaphase and injected into MII enucleated oocytes, provided high blastocyst formation and implantation rates, but the postimplantation development was as low as nuclear transfer with unsynchronized ES-cells (88, 103) indicating that parameters other than the cell cycle stage are involved in the long-lasting effects of cloning.

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Table 1. Effect of cell cycle stage of the donor nucleus on in vitro developmental nuclear transfer embryos in bovine and in murine species Species (type ofcells) Bovine (fibroblasts) Murine (ES-cells)

Cell cycle stage Metaphase GO Cycling Metaphase Gl G2

Embryos reconstructed 380 423 152 220 142 234

Cleaved embryos (%) 318 (81.7) 298 (70.4) 116 (76.3) 209 (95.0) 71 (50.0) 198 (84.6)

potential of

Blastocysts (% of reconstructed) 150 (39.5) 163 (38.5) 49 (32.0) 125 (56.8) 19 (13.4)* 33 (14.1)*

*Significantly different within column (P ~0.01).

Nuclear Remodeling and Nuclear Transfer Procedures Pregnancies have been established with blastocysts derived from injection of nuclei from fetal tibroblasts (51) or leukocytes (29) in the bovine species, but all of them were lost after 40 to 150 days of transfer. We have experimented with a protocol that involves microinjection of bovine fibroblast nuclei into enucleated oocytes. Using this protocol, we have obtained good rates of in vitro development into blastocysts (Table l), and some of them developed into live young (our unpublished data). In contrast, in mice, the same procedure of direct nuclear injection resulted in live pups derived from cumulus cells (86) or fibroblasts (87), whereas reconstituted mouse embryos by fusion resulted in a poor rate of development to the blastocyst stage and the birth of only one dead pup (41). In this species, a procedure of fusion has been successfully applied, but only after the serial transfer of the somatic nucleus first into an enucleated oocyte, then into a fertilized one-cell embryo (50, 63). In the pig (62, 65) and goat (3, 104), both microinjection of the donor nucleus and fusion of the donor cell have proved to be methods available for full-term development of cloned embryos. There is, at the moment, no identifiable reason for the differences observed between these species and procedures. There also seems to be species specificity in the timing of activation after transfer of the nucleus. In mice, the reprogramming of the genome of differentiated cells requires a prolonged exposure of the DNA to cytoplasmic factor(s) (86). This can be obtained by means of a retardation of activation after the injection of the donor nucleus, thus maintaining a high MPF activity and condensed chromatin directly exposed to the recipient cytoplasm. This probably accounts for better results obtained when injecting nuclei that were in metaphase as compared with the transfer of interphase nuclei (103; Table 1). In this case indeed, not only the nucleus is perfectly synchronized with the recipient oocyte, but also the chromatin is in direct contact of cytoplasmic factors, without the barrier of a nuclear membrane. The bovine species does not present such a clear-cut situation. The delayed activation (12, 94) seems to be as effkient as a simultaneous fusion-activation (85). Moreover, the use of donor nuclei in metaphase did not improve development to the blastocyst stage, thus suggesting that direct and immediate exposure of chromatin to M-II cytoplasm does not ensure a better remodeling than exposure of chromatin maintained under the nuclear envelop (Tablel).

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When examining the chromatin and nuclear envelope of tibroblast nuclei after fusion into M-II cytoplasts, we observed that only a minority of nuclei underwent PCC (18% ; n =57); most of them apparently were intact. We obtained up to 30% in vitro development to the blastocyst stage and have had 16 calves born with this procedure. This indicates that a prolonged chromatin condensation may not be an essential step in somatic cell nuclear reprogrammation in the bovine and confirms that reprogramming of somatic cell nuclei was possible when using a recipient oocyte that does not induce PCC (84). An opposite situation has recently been described with cumulus cells as a source of nuclei, suggesting that other environmental parameters may interact with either the cytoplast contents or the donor nucleus (82). Nuclear Remodeling and Memory of Cell Fate Chromatin is a highly dynamic structure that is continuously remodeled and modified in response to developmental and physiological signals. The building blocks of chromatin, nucleosomes, ensure the correct packaging of DNA around small basic proteins, the histones, for the control of gene expression (97), replication, and chromosome segregation (22). Two kinds of chromatin remodeling activities exist in all eukaryotic nuclei: ATP-dependent chromatin remodeling and covalent modifications of the histone NH2-terminus that protrudes from the nucleosome. The packaging of DNA can be altered by energy- (ATP-) dependent cellular machineries, such as the multiprotein SWI/SNF complex, initially identified both by genetics and biochemistry studies in yeast (review: 79). In mammalian cells, two proteins closely related to this complex, Brm (brahma) and Brgl, are required for transcriptional regulation. Several lines of evidence indicate that these proteins play a specific role in the regulation of cell growth through interactions with the Retinoblastoma Rb protein (review: 59), a major cell cycle regulator that controls the GliS transition as well as progression through S phase. Inactivation of the mouse Brgl gene is lethal before implantation; mutant mice for Brm are viable and fertile but exhibit a 10 to 15% increase in their body mass relative to wild-type littermates (70). This marked and unexpected difference in the phenotype of the respective null mutants is probably related to a different role of Brm and Brgl in chromatin remodeling throughout the preimplantation stages. This is supported by our previous observation that the Brgl protein is present in large amounts during that period, whereas Brm protein levels drop markedly at the four cell-stage, become barely detectable at the eight-cell and morula stages, and start to be reexpressed at the blastocyst stage but only in the inner cell mass and not in the trophoblast (53). More recently, we provided evidence that Brm expression during development is first restricted to mesodermal tissues involved in early vasculogenesis and heart morphogenesis while Brgl remains ubiquitously expressed (17). Early vasculogenesis is an essential process to the establishment of functional relationships between the embryo and the uterus. The fact that deficiencies in chorioallantoic vascularization are frequently observed during the gestational period of cloned sheep (21) and cattle (our unpublished observations) could be considered a direct consequence of some earlier defective action of the SWVSNF ATPase on the remodeling of a foreign chromatin. Because SWVSNF is required to induce short stretches of nucleasehypersensitive DNA in gene promoters (30), comparative analysis of nuclease-hypersensitive sites present on candidate promoters from normal and cloned embryos could offer an experimental means of detecting primary alterations in ATP-dependent chromatin remodeling.

