Principles of Cloning || Nuclear Origins and Clone Phenotype

21Principles of Cloning. DOI: © 2012 Elsevier Inc. All rights reserved.

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in a different temporal, cellular, or molecular context. SCNT also provides an opportunity to evaluate outcomes of interactions that do not occur during normal develop-ment, as a means of hypothesis testing. In this chapter we review some of the lessons learned from SCNT about nor-mal development, and look forward to mysteries yet to be solved.

SCNT provides an opportunity to evaluate outcomes of interactions that do not occur during normal development as a means of hypothesis testing.

ABERRANT PROPERTIES OF CLONED EMBRYOS

Gene Expression

The original objective of the fantastical experiment was to test whether developmental potency was retained with cellular specialization. As a result, one of the earliest

Nuclear Origins and Clone Phenotype: What Cloning Tells Us about Embryonic DevelopmentDasari Amarnath1 and Keith E. Latham2

1Taconic Farms, Germantown, NY, USA, 2Department of Animal Science, College of Agriculture, Michigan State University, East Lansing,

Michigan, USA

INTRODUCTION

Spemann’s “fantastical experiment” of adult somatic cell nuclear transfer (SCNT or “cloning”), originally proposed in the early 1900s as a way to test retention of genomic potency during cellular specialization, has now been undertaken in a wide range of species, using a wide range of nucleus donor–recipient cell combinations and a wide range of methodologies. These methodologies are continu-ing to evolve as practitioners seek to identify approaches to enhance the success of cloning, driven in large measure by significant potential pay-offs in areas such as livestock propagation, enhancement of population characteristics, transgenesis and the biopharmaceutical industry, endan-gered species’ preservation, and development of stem cell-based clinical applications. From the earliest applica-tions of SCNT, and continuing through these diverse meth-odological changes, SCNT continues to yield surprising results that inform us about molecular and cellular aspects of normal oocytes and embryos. SCNT provides a means of recapitulating early events, or eliciting those events

Chapter 3

Chapter OutlineIntroduction 21Aberrant Properties of Cloned Embryos 21

Gene Expression 21X-Chromosome Reactivation 22Loss of Imprinting 23Maternal mRNA Regulation 23Altered Metabolism and Physiology 24Altered Spindle Properties and Increased Aneuploidy 25

Effects of Developmental Stage of Donor Nucleus 25Effects of Cell Type of Donor Nucleus 26

Culture Requirements 26Effect of a Transformed Donor Cell State 26Cloning from Frozen and Dead Cells 26

Effects of Donor Nucleus Cell Cycle Stage 27Effects of Recipient Cell Type and Stage 27Effects of Genotype and Sex of Donor Nucleus 28Effects of Species Origin of Donor and Recipient – Challenges of Inter-Species Nuclear Transfer 28Remediation of Aberrant Properties 29

Epigenome-Modifying Drugs 29Procedural Modifications 30Enhancing Oocyte Composition 31

Mysteries yet to be Solved 31Acknowledgements 32References 32

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PART | I Basics22

assessments made in cloned embryos was to what degree the cloned embryo recapitulated expression of early molecular markers, using donor nuclei from embryonic stages. Early studies revealed acquisition of proteins by the transplanted nuclei (see, for example, Prather et al. (1991)), cell cycle progression and cleavage, and then re-expression of embryonic marker proteins, such as the Transcription Requiring Complex (70-kDa TRC) in mouse embryos (Latham et al., 1991a). Zygote cytoplasm could likewise elicit such changes, despite limited developmental potential, indicating that reprogramming to reactivate some genes was not specific to the oocyte. Collectively, these early studies revealed that silenced genes could be reacti-vated after nuclear transfer. Conversely, silencing of donor cell markers was evaluated as another measure of repro-gramming success.

SCNT generally yields limited results with respect to embryonic development, in terms of both the number of embryos developing and the maximum stage attained. As a result, there has been considerable interest in defining why this occurs. One often-studied aspect of SCNT embryos and their derivatives has been to evaluate gene expression profiles and compare them to normal expression profiles in order to assess the extent of reprogramming. Comparisons have been made examining adult and fetal tissues and cells, placentae, blastocysts, and embryonic stem cells derived from blastocysts. A difficulty with such compari-sons, however, is that a considerable amount of selection and embryo demise occurs by the time these stages are attained, so that the results do not fully reflect what occurs in the majority of cloned constructs.

Gene expression profiling of cloned mouse embryos at the one-cell and two-cell stages provided the first assess-ment of the degree of reprogramming in a whole popula-tion at an early stage before substantial embryo demise (Vassena et al., 2007). This analysis revealed that a small number of transcribed genes were mis-regulated at the one-cell stage prior to the major transcriptional activa-tion event, but that many more genes were mis-regulated at the late two-cell stage. Accounting for genes that were also affected in parthenogenetic control embryos and thus not specifically affected in cloned embryos, there remained nearly 1200 genes that were mis-regulated. Major catego-ries of affected genes related to transcription, regulation of transcription, oxidoreductase and electron transport activi-ties, and ion transport. The prominent effect on transcrip-tion factor genes raised the interesting possibility that, as genes that reside at the top of regulatory hierarchies in differentiated cells, transcription factor genes may be resistant to reprogramming, and that the continued mis-expression of these key regulators contributes to the mis-regulation and mis-expression of many downstream genes. Pathway analysis indicated that many of the affected genes indeed reside together in regulatory gene networks.

Whole transcriptomes in cloned embryos reveal that, while some nuclear reprogramming occurs, many genes are slow to be reprogrammed, or are not reprogrammed at all, which leads to aberrant characteristics of cloned embryos.