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The fact that the SWI/SNF complex is particularly important for those genes that are transcribed in late anaphase, when the mitotic condensation of chromatin is still not fully reversed (46), could help with screening. Acetylation is the covalent modification of histone NH2-termini that has been extensively characterized. Its main effects are to make chromatin accessible to DNA-binding proteins by destabilizing the nucleosome and the chromatin fiber (52). Thus, histone acetylation provides a critical link between chromatin structure and transcriptional output. We previously have shown that the hyperacetylated pool of histone H4 present in mouse oocytes at fertilization is linked to paternal but not maternal chromatin, whereas maternal chromatin became hyperacetylated in parthenogenetically activated oocytes (1). This observation is in concordance with the fact that, immediately after fertilization, the maternal pronucleus is more transcriptionally repressive than the paternal one (2). How the pattern of histone acetylation is affected after somatic cell nuclear transfer is still not known but could clearly depend on the strategy used for embryo reconstruction. Manipulation of the acetylation status of nuclei before their transfer into enucleated cytoplasts (for instance an induced global hyperacetylation through exposure of donor cells to the deacetylase inhibitor Trichosatin A) could provide a means of analyzing how a specific and early perturbation of chromatin could affect the expression of genes further along in development. EPIGENETIC REPROGRAMMING

OF DONOR DNA AFTER NUCLEAR TRANSFER

Epigenetic information provides instructions on how, where, and when the genetic information should be used. They do not involve changes to the DNA code and can persist through one or more generation. Chromatin remodeling induced by nuclear transfer is frequently associated with some deregulation of gene expression and recent observations point to the importance of DNA methylation in this process. Aberrant Gene Expression in Embryos Cloned with Somatic and Embryonic Cells A complete reprogramming of a somatic cell nucleus by the recipient cytoplast would result in an embryo with a similar profile of gene transcription as that seen in vivo or even after IVF. Expression profiles of individual genes provide a primary evaluation on the extent to which the donor genome is correctly reprogrammed. It has recently been shown, for instance, that transcription of several genes coding for growth factors and cytokines can be persistently modified in bovine nuclear transfer embryos (16) whereas other metabolic genes taken as candidates to explain abnormal fetal development were seen to be expressed at normal levels (96). Moreover the embryonic pattern of transcription can be affected not only by the nuclear transfer technique itself but also by the culture conditions used to carry the reconstructed embryos (60, 98). For instance, LOS is often attributed to the culture system and more specifically the presence of serum. It has been demonstrated recently that not only differences in culture medium (24), but serum supplementation (44) and serum starvation (S Baqir and L Smith, personnal communication) can result in aberrant expression of imprinted genes, which is associated more often with alteration in the methylation pattern. Although these gene-specific approaches are informative, they will rapidly become tedious because the expression profiles of many candidate genes are probably simultaneously (and differently from one embryo to the other) affected upon nuclear transfer.

Theriogenology

More global measurements of epigenetic reprogramming associated with nuclear transfer have started to be used to characterize the “switching off’ of those genes transcribed in the donor somatic cell but not normally transcribed in early embryonic development (20). Recent advances in genomics will however probably allow exhaustive comparisons of the expression profile of thousands of transcripts expressed from normal or nuclear transfer embryos. This is a challenging task which, until recently, has been hampered by two main obstacles: the lack of species-specific cDNA collections at appropriate developmental stages and the amount of starting biological material available with mammalian embryos. Concerning the first point, two arrayed cDNA collections have recently been established in cattle (76, 83). The first is drawn from pooled cDNA libraries of bovine tissues or organs, but the representation of embryonic stages is rather poor. The second has been established from a single somatic cell nuclear transfer-derived fetus at 60 days of age (83). As for the limited amount of material available within embryos, the obstacle is that the RT-PCR procedures designed for a whole amplification of the initial population of cDNAs creates distortion in the relative prevalence of transcripts, which rapidly impairs further analysis. Technical improvements of classical methods (69 and our recent unpublished results), together with new strategies designed to amplify or subtract the starting material (5, 66, 91) may represent major technical advances. Strategies in which the array sizes are reduced will also allow (up to a certain extent) the use of non-amplified material (7). A network of laboratories is now establishing new cDNA collections obtained from early bovine embryos. The objective is to identify novel genes involved in early bovine embryogenesis and to make this information available to the scientific community (http://www.cstinc.com/begc). The outcome of this collaboration will be a genome-wide profiling of gene expression during bovine embryonic development. From there, transcript maps could be established for global analysis of bovine nuclear reprogramming. This approach will also allow researchers to define the appropriate culture conditions for donor cells without the restriction imposed by the choice of candidate genes (81). Time-Dependent

Methylation Events in Cloned Embryos

A molecular hypothesis that is put forward to explain the common mechanisms underlying growth and other abnormalities associated with nuclear transfer is DNA methylation. This DNA methylation is one of the best-studied epigenetic modifications in all organisms that are associated with compact and inactive chromatin stucture (8). Mammalian somatic cells show elevated DNA methylation levels with low methyltransferase activity (54, 58), whereas embryonic cells and gametes are less methylated, and sperm are more methylated than oocytes (35). As a consequence, the genome of the embryo is hypomethylated and subsequently undergoes global de novo methylation (review: 67). Using antibodies against Smethylcytosine, we observed that nuclear transfer bovine preimplantation embryos fail to reproduce the distinguishable parental chromosome methylation pattern observed with normal (in vitro produced) embryos; rather, they maintain their somatic pattern during several cleavages before chromosomes become undermethylated on euchromatin (9). Aberrant methylation profiles of specific genomic regions of nuclear transfer embryos have also been evidenced in cattle (37) and could thus be considered a consequence of the abnormal timing of global chromatin methylation that we have evidenced. The onset of zygotic transcription must be delayed for chromatin remodeling during normal development and a complex process of translational recruitment of maternal messengers exquisitely controls the

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occurrence of this event (91). Because time-dependent DNA methylation is involved in a critical layer of control for enhancing transcriptional silencing (8), its time-dependant variations may play a determinant role in setting up the correct pattern of gene activities after nuclear transfer. Transient changes in the localization of the methyltransferase, Dnmtl , during the four-cell stage of mouse embryos and nucleocytoplasmic trafficking of Dnmts during mouse preimplantation development (71) confirm the importance of these time-dependent events. From a practical point of view, the above approaches could constitute a means to screen for the nuclear transfer techniques that would mimic the epigenetic remodeling occurring in normal embryos. Quantifications of global or specific patterns of DNA methylation could, for instance, be associated with manipulations of calcium transients at egg reconstruction using procedures shown to generate long-lasting effects on development (64) or with a prolonged exposure of donor nuclei to a zygotic cytoplast through re-cloning. However, DNA methylation does not interfere directly with transcription but rather marks the DNA, creating a repressive state in association with histone deacetylases (HDs) and chromatin remodeling machineries (review: 72). In fact, the two epigenetic processes were brought together recently by the identification of MeCP2, a methyl binding protein that recruits HDs through a co-repressor (Sin3A), creating a transcriptionaly silenced complex (37). Given the biological nature of imprinted gene expression, which is frequently correlated with the methylation status of their prompters, the interaction between both silencing mechanisms (DNA methylation and histone deacetylation) is somehow inevitable. This compounds an additional difficulty to the molecular analysis of functional changes in chromatin structure. Moreover, widespread dysregulation of gene expression has now been observed in cloned mice even when surviving to adulthood (36). This has lead to the conclusion that mammalian development may be rather tolerant to epigenetic abnormalities. Finally, in that work, a high variability in imprinted gene expression was observed between embryos, but no correlation could be established with their abnormal development (36). Imprinted gene expression is highly dependent on methylation. Thus, extensive characterization of the methylation status at several loci will probably confirm the epigenetic instability of nuclear transfer embryos. IMPORTANCE OF DONOR CELLS FOR SUCCESSFULL CLONING Donor Cells and Development of Nuclear Transfer Embryos Various differentiated cell types have been used as sources of nuclei for cloning domestic and laboratory animals. Cumulus and granulosa cells have, until now, extensively been used in mice, cattle, and pigs (65, 86, 94). Almost all cell types tested so far have resulted in live offspring, although with great differences in their cloning efficiency. These differences are frequently observed between subcultures (batches) derived from the same biopsy (thus from the same genotype) as shown in Table 2A for the bovine species. Differences have also been evidenced between mouse embryonic cell lines and between clonal cell lines of tibroblasts derived from the same pig fetus (36, 49). A recent report in the mouse shows that ES cell nuclear transfer blastocysts can develop to term at a lo- to 20-fold higher efficiency than those from somatic cells (25). This suggests that undifferentiated embryonic cells might serve as more effective nuclear donors than somatic cells because their epigenetic state may more closely resemble that of early embryos. In this work, however, there was no close synchrony between donor and recipient cell cycles at embryo

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reconstruction. Using donor chromatin at the metaphase stage, we found (Table 2B) that ES cell nuclear transfer can develop into blastocyts at a 1O-fold higher efficiency than somatic cells, but their subsequent development into fetuses was about 3-fold lower. Thus, conclusions on the reprogramming requirements of ES cells should be considered cautiously. As mentioned previously, ES cells have a high methylation activity, whereas that of somatic cells is low or undetectable (54). To what extent the extensive demethylation of chromatin that occurs during preimplantation development interferes with the remethylation of the genome during postimplantation stages remains to be determined. Several other factors may also be involved. Donor Cell Age. No clear relationship exists between the age of adult donors and the potential of development to term of the reconstructed embryos. This is well exemplified by the successful cloning achieved with a 2 1-yr-old Brahman bull and a 17-yr-old Japanese Black Beef bull (33, 48). Although the age of donors can affect the growth of cells in vitro, it does not affect the potential of reconstructed embryos for development to the blastocyst stage (34, 42) or to term (Table 2A). The in vivo development of embryos, however, is generally higher when derived from fetal instead of adult cells (3 1, 42) Cell Cvcle Stage. A GO-induced cell cycle phase of donor cells, initially considered as a requirement for a successful cloning (95) is today widely used before embryo reconstruction (3, 40, 86, 94, 102). The GO stage that is generally induced by serum starvation of donor cells has, however, apparently no positive effect on the developmental potential of nuclear transfer embryos in cattle, pigs, and even mice (12, 48, 63). In cattle, no difference in the rate of blastocysts obtained either with serum or non-serum starved cells could be evidenced by several groups (33,48, 85). Published data in that species rather suggest that serum starvation results in a higher rate of blastocyst development only when fetal cells are used as sources of nuclei (33, 102). This could be related to a higher sensibility to contact inhibition and a longer cell cycle for cultures derived from adult tissues (our unpublished observations). As a consequence, a higher proportion of cells are found in the Gl phase, even when cultures are at low confluence (39).

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Table 2. Developmental genotype

potential

of embryos reconstructed

from donor cells of the same

A. Cattle. Fibroblasts obtained from the same donor but from different biopsies and subcultures; fetus examined at Day 35 of pregnancy Type of donor cell (number of passages) Biopsy at 9 month (passages 4 - 8) Subculture A (passage 4) Subculture B (passages4 -8) Subculture C (passages 4- 8) Subculture D (passages 6-7) Biopsy at 12 month (passages 4 -8) Biopsy at 18 month @assages4-8)

Blastocysts per embryo reconstructed 39/217 (17.9)

Fetuses per embryo transferred 13/32 (40.6)

Live young per embryo transferred 2 I32 (6.2)

2/26 (7.7)

l/2 (50.0)

0

4/76 (5.3)

3/4 (75.0)

l/4 (25.0)

20167 (29.8)

5/19 (26.3)

0

13/48 (27.1)

4/7 (57.1)

l/7 (14.2)

43/99 (43.4)

4/24 (16.6)

l/24 (4.1)

114/452 (25.2)

17/58 (29.3)

3 (5.1)

B. Mouse. ES cells and adult fibroblasts from the same genotype (129ISvPas); embryos reconstructed from metaphase nuclei and transferred at the two-cell stage; foetus examined at Day 7.5 of pregnancy Typeof donor cell (No. of passages)