Such studies of whole transcriptomes in cloned embryos reveal that, while some nuclear reprogramming occurs, many genes are slow to be reprogrammed, or are not repro-grammed at all, which leads to aberrant characteristics of cloned embryos. This limitation in reprogramming suggests that while the oocyte is unique in its ability to reprogram nuclei, its ability to do so must be constrained in signifi-cant ways. This might include limitations on what kinds of chromatin or epigenetic marks can be reprogrammed, dif-ferences in the genomic substrates between somatic cells and gamete genomes that present barriers to reprogram-ming, requirements that must be met for access to epigenetic marks in order to reprogram them, stage-dependent acqui-sition of essential factors that mediate some steps in repro-gramming, and possibly a lack of expression of other factors that are needed to modify chromatin-associated proteins in the donor nuclei.

X-Chromosome Reactivation

The regulation of the X chromosome presents an interest-ing challenge in cloning. During normal development, one of two X chromosomes in female cells is chosen to undergo silencing by heterochromatization. The process initiates at the X inactivation center (Xic) in the early embryo, and inactivation spreads from that point, so that genes nearest the Xic become monoallelic first, followed by more dis-tal genes (Latham, 1996; Latham and Rambhatla, 1995). In mice, imprinting of the Xist gene leads to preferential paternal X-chromosome inactivation early, and in extra-embryonic cells, but this is lost and inactivation becomes nominally random in somatic cells (genetic factors can render the process non-random). This is reflected in prefer-ential repression of paternal genes near the Xic during pre-implantation development followed by repression of more distal genes. With cloning, either the inactivated X chromo-somes could remain silent, with uniform allelic inactiva-tion in all cells of the clone, or the inactive X chromosome could become reactivated, allowing randomized inactiva-tion to occur during development.

In mice, initial studies indicated that X-chromosome reactivation occurs (Eggan et al., 2000). Subsequent stud-ies revealed that cloned embryos reactivated X chromo-somes, but continued to display a gradient of inactivation, such that bi-allelic expression was observed more often for more distal genes than for genes near the Xic (Nolen et al., 2005). Lack of reactivation was reported in one clone; however, most displayed some degree of reactivation, and

Chapter | 3 Nuclear Origins and Clone Phenotype: What Cloning Tells Us about Embryonic Development 23

(Mann et al., 2003). Imprinting abnormalities were also reported for clones in other species (Suzuki et al., 2011; Suzuki et al., 2009; Yang et al., 2005; Wei et al., 2010; Young et al., 2003) and may contribute to abnormal pla-centa growth and development (Guillomot et al., 2010), though some expressed imprinted genes may not be prob-lematic (Arnold et al., 2006).

Aside from possible culture effects, failure to display correct gene imprinting in clones appears to be in part due to incorrect expression and localization of DNA methyltrans-ferase DNMT1 isoforms to cleavage-stage nuclei (Chung et al., 2003). This suggests that the normal localization of these proteins may be directed by specific architectural aspects of embryonic nuclei, which are lacking in somatic cell nuclei even after nuclear remodeling to reach cleavage stages. The nature of these differences is not known.

Another challenge facing cloned embryos is the loss of epigenetic information that occurs during normal develop-ment. Since our first discovery of stage-specific regulation of imprinted genes in pre-implantation embryos (Latham et al., 1994b), it has become widely appreciated that many imprinted genes display stage-specific regulation, cell-type specific regulation, or a combination of the two. Of partic-ular relevance to cloning are genes that display imprinting in the extra-embryonic lineages but not in somatic lineages, which comprise about 20% of imprinted genes (Kuzmin et al., 2008). An illustrative example is the Sfmbt2 gene, which is imprinted and expressed from the paternal allele in early embryos and placentae, but biallelically expressed in somatic cells (Kuzmin et al., 2008). This gene is overex-pressed in cloned two-cell stage mouse embryos (Vassena et al., 2007), consistent with loss of imprinting in the somatic donor cells. A loss of imprinting for these genes in somatic lineages could create a lack of placenta-specific imprinted gene regulation, combined with loss of imprinting at other genes, and contribute to the abnormal physiology seen in cloned placentae.

By observing phenotypic effects in cloned embryos, par-ticularly in the placenta, clones provide new glimpses into the roles of specific imprinted genes, and their imprinted status, in normal development. Additionally, further studies of how cloned embryos regulate genes that display loss of imprinted regulation in somatic cells but imprinted regula-tion in the placenta will provide new insight into the degree to which imprinting may be retained or lost in somatic lineages for different imprinted genes during the shift to biallelic expression.

Maternal mRNA Regulation

Analysis of cloned embryo transcriptomes at early cleavage stages reveals that they regulate their maternal mRNA popu-lations abnormally. A small number of maternal transcripts are incorrectly regulated at the one-cell stage in cloned

there was considerable variability amongst individual cells (Nolen et al., 2005). The placenta of one cloned embryo displayed bi-allelic X-chromosome expression and was enlarged. Overall, these results indicated that, while reacti-vation occurs in cloned embryos, X-chromosome regulation is inconsistent. Aberrant X-linked gene expression was also seen in deceased cloned piglets (Jiang et al., 2008). Culture of donor cells with S-adenosylhomocysteine enhances clon-ing outcome in part by facilitating X-chromosome reactiva-tion (Jeon et al., 2008). Moreover, inhibiting Xist expression or activity enhances cloning outcome, and it has been sug-gested that cloned embryo demise may be in large part pre-ordained by early X-chromosome inactivation status (Inoue et al., 2010; Matoba et al., 2011). Interestingly, nega-tive effects of continued expression of Xist from the active X chromosome were compounded by a failure to reverse repressive histone modifications in the early embryo (Inoue et al., 2010). Normalized X-chromosome inactivation also appears to be achieved through the derivation of cultured embryonic stem cell lines from cloned embryos (Shibata et al., 2008), which may reflect a strong selection on sur-viving cells. Chapter 15 of this volume provides an in-depth discussion of X-chromosome regulation in clones.