Blastocysts (%) per embryo reconstructed

Implantationsites per embryo transferred

Fetuses per:

ES

54008 (50.0)

38/221 (17.1)

Embryo transferred 8/221 (3.6)

(passages 12-14) Fibroblasts (passages 2-4)

4176 (5.3)

35/402 (8.7)

51402 (1.2)

Blastocyst (%)* (7.2) (22.6)

*Estimation from the blastocyst rate in vivo (transient fosters). Time in Culture. Freshly isolated cells or cells from primary cell cultures are generally used as donor nuclei. An established bovine epithelial cell line provided donor nuclei totally unable to direct embryonic development as compared with those derived from primary culture of the same tissue (mammary gland, 101). The cloning competence of donor cells did not appear to be compromised after culture of fibroblasts over 15 passages (48, our unpublished observations). This is in agreement with our observation that cells from adult skin had remarkably stable karyotypes even when kept in culture for 40 cell doublings (about 15 passages). Mammalian cells in vitro are, however, prone to undergo mutations, loss of imprints, and alteration in methylation status as shown recently with mouse ES-cells from the same sub-clone (36) and a dramatic decrease (about 40%) in the expression of imprinted genes at passages above 36 (S Baqir and L Smith, personnal communication). This may contribute to the drop in implantation

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observed when mouse nuclear transfer embryos are derived from ES-cells used after more than 19 passages (103). Manipulation of Cells During Culture. Controlled genetic modifications of farm animals through targeted gene insertion will have a profound impact on selection and breeding. This objective has recently been brought to reality with sheep by combining in vitro manipulations of donor cells (for targeted insertion at the alpha-l procollagen locus) with nuclear transfer (57). In this work, 14 live-born lambs were produced of which 7 died 30 hrs after birth and 4 within the following 3 months. This very high rate of postnatal mortality could result from the extensive manipulations of donor cells required for targeting a given gene. These manipulations would rapidly compromise the reprogramming potential of nuclei. Recent results, however, suggest a more complex situation (19). This group has successfully inactivated the alpha-( 1,3)-galactosyl transferase (GGTAl) and the prion (PrP) genes also in the sheep: from 120 embryos transferred, 4 animals were born, but 3 died soon after birth and the remaining one after 12 days. These failures could reflect the fact that each of these genes is required for normal fetal development. Such an interpretation should, however, be considered with caution because, in the mouse, the same null mutations have been shown to lead to viable offspring. Moreover, when the authors used as controls the same source but of non-transfected cells (that they had previously submitted to the same culture conditions), they also ended up with negative result. Thus, the association of in vitro manipulation of donor cells with nuclear transfer for controlled genetic modification appears to be highly dependent on specific but fortuitous combination of donor cell genotype, culture conditions (media, concentration of cells at each passage), and cell manipulation (selection pressure exerted on the cell population during screening for genetic recombination). This is to some extent confirmed by results in the mouse, where heterozygocity of several donor genotypes was shown to lead to high postnatal death of clones, whereas long-term in vitro culture or gene targeting performed on another donor genotype did not (25). Definition of the cellular and molecular basis required for such a successful combination will probably remain a tremendous challenge for at least the coming decade. Improvement of Nuclear Reprogramming Through Pre-Treatment of Donor Cells Would it be possible to make the somatic nucleus easier to reprogram following its introduction into the recipient oocyte? In experiments using Xenopus egg extracts, there are several example of reactivation of DNA replication into nuclei from terminally differentiated cells (55, 92); although all nuclei undergo an apparent similar remodeling (decondensation, importation of nuclear proteins, accumulation of lamin), initiation of replication is observed only for those nuclei that are previously permeabilized before exposure to extracts (55). An indirect proof of successful reprogramming would thus be to assess the pattern of DNA replication after the transplantation of a decondensed nucleus into enucleated cytoplasm. When in culture, a large proportion of mammalian adult somatic cells are not cycling because of contact inhibition (39). Using serum starved cattle tibroblasts we observed that reversion of this GO-like stage made cells re-enter a new cycle only after 16 to 18h of switching to a serum-fed condition. Taking this into account, we designed a series of experiments in which nuclei were submitted, prior to nuclear transfer, to a moderate protease treatment associated with the permeabilitization of the donor cell membrane. Improvement for both in vitro and in vivo development of embryos reconstituted with these donor cells was obtained for three replicate experiments performed with two different genotypes (Table 3). Although the cellular and

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molecular effects of such a treatment remains to be characterized, our preliminary observations indicate that some changes in the pattern of replication of those treated nuclei do occur that somewhat positively affect their functional reorganization upon introduction into recipient oocytes. Reprogramming of somatic cell chromatin involves a tremendous exchange of components that affect replication (23, 45, 56). In our experiment, pre-treatment may prepare nuclei to improve their reprogramming efficiency by somatic factors because we transfer them into aged or pre-activated oocytes where they remain for hours in an uncondensed state (82,84). Table 3. Effect of treatment of donor fibroblast on development of nuclear transfer embryos in bovine Donor cells Treated Not treated Total

No. of blastocystes transferred (% of reconstruct.) 56 (24.1) 52 (16.3) 108 (19.6)

No. of pregnanciesiNo. W) Day 35 Day 60 16138 15138 (42.1) (39.5) 101.36 8/36 (27.8) (22.2) 26174 23174 (35.1) (31.1)

of recipients No. of offspring W) Day ~90 7138 4 (18.4) (7.1) 4136 (11.1) f3.8) 1l/74 (14.9) ps.5)

CONCLUSION A major limitation to the development of nuclear transfer technologies today is the occurrence of long-lasting effects that frequently alter the viability and affect the welfare of somatic cell clones. The nuclear transfer procedure itself appears to be only partially responsible for these developmental failures, and there is now growing evidence that defective gene expression may result from epigenetic changes affecting the donor cells. Cytosolic extracts obtained from mammalian cells provide an elegant means for dissecting the cell cycle-dependent biochemical mechanisms of somatic cell nuclear remodeling (77). Meanwhile, manipulating the post-transcriptional regulation of cells (for instance their methylation status) will probably help to unravel the mechanisms of epigenetic remodeling of a foreign chromatin by the surrounding cytoplasm. However, cloning requires, at least at present, the exposure of a foreign nucleus to the cytoplasm of an enucleated oocyte. Because of technical difficulties mainly related to the low amount of material available, the specific role exerted by oocyte cytoplasm on nuclear reprogramming remains largely unknown. Although post-transcriptional regulations in most somatic cells are generally complementary to transcriptional control of gene expression, the distinctive feature of an oocyte’s cytoplasm is to simultaneously manage post-transcriptional regulation of genes expressed in one organism (the mother where oogenesis takes place) together with the transcriptional control of a genetically different organism (the embryo). These events are tightly time-dependent. For instance, the activation of the embryonic genome is progressive and should neither occur too early, because it would result in the random expression of genes, the structural regulation of which is still not definitive, nor too late because zygotic transcripts have to take over the maternal transcripts and proteins (91). This makes the oocyte perhaps a unique cell for ensuring the full reprogramming of a somatic cell nucleus.

The new approaches of genomics associated with induced targeted mutation through transgenesis will allow an integrated analysis of the multilevel regulations exerted by the oocyte on a foreign chromatin. A growing number of physiologically normal ruminants and rodents obtained from somatic cell nuclear transfer are now present in different laboratories all over the world. They testify that cloning can be innocuous to animals. Bringing this technology to routine practice will, however, require greater knowledge and understanding of the basic biological processes underlying epigenetic control of nuclear activities. An important issue at present is to limit the production of aberrant phenotypes that may result in significant insult to the nature and welfare of animals. This would probably require the adoption of safeguards by the scientific community to overcome the social reluctance to animal cloning largely dominant today in many countries.

REFERENCES

1.

2. 3.

4. 5. 6.

7.

8. 9.

Adenot PG, Szollosi MS, Chesne P, Chastant S, Renard JP. In vivo aging of oocytes influences the behaviour of nuclei transferred to enucleated rabbit oocy-tes. Mol Reprod Dev 1997;46:325-336. Aoki F, Worrad DM, Schultz RM. Regulation of transcriptional activity during the first and second cell cycles in the preimplantation mouse embryo. Dev Biol 1997; 18 1:296-307. Baguisi A, Behboodi E, Melican DT, Pollock JS, Destrempes MM, Cammuso C, Williams JL, Nims SD, Porter C, Midura P, Palacios MJ, Ayres SL, Denniston RS, Hayes ML, Ziomek CA, Meade HM, Godke RA, Gavin WG, Overstrom EW, Echelard Y. Production of goats by somatic cell nuclear transfer. Nature Biotech 1999; 17:456-46 1. Barker DJ and Clark PM. Fetal undernutrition and disease in later life. Rev Reprod 1997;2:105-112. Baugh LR, Hill AA, Brown EL, Hunter CP. Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 2001;29:E29. Behboodi E, Anderson GB, Bondurant RH, Cargill SL, Kreuscher BR, Medrano JF, Murray JD. Birth of large calves that developed from in vitro-derived embryos. Theriogenology 1995;44:227-232. Bertucci F, Houlgatte R, Benziane A, Granjeaud S, Adelaide J, Tagett R, Loriod B, Jacquemier J, Viens P, Jordan B, Bimbaum D, Nguyen C. Gene expression profiling of primary breast carcinomas using arrays of candidate genes. Hum Mol Genet 2000;12:9:2981-2991. Bird AP, Wolffe AP. Methylation-induced repression--belts, braces, and chromatin. Cell 1999; 99:45 1-454. Bourc’his D, Le Bourhis D, Patin D, Niveleau A, Comizzoli P, Renard J-P, ViegasPequignot E. Delayed reprogramming of chromosome methylation patterns in bovine cloned embryos. Curr Biol, 2001 (in press).

Theriogenology

10. Campbell KHS, Loi P, Otaegui PJ, Wilmut I. Cell cycle co-ordination

11. 12.

13. 14.

15. 16. 17.

18.

19.

20. 21. 22. 23.

24.

25.

26. 27.

217

in embryo cloning by nuclear transfer. Rev Reprod 1996; 1:40-46. Campbell K, MC Whir J, Ritchie W, Wilmut I. Sheep cloned by nuclear transfer from a cultured cell line. Nature 1996;380:64 - 66. Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce de Leon FAP, Rob1 JM. Cloned transgenic calves produced from nonquiescent fetal fibroblast. Science 1998;280:1256-1258. Collas P and Rob1 JM. Relationship between nuclear remodelling and development in nuclear transplant rabbit embryos. Biol Reprod 1991;45:455-465. Collas P, Pinto-Corriea C, Ponce de Leon FA, Rob1 JM. Effect of donor cell cycle stage on chromatin and spindle morphology in nuclear transplant rabbit embryos. Biol Reprod 1992;46:501-511. Czolowska R, Sziilliisi D, Szolliisi MS. Changes in embryonic 8-cell nuclei transferred by means of fusion to mouse eggs. Int J Dev Biol 1992; 36:543-553. Daniels R, Hall V, Trotmson AO. Analysis of gene transcription in bovine nuclear transfer embryos reconstructed with granulosa cell nuclei. Biol Reprod 2000;63: 1034-1040. Dauvillier S, Ott MO, Renard JP, Legouy E. BRM (SNF2alpha) expression is concomitant to the onset of vasculogenesis in early mouse postimplantation development. Mech Dev 2001;101:221-225. Dean W, Bowden L, Aitchison A, Klose J, Moore T, Meneses JJ, Reik W, Feil R. Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: Association with aberrant phenotypes. Developmentl998; 125:2273-2282. Denning C, Burl S, Ainslie A, Bracken J, Dinnyes A, Fletcher J, King T, Ritchie M, Ritchie WA, Rollo M, De Souza P, Travers A, Clark, J. Deletion of the alpha (1,3) galactosyl transferase (GGTAl) gene and the prion protein (PrP) gene in sheep. Nature Biotech 2001;19:559-562. De Sousa PA, Winger Q, Hill JR, Jones K, Watson A, Westhusin ME. Reprogramming of tibroblast nuclei after transfer into bovine oocytes. Cloning 1999; 1:63-69. De Sousa PA, King T, Harkness L, Young LE, Walker SK, Wilmut I. Evaluation of gestational deficiencies in cloned sheep fetuses and placentae. Biol Reprod 2001;65:23-30. Demeret C, Vassetzky Y, Mechali M. Chromatin remodelling and DNA replication: From nucleosomes to loop domains. Oncogene 2001; 20:3086-3093. Dimitrov S, Wolffe AP. Remodeling somatic nuclei in Xenopus laevis egg extracts: Molecular mechanisms for the selective release of Histone Hl and Hl’ from chromatin and the acquisition of transcriptional competence. EMBO J 1996;15:5897-5906. Doherty AS, Mann MR, Tremblay KD, Bartolomei MS, Schultz RIM. Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo. Biol Reprod 2000; 62:1526-1535. Eggan K, Akutsu H, Loring J, Jackson-Grusby L, Klemm M, Rideout WM, Yanagimachi R, Jaenisch R. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. PNAS 2001; 98:6209-6214. Farin PF, Farin CE. Transfer of bovine embryos produced in vivo or in vitro: Survival and fetal development. Biol. Reprod. 1995;52:676-682. Fulka J, First NL, Moor RM. Nuclear transplantation in mammals. Remodeling of transplanted nuclei under the influence of maturation promoting factor. BioEssays 1996;18:835-840.