Taken together, these observations on X-chromosome reactivation in cloned embryos indicate that reactivation most likely occurs over several cell divisions, via stochastic events that lead to changes in the epigenetic state of the Xist gene, and that this allows subsequent changes in the epigenetic state of the X chromosome in a graded fashion, reversing the epigenetic changes that occur during normal develop-ment. However, this process is very inefficient. The inability of early-stage cloned embryos to regulate X-chromosome reactivation, counting, and inactivation in a controlled man-ner may reflect a difference in X-chromosome repression in the early embryo compared to somatic cells. Mechanisms to reverse inactivation may therefore not act efficiently upon inactivated X chromosomes imported from somatic cells. Additionally, reliance on passive processes coupled to DNA replication rather than active processes could contribute to the X-chromosome inactivation mosaicism observed in cloned embryos.

Loss of Imprinting

In recent years it has become apparent that embryo cul-ture can adversely affect the faithful retention of epigenetic information during early embryogenesis (Doherty et al., 2000; Mann et al., 2004; Banrezes et al., 2011; Watkins et al., 2008; Kwong et al., 2006). This has led to concerns about whether loss of imprinting information occurs sys-tematically in cloned embryos. Mouse cloned blastocysts indeed display disruptions in imprinted gene expression and methylation, and only 4% displayed a normal blasto-cyst mode of expression for five imprinted genes studied

PART | I Basics24

mouse embryos, and a much larger number at the two-cell stage (Vassena et al., 2007). Interestingly, it is well known that inhibiting RNA polymerase II transcription also affects maternal mRNA regulation (Rambhatla et al., 1995). These observations highlight an important interaction between nucleus and cytoplasm in the early embryo that remains to be understood, and suggest that nuclear transfer technology can play a pivotal role in discovering underlying mecha-nisms of this interaction and the consequences when the dialog is altered.

Altered Metabolism and Physiology

The early cleavage-stage embryo differs dramatically from the somatic cells to which it will eventually give rise. The differences are numerous, and notable ones include the fol-lowing: the cell cycle and its regulation are fundamentally different (e.g., cleavage without growth); two-dimensional protein gels reveal strikingly different gene expression pro-files (Latham et al., 1991b); carbon substrate preferences are shifted toward small molecules such as pyruvate and lac-tate, and oxygen consumption is low initially (Leese, 2012); amino acid transporters, glucose transporters, ion transport-ers, etc. differ fundamentally in expression (Van Winkle, 2001; Baltz et al., 1993; Baltz et al., 1991a,b; Pantaleon et al., 2001; Leppens-Luisier et al., 2001; Chi et al., 2000; Carayannopoulos et al., 2000; Carayannopoulos et al., 2004; Moley et al., 1998; Morita et al., 1994); mechanisms of osmoregulation and pH regulation differ (Baltz, 2001; Edwards et al., 1998; Zhao and Baltz, 1996); mitochondrial ultrastructure is unique (Sathananthan and Trounson, 2000; Hillman and Tasca, 1969).

One consequence of the inefficient reprogramming in cloned embryos is that they display a number of somatic cell-like features.

One consequence of the inefficient reprogramming in cloned embryos is that they display a number of somatic cell-like features (Gao et al., 2003). This includes differ-ences in metabolism and physiology as compared to ferti-lized embryos. This effect can be manifested quite early. Cloned mouse embryos progress more efficiently from one-cell to two-cell stages with glucose present (Chung et al., 2002). Normal embryos have no requirement for glucose at this stage. Cloned embryos also display enhanced glucose uptake and precocious glucose transporter localization to the cell surface (Gao et al., 2003). Moreover, cloned embryos prefer higher oxygen concentrations than normal embryos. These observations indicate that cloned embryo physiology and metabolism are more somatic-like than embryo-like.

Compounding the effects of altered gene expression on metabolism are potential aberrations in mitochondria

distribution and activity. Two general kinds of aberra-tions may exist. The first relates to aberrant regulation of endogenous mitochondria. In the normal oocyte and early embryo, mitochondria have unique ultrastructures and have primitive internal architectures with few cristae, indicative of limited capacity for oxidative phosphoryla-tion. This changes as development proceeds and the meta-bolic activity of the embryo changes. Interestingly, cloned embryos display enhanced expression of many mRNAs related to oxidative phosphorylation (Vassena et al., 2007), which could contribute to an accelerated up-regulation of oxygen-requiring oxidative phosphorylation processes, accounting for increased need for glucose and oxygen.

The second aberration affecting mitochondria relates to the fate of donor cell mitochondria, and how their activity might contribute to phenotypic effects. Sperm mitochondria are typically eliminated through oocyte mechanisms that recognize ubiquitinated proteins and target these organelles for destruction (Sutovsky et al., 1999). Donor cell mito-chondria are not modified in this manner, and hence persist. This contributes to inter-species nuclear transfer embryo abnormalities and affects intra-species nuclear transfer out-comes. Donor cell mitochondria undergo abnormal distribu-tion within the embryos and can display unequal segregation to daughter cells during cleavage, and the distribution of mitochondria with different morphologies is altered in cloned embryos (Zhong et al., 2008). Because dynamic mitochondrial redistributions spatially within the cell (e.g., movement to the perinuclear region) may be associated with embryo quality, this change may affect cell physiology in cloned embryos (Zhong et al., 2008).

Donor cell mitochondria undergo abnormal distribution within the SCNT embryos and can display unequal segre-gation to daughter cells during cleavage; the distribution of mitochondria with different morphologies is also altered in cloned embryos.

The alterations in mitochondrial function may affect the regulation of apoptosis. The apoptotic index can be elevated in cloned embryos compared to normal embryos (Park et al., 2004). Culture of cloned embryos with IGF1 reduces the apoptotic index (Kim et al., 2006). Likewise, treatment with histone deacetylase inhibitors such as trichostatin A or oxamflatin inhibit apoptosis in cloned embryos by sup-pressing pro-apoptotic gene expression and/or activating expression of anti-apoptotic genes (Su et al., 2011; Cui et al., 2011). Apoptosis can also be inhibited with alpha-tocopherol and L-ascorbic acid, which act as antioxidants and provide a protective effect (Jeong et al., 2006).

Taken together, these observations highlight the value of cloned embryos as tools for understanding the mecha-nisms that regulate essential metabolic and physiological


processes in the early embryo. The cloned embryo also provides a valuable tool for studying how these processes are coordinated specifically with nuclear processes, as well as DNA replication and cell cycle transit.

Altered Spindle Properties and Increased Aneuploidy

Removal of crucial spindle-associated proteins was first proposed to be a potential cause of SCNT embryo demise in non-human primate embryos (Simerly et al., 2003). Subsequent studies in mouse embryos, however, revealed that most proteins that are depleted by removing the spin-dle–chromosome complex (SCC) can be restored to nor-mal levels within a matter of a few hours (Miyara et al., 2006). This requires the presence of a genome, indicating that the ability to form a new SCC is linked to this recov-ery. However, some proteins remain deficient on the SCC, including calmodulin (Miyara et al., 2006) and clathrin heavy chain, aurora kinase, and other proteins (Han et al., 2010). In the case of calmodulin, the deficiency is a failure of the newly formed spindle to acquire the protein. A sup-ply of the protein remains after SCC removal and its lev-els can be restored after SCNT. Additionally, the deficiency is also observed in tetraploid constructs prepared without SCC removal, and the deficiency is not seen with embry-onic donor cell nuclei. Tetraploidy was observed in a small percentage of cells in some clones (Nolen et al., 2005). Aneuploidy was reported in larger fractions of cells in another study (Mizutani et al., 2012). Immunofluorescent visualization of the SCC and associated proteins coupled with fluorescent DNA staining invariably reveals a dra-matic delay in congression of chromosomes on the newly formed spindle after SCNT (Miyara et al., 2006; Han et al., 2010). These observations led to the striking realization that the donor nucleus controls formation of the new SCC by some undetermined mechanism, and that the origin of the donor nucleus affects the nature of this process.

EFFECTS OF DEVELOPMENTAL STAGE OF DONOR NUCLEUS

A widespread observation with SCNT in both amphibians and mammals is that success declines dramatically with developmental stage of the donor genome. Cloning was initially successful with blastomere-stage nuclei in mam-mals (Smith et al., 1988; Stice and Robl, 1988; Prather et al., 1987; Tsunoda et al., 1987; First and Prather, 1991), and is more efficient with embryonic stem cell donors than with fetal- or adult-stage donors (Wakayama et al., 1999). Cloning in amphibians revealed that chromosomal abnor-malities were more common with more advanced-stage donor nuclei due to an inability of advanced-stage nuclei to decondense fully in the ooplasm (Di Berardino and

Hoffner, 1970). Historically, one of the burdens of demon-strating developmental totipotency in differentiated somatic cells has been to confirm that the clone was derived from a differentiated cell rather than a rare stem cell present in the donor cell population. In the frog, cloning with later-stage nuclei is less efficient than cloning with earlier-stage nuclei (Di Berardino and King, 1967), and cloning with nucleated hemoglobin-expressing and thus obviously differentiated erythrocyte nuclei failed to yield live adults even with many rounds of serial NT (Orr et al., 1986). Other studies in the frog reported successful cloning after using other marker approaches (Gurdon and Byrne, 2004). In mice, success-ful cloning with known differentiated donor nuclei has been achieved, but involved ES cell derivation and tetraploid complementation as facilitating techniques (Hochedlinger and Jaenisch, 2002; Eggan et al., 2004). Differentiated natural killer T cells with rearranged TCR genes were used successfully for cloning by direct nuclear transfer (Inoue et al., 2005).

One striking effect of donor stage on outcome relates to spindle abnormalities. Clone constructs made with mouse embryonic fibroblasts or adult cumulus cells display defi-ciencies in spindle protein composition, but those made with blastomere nuclei or ES cell nuclei do not (Miyara et al., 2006). Loss of imprinting also could contribute to stage-specific donor cell effects as described above. Additionally, it was suggested that nuclei of different stages may contribute different amounts of essential factors to overcome depletion of factors during the cloning procedure (Egli and Eggan, 2010).

Cloning technology may be a useful tool for studying the regulation of telomerase expression and telomere length.