218

28.

29. 30.

31.

32.

33.

34.

35. 36.

37

38. 39.

40 41.

42. 43.

Theriogenology

Gall L, Dedieu T, Chesne P, Ruffini S, Sevellec C, Peynot N, Renard JP, Heyman Y, Bovine embryo cloning: Characterization of the recipient cytoplasts by phosphorylation patterns and kinase activities. Develop Growth Differ 1996;38:517-525. Galli C, Duchi R, Moor R, Lazzari G. Mammalian leucocytes contain all the genetic information necessary for the development of a new individual. Cloning 1999; 1: 16 1- 170. Gregory PD, Schmid A, Zavari M, Mtinsterkijtter M and Wolfram H&z. Chromatin remodelling at the PH08 promoter requires SWI-SNF and SAGA at a step subsequent to activator binding. EMBO J 1999; 18:6407-64 14. Heyman Y, Chavatte-Palmer P, LeBourhis D, Camous S, Vignon X, Renard JP. Frequency and occurrence of late gestation losses from cattle cloned embryos. 2002; Biol. Reprod (in press). Hill JR, Roussel AJ, Cibelli JB, Edwards JF, Hooper NL, Miller MW, Thompson JA, Looney CR, Westhusin ME, Rob1 JM, Stice SL. Clinical and pathological features of cloned transgenic calves and fetuses (13case studies). Theriogenology 1999;5 1: 145 1- 1466. Hill JR, Winger Q, Long C, Looney C, Thompson J, Westhusin ME. Development rates of male bovine nuclear transfer embryos derived from adult and fetal cells. Biol. Reprod 2000;62:1135-1140. Hill JR, Burghard RC, Jones K, Long C, Looney C, Shin T, Spencer T, Thompson J, Winger Q, Westhusin ME. Evidence for placental abnormality as a major cause of mortality in first-trimester somatic cell cloned bovine fetuses. Biol. Reprod 2000;63: 17871794. Howlett SK, Reik W. Methylation levels of maternal and paternal genomes during preimplantation development. Development 1991; 113 : 119-27. Humpherys D, Eggan K, Akitsu H, Hochedlinger K, Rideout WM, Biniszkiewicz D, Yanagimachi R, Jaenish R. Epignetic instability in ES cells and cloned mice. Science 2001; 293:95-97. Jones PL, Veenstra GJ, Wade PA, Vermaak D, Kass SU, Landsberger N, Strouboulis J, Wolffe AP. Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription Nat Genet 1998; 19: 187-l 9 1. Kang YK, Koo DB, Park JS, Choi YH, Chung AS, Lee KK, Han YM Aberrant methylation of donor genome in cloned bovine embryos. Nat Genet 200 1;28: 173- 177. Kasinathan P, Knott JG, Moreira P, Burnside A, Jerry DJ, Rob1 JM. Effect of fibroblast donor cell age and cell cycle on development of bovine nuclear transfer embryos in vitro. Biol Reprod 2001; 64:1487-1493. Kato Y, Tani T, Sotomaru Y, Kurokawa K, Kato J, Doguchi H, Yasue H, Tsunoda Y. Eight calves cloned from somatic cells of a single adult. Science 1998;282:2095-2098. Kato Y, Yabuuchi A, Motosugi N, Kato J, Tsunoda Y. Developmental potential of mouse follicular epithelial cells and cumulus cells after nuclear transfer. Biol Reprod 1999;61:1110-1114. Kato Y, Tani T, Tsunoda Y. Cloning of calves from various somatic cell types of male and female adult, newborn and fetal cows. J Reprod Fertil2000;120:231-237. Keefer CL, Baldassare H, Keyston R, Wang B, Bhatia B, Bilodeau AS, Zhou JF, Leduc M, Downey BR, Lazaris, Karatzas C.N. Generation of dwarf goat (Capra hircus) clones following nuclear transfer with transfected and non transfected fetal fibroblasts, and in vitro-matured oocytes. Biol Reprod 2001;64:849-856.

Theriogenology

44.

45. 46. 47. 48.

49.

50. 51.

52. 53. 54.

55.

56.

57.

58.

59. 60.