One outcome of cloning approaches has been the oppor-tunity to assess effects on telomere length and potential proliferative lifespan. Cloned sheep display shorter tel-omeres than age-matched controls (Shiels et al., 1999a; Alexander et al., 2007), and Dolly displayed telomere lengths appropriate for the nuclear donor (Shiels et al., 1999b); however, normal breeding of cloned sheep restores normal telomere lengths (Alexander et al., 2007). Cloned pigs display telomeres similar to age-matched controls (Jiang et al., 2004; Kurome et al., 2008) and serial clon-ing does not erode telomere length (Kurome et al., 2008). Telomere length is sensitive to donor aging phenotype (Jeon et al., 2012). Another study reported lengthening of telomeres and prolongation of replicative lifespan in bovine cloning (Lanza et al., 2000). This appears to vary with donor cell type, and it has been suggested that nuclear transfer may induce telomere expansion (Miyashita et al., 2002). This may arise from elevated telomerase expression (Jeon et al., 2005). Hence, the species dependence reported

PART | I Basics26

for fates of telomeres may reflect a combination of cell type as well as species-specific induction (or a lack of it) of telomerase activity. This raises the intriguing notion that cloning technology may be a useful tool for studying the regulation of telomerase expression and telomere length.

EFFECTS OF CELL TYPE OF DONOR NUCLEUS

The preceding sections mention several instances wherein donor cell type affects cloning outcome. The potential rea-sons for such effects are many. In this section, we focus on the effects of three aspects of donor cell phenotype on outcome.

Culture Requirements

Successful cloning requires reprogramming of nuclei, at least to a state that is compatible with early embryogenesis. This is a phenomenal feat, given that normal embryos and somatic cells differ dramatically in many respects, includ-ing homeostatic regulation, cell cycle regulation, and culture requirements. It has become apparent that reprogramming does not happen in a single instant, but rather is a pro-tracted process that occurs over many days, likely through-out pre-implantation development, and possibly extending into early post-implantation development until just before gastrulation. As a result, providing cloned embryos with an optimized environment during the reprogramming period could comprise essentially a practical impossibility, as the cloned embryo needs would be continually changing with the course of reprogramming. Moreover, because repro-gramming is slow, the initial demands of the cloned embryo could vary with initial gene expression pattern of the donor nucleus. And overall, a significant dilemma in cloning is how to achieve greater reprogramming so that cloned embryo physiology is most compatible with the oviduct or uterus by the time embryo transfer must be performed.

Our early studies of culture requirements in cloned mouse embryos revealed that cumulus cell cloned con-structs developed better with initial culture in less highly optimized embryo culture media followed by a switch to more optimized medium (Chung et al., 2002). We later found that clones develop at a greater efficiency in the somatic cell culture formulation MEMα (minimal essen-tial medium alpha), as compared to the embryo-optimized KSOM (potassium simplex optimized medium) formula-tion (Gao et al., 2004). This was observed when dimethyl-sulfoxide was excluded from the procedure (Chung et al., 2002) (DMSO is used as a solvent for cytochalasin B, but is well known to modulate gene expression via non-specific and poorly defined mechanisms). Effects of media used for oocyte and donor cell suspension are also seen (Gao et al., 2004). In contrast, myoblast-derived cloned constructs

developed better in a culture medium that is optimized for myoblasts (Gao et al., 2003). For other species, other media systems are reported to yield enhanced success rates (Simon et al., 2006; Wang et al., 2011; Jang et al., 2008). Collectively, these results with cloned embryos highlight unique requirements of fertilized embryos for in vitro cul-ture. Further discussion of culture media for mammalian embryos is provided in Chapter 8. Additional discussion of effects of donor cell type is provided in Chapter 12.

Effect of a Transformed Donor Cell State

Whether cancer cells can be reprogrammed to an embry-onic state has been a compelling question for many years. Robert McKinnell demonstrated that frog Lucké renal car-cinoma nuclei transferred into enucleated eggs results in prefeeding swimming tadpoles, and significant develop-mental potential can be demonstrated by grafting to host tadpoles (McKinnell, 1979; Lust et al., 1991; McKinnell et al., 1969). Virally induced pronephric carcinoma cell nuclei can also support development to post-neurula stage and larval stages free of tumors (DiBerardino et al., 1983). King and DiBerardino attempted frog cloning using renal carcinoma (primary and cultured donor cells) and intraocular tumors, but achieved maximum development to abnormal larval stages (King and DiBerardino, 1965). These studies indicated that tumor cell nuclei can be repro-grammed but that their potentials remain restricted.

Cloning outcomes in mice using cancer cells as nucleus donors were reported (Hochedlinger et al., 2004). Nuclei from leukemia, lymphoma, and breast cancer supported pre-implantation development but embryos failed to pro-gress beyond the blastocyst stage. Clones made with RAS-inducible melanoma nucleus donors yielded ES cells, which in chimeric animals promoted earlier onset and expanded tumor phenotypes. Cloning with embryonal car-cinoma (EC) cells yielded ES cells that displayed similar results in chimeric embryos as obtained with original EC cells (Blelloch et al., 2004).

Genetic lesions can contribute to limited developmen-tal potentials of tumor cell clones (Blelloch et al., 2004). The degree to which un-reprogrammed epigenetic restric-tions apply has not been extensively tested. These studies suggest that cloning by transfer of tumor cell nuclei pro-vides a useful model system for identifying genes that may support or restrict developmental potential, by identifying either genetic or epigenetic aberrations.

Cloning from Frozen and Dead Cells

One possible application of cloning technology that has captured the imagination is the possibility of cloning with nuclei from deceased or frozen bodies. One early study reported cloning of an endangered species by inter-species


nuclear transfer using cells from a deceased individual (Loi et al., 2001). More recently, cloning with nuclei from frozen mouse bodies was reported, with the interesting result that brain cells seemed most likely to support devel-opment (Wakayama et al., 2008). These results provide intriguing evidence that even nuclei in frozen tissues can be sufficiently intact to support development. The cell-type effects may reflect a difference in nuclear architecture, nuclease content, or presence of other macromolecules that may act as endogenous cryoprotectants. These results also suggest that this experimental approach may be useful for probing DNA repair activities and efficiencies and other aspects of genome processing in cloned constructs, possi-bly informing us about the normal capabilities of oocytes and early embryos at these stages.

EFFECTS OF DONOR NUCLEUS CELL CYCLE STAGE

Cell cycle synchrony between the donor cell nucleus and recipient oocyte is an essential prerequisite for success-ful nuclear reprogramming and full-term development in SCNT (Wilmut et al., 1997). Donor cells synchronized to the G0, G1, and M phases are routinely used for somatic cell nuclear transfer into metaphase II oocytes to prevent DNA damage and uncoordinated DNA replication of the donor cell genome. The cell cycle stage of donor cells must also be compatible with high MPF activity in MII oocytes. High MPF activity in MII oocytes induces nuclear envelope breakdown (NEBD) of the donor cell nucleus and premature chromosomal condensation (PCC) (Campbell et al., 1996; Tani et al., 2001). The degree of PCC observed is depend-ent on the MPF activity (Kwon et al., 2008), the duration of exposure to MPF activity, and the cell cycle stage of the donor cell nucleus.

The cell cycle stage of donor cells must also be compatible with high MPF activity in MII oocytes.

When donor cells at the GO/G1, G1, G2, and M phases undergo PCC, chromatin condenses into elongated chromo-somes with single or double sister chromatids. However, PCC of S-phase nuclei leads to a typical pulverized appear-ance, with a high incidence of chromosomal abnormalities (Tani et al., 2001; Collas et al., 1992). PCC is believed to be essential for promoting nuclear reprogramming in SCNT. While the precise mechanism by which PCC facilitates nuclear reprogramming is not well known, NEBD and PCC may help in facilitating release of chromatin-bound somatic factors and increase the accessibility of donor cell chroma-tin to oocyte factors involved in nuclear reprogramming and DNA synthesis (Choi and Campbell, 2010). Following PCC, nuclei reform and DNA replication can commence. While

a second polar body containing half the chromosomal con-tent is usually extruded after fertilization to establish normal ploidy, chromosome allocation to a polar body in SCNT constructs is random. To avoid aneuploidy, donor cells at G0/G1, G1 with 2N chromosomal content are usually pre-ferred over donor cells at S or G2, and polar body extrusion is prevented to maintain diploidy. Donors at M phase can be used if polar body extrusion is allowed. Therefore donor cells at G0/G1 and G1 phases with 2N chromosomal content are usually preferred over donor cells at G2 and M phases with 4N chromosomal content. Failure of second polar body extrusion with G2- and M-phase donor cells leads to the formation of tetraploid embryos. Further discussion of cell cycle aspects of cloning is provided in Chapter 14.

EFFECTS OF RECIPIENT CELL TYPE AND STAGE

Cloning has been most successful using oocytes as recipi-ents. Early studies using zygote recipients indicated that cloning would be very challenging with fertilized recipi-ent cytoplasts (McGrath and Solter, 1984), even though embryonic marker genes could be reactivated (Latham et al., 1991a). One aspect that may limit success of zygotic cytoplasts as recipients relates to the normal progression of events that create the potential for gene transcription and regulation. The ability to transcribe genes in the mouse embryo is acquired with passage through the first S phase, and the ability to regulate transcription is acquired with pas-sage through the second S phase (Davis et al., 1996; Aoki et al., 1997). Embryonic eight-cell donor nuclear transfer studies revealed that the early zygote cytoplasm is tran-scriptionally repressive, so that the embryonic 70-kDa TRC marker and other marker proteins indicative of the two-cell stage are reactivated using late zygote but not early zygote recipients (Latham et al., 1992, 1994b). This was in part due to a failure to reprogram the donor nucleus, but, more importantly, early zygote cytoplasm imposed an irreversible repression upon some genes already expressed in the eight-cell donor embryos, and these genes were not reactivated upon development to the two-cell stage. The basis for the irreversible gene silencing effects of early zygote cytoplasm is unknown, but seems to distinguish this stage of recipient from the earlier MII-stage oocyte, for which cloning has been successful, and suggests that significant changes asso-ciated with fertilization and oocyte activation may modify the cytoplasm to reinforce a state of transcriptional silence. Presumably, this early state is necessary to prevent inap-propriate gene expression from occurring until the ability to regulate gene transcription has been acquired.

Mitotic-phase zygote cytoplasm has been used suc-cessfully for cloning, whereas earlier stage zygotes have not been successful as recipients. This was interpreted in the context as possible retention of materials that might

PART | I Basics28

otherwise be lost through enucleation (Egli et al., 2007). Indeed, a prior study (Greda et al., 2006) suggested that par-tial disruption of the pronucleus prior to removal increased the ability of the interphase cytoplast to support develop-ment. Subsequent studies showed, once again, abnormal gene transcription with interphase recipients (Egli et al., 2009), and indicated that some transcription factors such as SMARCA4 may be lost with pronucleus removal. A loss of factors sequestered to the nuclear compartment, however, would not account for repression of actively transcribed genes. Thus it appears that a combination of processes (gene repression and loss of essential proteins via enucleation) may restrict the ability of the interphase zygote cytoplasm to support development after nuclear transfer. The results so far indicate that there are likely stage-dependent changes in both cytoplasm and nucleoplasm that affect nuclear function, and that significant differences exist between nucleoplas-mic components of zygotes as compared to somatic cells. Understanding these differences would likely improve our understanding of reprogramming processes that occur during normal development as the embryonic genome is created.