219

Khosla S, Dean W, Brown D, Reik W, Feil R. Culture of preimplantation mouse embryos affects fetal development and the expression of imprinted genes. Biol Reprod 2001; 4:9 18926. Kikyo N, Wolffe AP. Reprogramming nuclei: Insights from cloning, nuclear transfer and heterokaryons. J Cell Sci 2000; 113: 1 I-20. Krebs JE, Fry CJ, Sarnuels ML, Peterson CL. Global role for chromatin remodeling enzymes in mitotic gene expression. Cell 2000;102:587-598. Kruip TAM, den Daas JHG. In vitro produced cloned embryos: Effects on pregnancy, parturition and offspring. Theriogenology 1997; 278:2 130-2 133. Kubota C, Yamakuchi H, Todoroki J, Mizoshita K, Tabara N, Barber M, Yang X. Six cloned calves produced from adult fibroblast cells after long-term culture. Proc. Natl. Acad. Sci. 2000;97:990-995. Ktihholzer B, Hawley RJ, Lai L, Kolber-Simonds D, Prather RS. Clonal lines of transgenic fibroblast cells derived from the same fetus result in different development when used for nuclear transfer in pigs. Biol Reprod 2001; 64:1695-1698. Kwon OY and Kono T. Production of identical sextuplet mice by transferring metaphase nuclei from four-cell embryos. Proc Nat1 Acad Sci USA 1996;93:13010-13013. Lacham-Kaplan 0, Diamente M, Pushett D, Lewis I, Trounson A. Developmental competence of nuclear transfer cow oocytes after direct injection of fetal fibroblast nuclei. Cloning 2000;2:55-62. Lee DY, Hayes JJ, Pruss D, Wolffe AP. A positive role for histone acetylation in transcription factor access to nucleosomal DNA. Cell 1993;72:73-84. Legouy E, Thompson EM, Muchardt C, Renard JP. Differential preimplantation regulation of two mouse homologues of the yeast SW12 protein. Dev Dyn 1998;212:38-48. Lei H, Oh SP, Okano M, Juttermann R, Goss KA, Jaenisch R, Li E. De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells. Development 1996;122:3195-3205. Leno G, Munshi R. Reactivation of DNA replication in nuclei from terminally differentiated cells: Nuclear membrane permeabilization is required for inititaion in Xenopus egg extract. J Exp Cell Res 1997;232:412-419. Lu ZH, Sittman DB, Brown DT, Munshi R, Leno GH. Histone Hl modulates DNA replication through multiple pathways in Xenopus egg extract. J Cell Sci 1997; 110:27452758. 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;405:1066-1069. Monk M, Boubelik M, Lehnert S. Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development 1987; 99:371-382. Mutchardt C and Yaniv M. ATP-dependent chromatin remodelling: SWI/SNF and Co. are on the job. J Mol Biol 1999; 293:187-198. Nieman H, Wrenzycki C. Alteration of expression of developmentally important genes in preimplantation bovine embryos by in vitro culture conditions: implications for subsequent development. Theriogenology 2000; 53:21-34.

220

61.

62. 63. 64. 65.

66.

67. 68. 69 70.

71.

72. 73.

74.

75.

76

77.

Theriogenology

Ogura A, Inoue K, Ogonuki N, Noguchi A, Takano K, Nagano R, Suzuki 0, Lee J, Ishino F, Matsuda J. Production of male cloned mice from fresh, cultured, and cryopreserved immature Sertoli cells. Biol. Reprod 2000; 62:1579-1584. Onishi A, Iwamoto M, Akita T, Mikawa S, Takeda K, Awata T, Hanada H, Perry AC. Pig cloning by microinjection of fetal fibroblast nuclei. Science 2000;289:1188-1190. Ono Y, Shimozawa N, Ito M, Kono T. Cloned mice from fetal fibroblast cells arrested at metaphase by a serial nuclear transfer. Biol Reprod 2001;64:44-50. Ozil JP, Huneau D Activation of rabbit oocytes: The impact of the Ca2+ signal regime on development. Development 2001;128:917-928. Polejaeva IA, Chen SH, Vaught TD, Pag RL, Mullins J, Ball S, Dai Y, Boone J, Walker S, Ayares D, Colman A, Campbell KH. Clone pigs produced by nuclear transfer from adult somatic cells. Nature 2000;407:86-90. Rast JP, Amore G, Calestani C, Livi CB, Ransick A, Davidson EH. Recovery of developmentally defined gene sets from high-density cDNA macroarrays. Dev Biol 2000;228:270-286. Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development Science 2001;293:1089-1093. Renard JP, Chastant S, Chesne P, Richard C, Marchal J, Cordonnier N, Chavatte P, Vignon X. Lymphoid hypoplasia and somatic cloning. The Lancet 1999;353:1489-1491. Revel F, Renard JP, Duranthon V. PCR-generated cDNA libraries from reduced numbers of mouse oocytes. Zygote 1995;3:241-250. Reyes JC, Muchardt C, Yaniv M. Components of the human SWI/SNF complex are enriched in active chromatin and are associated with the nuclear matrix. J Cell Biol 1997;137:263-274. Rougier N, Bourc’his D, Gomes DM, Niveleau A, Plachot M, Paldi A, Viegas-Pequignot E. Chromosome methylation patterns during mammalian preimplantation development Genes Dev 1998;15:2108-2113. Rountree MR, Bachman KE Herman JG, Baylin SB. DNA methylation, chromatin inheritance, and cancer. Oncogene 2001;20:3156-3165. Sasser RG, Ruder CA, Ivani KA, Butler JE, Hamilton WC. Detection of pregnancy by radioimmunoassay of a novel pregnancy-specific protein in serum of cows and a profile of serum concentrations during gestation. Biol Reprod 1986;35:936-942. 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 Iibroblasts. Science 1997;278:2130-2133. Sinclair KD, McEvoy TG, Maxfield EK, Maltin CA, Young LE, Wilmut I, Broadbent PJ, Robinson JJ. Aberrant fetal growth and development after in vitro culture of sheep zygotes. J Reprod Fertil 1999;116:177-186. Smith TP, Grosse WM, Freking BA, Roberts AJ, Stone RT, Casas E, Wray JE, White J, Cho J, Fahrenkrug SC, Bennett GL, Heaton MP, Laegreid WW, Rohrer GA, ChitkoMcKown CG, Pertea G, Holt I, Karamycheva S, Liang F, Quackenbush J, Keele JW. Sequence Evaluation of Four Pooled-Tissue Normalized Bovine cDNA Libraries and Construction of a Gene Index for Cattle. Genome Res 200 1; 11:626-630. Steen RL, Martins SB, Tasken K, Collas P. Recruitment of protein phosphatase 1 to the nuclear envelope by A-kinase anchoring protein AKAP149 is a prerequisite for nuclear lamina assembly. J Cell Biol 2000;150:1251-1262.