EFFECTS OF GENOTYPE AND SEX OF DONOR NUCLEUS

Early attempts at SCNT preferred female animals for clon-ing due to potential industrial applications of the emerg-ing technology, and initially better term development of cloned embryos made with female-derived versus male-derived donor nuclei (Wakayama et al., 1998; Wakayama and Yanagimachi, 1999). However, the developmental inef-ficiency of male SCNT embryos was not a consequence of sex effect in the above studies but due to the type of donor cell used and their cell cycle stage (Ogura et al., 2000). The reprogramming efficiency of donor cells is dependent on the epigenetic state (type) of the donor cell, and the cell cycle stage, species, and genotype of the donor cells. Species-specific characteristics can present procedural challenges. For example, inadequate control over oocyte activation con-tributes to cloning challenges in rats (Chebotareva et al., 2011). Fibroblasts of fetal and adult origin are preferred donor cells for SCNT in farm animals, but they are not very good donor cells in mouse. Furthermore, the genotype of the donor cells has significant influence on the outcome of SCNT. Cumulus cells from F1 (B6D2F1 or B6C3F1) strains of mice supported better in vitro development of SCNT embryos to morula/blastocysts than those from inbred strains (C3H/He or B6 or DBA/2) (45% vs 29%; see also (Gao et al., 2004)). Tail-tip fibroblasts from their male coun-terparts were not different but were developmentally less efficient (13% vs 8%). The effects of sex or genotype of the donor cells, while influencing the post-implantation devel-opment of cloned embryos (Wakayama and Yanagimachi, 2001; Inoue et al., 2003), has no effect on the efficiency of

establishment of ntES cells lines from the SCNT blastocyts (Wakayama et al., 2005). While cumulus and immature Sertoli cells from male and female B6D2F1 mice, respec-tively, were not different in their ability to support post-implantation development of SCNT embryos, Sertoli cells were found to be significantly more efficient than cumulus cells from the B6 × 129 genotype. Interestingly, Sertoli or cumulus cells from a B6 × JF1 genotype failed to pro-duce cloned pups (Inoue et al., 2003). Other studies have revealed a significant effect of donor cell genotype on out-come. Transplanting nuclei from a female into her own eggs yielded enhanced rates of success in cattle (Yang et al., 2006). Interactions between donor-cell and recipient-cell genotypes can arise (Gao et al., 2004). Others have reported reduced success with certain strains of mice or with inbred and outbred strains versus hybrid genotypes (Wakayama and Yanagimachi, 2001; Kishigami et al., 2007). Earlier studies indicated interactions between embryo sex and culture envi-ronment during pre-implantation development (Banrezes et al., 2011; Peippo et al., 2001; Jimenez et al., 2003); however, this is not observed universally. A possible effect of sperm injection on the sex ratio of developing human embryos was reported (Dumoulin et al., 2005). Maternal factors may interact with the embryonic epigenome in a sex-dependent manner (Kwong et al., 2006) and other manipulations can have selective effects on female versus male progeny (Cheng et al., 2009). Some of the effects of sex on normal embryonic development may thus reflect an interaction between culture environment, maternal physiol-ogy, and genotype, with a potential effect, for example, on X-chromosome inactivation that could affect embryonic metabolism. Detailed analysis of X-chromosome expression states in cloned embryos offers a potential means of evaluat-ing these interactions during pre-implantation development.

EFFECTS OF SPECIES ORIGIN OF DONOR AND RECIPIENT – CHALLENGES OF INTER-SPECIES NUCLEAR TRANSFER

Successful cloning of a large number of species using a vari-ety of donor cells demonstrates the unique ability of oocytes to reprogram differentiated nuclear donor genomes to a toti-potent embryonic state. Despite sustained efforts, however, the overall efficiency of this technology has remained poor in domestic and wild animals. While there has been some significant improvement in pre- and post-implantation devel-opment of SCNT embryos in mice (Terashita et al., 2012), similar improvement in cloning of domestic animals has seen more modest incremental increases. However, even at these low efficiency rates, SCNT offers novel opportunities for conservation of endangered species of animals and pro-duction of tailor-made ES cells for human therapeutic appli-cations. Limited availability of oocytes from endangered species is a major constraint, and the alternative approach


of inter-species somatic cell nuclear transfer (iSCNT) using oocytes readily available from closely related domestic spe-cies as recipients has been proposed. Using iSCNT between species that are not closely related to and that cannot hybrid-ize naturally often fails to support development beyond the stage at which embryonic genome activation takes place in recipient (oocyte donor) species (Beyhan et al., 2007). The failure of iSCNT embryos to develop beyond early stages has been attributed to an array of issues, such as incomplete nuclear reprogramming (Wang et al., 2009), poor nuclear–cytoplasmic compatibility (Amarnath et al., 2011; Narbonne et al., 2011), nuclear–mitochondrial incompatibility (Jiang et al., 2011), defective nucleogenesis (Song et al., 2009) and incomplete embryonic genome activation (Lagutina et al., 2010), and failure of degradation of maternal transcripts (Wang et al., 2011). Global transcriptome analysis of iSCNT embryos revealed partial embryonic genome activation, fail-ure of stage-specific expression of several pluripotent genes, and impaired degradation of maternal transcripts (Wang et al., 2009; Wang et al., 2011; Chung et al., 2009). Similar results are seen in intra-species cloned mouse embryos (Vassena et al., 2007). The timely translation of maternal transcripts is essential for the maternal to embryonic tran-sition, and a failure of timely degradation of maternal tran-scripts is detrimental to further embryonic development (Alizadeh et al., 2005). Nuclear–cytoplasmic incompat-ibility had a dramatic inhibitory influence on embryonic and fetal development and expression of several genes involved in pluripotency; mitochondrial function in fertilized mouse zygotes fused with a small amount of pig cytoplasm (inter-species cytoplasm hybrids) was seen. Interestingly, mouse cytoplasm (90–95% of the total volume of cytoplasm) pre-sent in these pig–mouse cytoplasm hybrids could not miti-gate the inhibitory influence of pig cytoplasm (Amarnath et al., 2011). Nuclear–cytoplasmic incompatibility and developmental defects in iSCNT embryos is a consequence of inefficient cell signaling and differences in the concentra-tion of key cytoplasmic proteins between unrelated species in these hybrid embryos. Further treatment of iSCNT embryos with species-specific key proteins deficient in inter-species hybrid embryos in amphibians partially rescued the develop-ment of hybrid embryos (Narbonne et al., 2011).