Theriogenology

78. 79. 80.

81.

82.

83.

84.

85.

86.

87. 88. 89. 90.

91.

92.

93

94.

221

Stice SL, Strelchenko NS, Keefer CL, Matthews L. Pluripotent bovine embryonic cell lines direct embryonic development following nuclear transfer. Biol Reprod 1996;54: 100-l 10. Sudarsanam P, Winston F. The Swi/Snf family nucleosome-remodeling complexes and transcriptional control. Trends Genet 2000;16:345-351. Tamashiro KLK, Wakayama T, Blanchard RJ, Blanchard DC, Yanagimachi R. Postnatal growth and behavioral development of mice cloned from adult cumulus cells. Biol Reprod 2000; 63:328-334. Tanaka TS, Jaradat SA, Lim MK, Kargul GJ, Wang XH, Grahovac MJ, Pantano S, Sano Y, Piao YL, Nagaraja R, Doi H, Wood WH, Becker, Ko MSH. Genome-wide expression profiling of mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray. Proc. Natl. Acad. Sci. USA 2000;97:9127-9132. Tani T, Kato Y, Tsunoda Y. Direct exposure of chromosomes to nonactivated ovum cytoplasm is effective for bovine somatic cell nucleus reprogramming. Biol Reprod 2001;64:324-330. Taniguchi Y, Lejukole HY, Yamada T, Akagi S, Takahashi S, Shim&u M, Yasue H, Sasaki Y. Analysis of expressed sequence tags from a cDNA library of somatic nuclear transfer-derived cloned bovine whole foetus. Anim Genet 2001;32:1-6. Vignon X, Chesne P, LeBourhis D, Flechon JE, Heyman Y, Renard JP. Developmental potential of bovine embryos reconstructed from enucleated matured oocytes fused with cultured somatic cells. C R Acad. Sci. Paris, Life Sci. 1998;321:735-745. Vignon X, LeBourhis D, Adenot P, Marchal J, Lavergne Y, Laloi E. Production of bovine embryos by nuclear transfer of adult skin fibroblasts: The effect of serum starvation. Theriogenology 2000;53:245 (abst). Wakayama T, Perry ACF, Zuccotti M, Johnson KR, Yanagimachi R. Full-term development of mice from enucleated oocytes injected with cumulus cells nuclei. Nature 1998; 394:369-374. Wakayama T, Yanagimachi R. Cloning of male mice from adult tail-tip cells. Nature Genet 1999;22:127-128. Wakayama T, Rodriguez I, Perry ACF, Yanagimachi R, Mombaerts P. Mice cloned from embryonic stem cells. Proc. Natl. Acad. Sci. USA 1999; 96:14984-14989. Walker SK, Heard TM, Seamark RF. In vitro culture of sheep embryos without coculture: Successes and perspectives. Theriogenology 1992;37: 111-126. Walker SK, Hartwich KM, Seamark RF. The production of unusually large offspring following embryo manipulation: concepts and challenges. Theriogenology 1996;45: lll120. Wang Q, Latham KE. Translation of maternal messenger ribonucleic acids encoding transcription factors during genome activation in early mouse embryos. Biol Reprod 2000;62:969-978. Wangh L, DeGrace J, Sanchez JA, Gold A, Yeghiazarians Y, Wxeidemann K, Daniels S. Efficient reactivation of Xenopus erythrocyte nuclei in Xenopus egg extract. J Cell Sci 1995;108:2187-2196. Wells DN, Misica PM, Day TA. Tervit HR. Production of cloned lambs from an established embryonic cell line: a comparison between in vivo and in vitro- matured cytoplasts. Biol Reprod 1997;57:385-393. Wells DN, Misica PM, Tervit HR. Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells. Biol Reprod 1999;60:996-1005.

222

95. 96.

97. 98.

99. 100. 101.

102.

103.

104.

Theriogenology

Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KHS. Viable offspring derived from fetal and adult mammalian cells. Nature 1997;385:810-813. Winger Q, Hill J, Shin T, Watson AJ, Kraemer DC, Westhusin ME. Genetic reprogramming of lactate dehydrogenase, citrate synthase, and phophofructokinase mRNA in bovine nuclear transfer embryos produced using bovine fibroblast cell nuclei. Mol Reprod Dev 2000;56:458-464. Wolffe AP, Matzke MA. Epigenetics: Regulation through repression. Science 1999;286:481-486. Wrenzycki C, Wells D, Herrman D, Miller A, Oliver J, Tervit R, Niemann H. Nuclear transfer protocol affects messenger RNA expression patterns in cloned bovine blastocysts. Biol Reprod 2001; 65:309-317. Young LE, Sinclair K, Wilmut I. Large offspring syndrome in cattle and sheep. Rev Reprod 1998;3:155-163. Young LE, Fairbum HR. Improving the safety of embryo technologies: Possible role of genomic imprinting. Theriogenology 2000;53:627-648. Zakhartchenko V, Alberion R, Stojkovic M, Prelle K, Schemthaner W, Stojkovic P, Wenigerkind H, Wanke R, Dtichler M, Steinbom R, Mueller M, Brem G, Wolf E. Adult cloning in cattle: Potential of nuclei from a permanent cell line and from primary cultures. Mol Reprod Dev 1999;54:264-272. Zakhartchenko V, Durcova-Hills G, Stojkovic M, Schemthaner W, Prelle K, Steinbom R, Mueller M, Brem G, Wolf E. Effects of serum starvation and re-cloning on the efficiency of nuclear transfer using bovine fetal tibroblasts. J Reprod Fertil 1999;115:325-33 1. Zhou Q, Jouneau A, Brochard V, Adenot P, Renard JP. Developmental potential of mouse embryos reconstructed from metaphase embryonic stem cell nuclei. Biol Reprod 2001;65:412-419. Zou X, Chen Y, Wang Y, Luo J, Zhang Q, Zhang X, Yang Y, Ju H, Shen Y, Lao W, Xu S, Du M. Production of cloned goats from enucleated oocytes injected with cumulus cell nuclei or fused with cumulus cells. Cloning 2001;3:31-37.