Successful cloning of a large number of species using a variety of donor cells demonstrates the unique ability of oocytes to reprogram differentiated nuclear donor genomes to a totipotent embryonic state.

Inter-species somatic cell nuclear transfer embryos are inefficient in generation of ATP (Wang et al., 2009). The generation of ATP by the electron transport chain in mito-chondria is encoded by both nuclear and mitochondrial DNA. The efficiency of ATP generation is dependent on

the compatibility between the nucleus and mitochondria. Unimaternal transmission of mitochondria in embryos and offspring from natural fertilization ensures mitochondrial homoplasmy, whereas SCNT embryos and offspring can be heteroplasmic, with mitochondria introduced via the karyoplast used for SCNT persisting (Sutovsky et al., 1999; Sutovsky et al., 1996). While the existence of heteroplasmy in healthy cloned offspring indicates some level of compat-ibility between the two populations of mitochondria, het-eroplasmy may lead to impaired mitochondrial function, developmental defects as in cytoplasm transfer, and mito-chondrial diseases (Bowles et al., 2007). The chance of mitochondrial heteroplasmy in SCNT is very high when the recipient oocyte and donor cells are from diverse genotypes, as in iSCNT. In iSCNT embryos this is further complicated by the greater incompatibility between nucleus and mtDNA encoded mitochondrial proteins. Diverse outcomes have been reported for elimination versus retention of nuclear donor cell mitochondria (Latham, 2004). The overall effect of nucleus–mitochondrial incompatibility is significant for iSCNT embryos. Replacement of oocyte mitochondria of pigs with a mouse mitochondrial population in conjunction with use of stem cell extracts improved the in vitro development potential of mouse–pig iSCNT embryos (Jiang et al., 2011).

These results illustrate a unique ability of inter-species and inter-strain nuclear transplantation, coupled with mitochondrial and ooplasm transfer, to be employed to evaluate and understand nuclear–cytoplasmic and nuclear–mitochondrial compatibilities in early embryos. Stage-specific and stage-independent effects could be explored by comparing outcomes from donor nuclei from different stages. This would help to distinguish effects related to nuclear reprogramming deficiencies. The incorporation of mitochondrial and ooplasm transfer would allow the con-tributions of oocyte-encoded ooplasmic components and mitochondria to phenotype to be distinguished.

REMEDIATION OF ABERRANT PROPERTIES

The universal challenge of cloning in any species has been, and remains, overcoming the low rate of success. Some genetic manipulations, such as Xist gene deficiency or introduction of pluripotency genes, can increase efficiency, but genetic alterations that modify the resultant cloned embryo genome do not constitute true cloning. A great deal of effort has been invested during the past decade in improving the cloning methodology. There are three main areas of modification that we will consider.

Epigenome-Modifying Drugs

The first area, and the one that has borne the greatest yield in terms of enhanced term development rates, consists

PART | I Basics30

of the application of epigenome modifying drugs to enhance the speed and extent of nuclear reprogramming. Treatments have been applied to donor cells before nuclear transfer, or to cloned constructs after nuclear transfer. Chapter 13 provides additional information on the use of epigenome modifying drugs in cloning.

One early approach was to apply DNA hypomethylat-ing drugs in an effort to reactivate genes that might have become silenced (Tsuji et al., 2009; Enright et al., 2005; Enright et al., 2003). An inherent problem is the lack of specificity of drugs such as 5-aza-cytidine, and their poten-tial for erasing beneficial (e.g., imprinting) as well as harm-ful DNA methylation. One study reported that beneficial effects of a less toxic DNA methyltransferase inhibitor S-adenosylhomocysteine might be achievable, but that criti-cal developmental windows of treatment may need to be identified with respect to both stage and duration (Jafari et al., 2011).

An alternate category of epigenetic drugs that has the potential for reactivating silenced genes without remov-ing DNA methylation (so that imprinting information could in theory be retained) are drugs that modulate post-translational histone modifications, particularly the histone deacetylase inhibitors (HDACi). Early efforts yielded unsat-isfactory results (Tsuji et al., 2009), but more refined short-term treatments have proven to be more promising (Enright et al., 2003). Efficacy of these drugs has been realized by several measures, ranging from effects at early stages to effects on placenta, term development rates and phenotypes of cloned offspring (Enright et al., 2003). Trichostatin A (TSA) (Kishigami et al., 2007; Kishigami et al., 2006) and Scriptaid (Van Thuan et al., 2009; Zhao et al., 2009) permit cloning using donor genotypes that are otherwise refrac-tory to success, increases overall success rate, and improves inter-species cloning success (Lee et al., 2010).

One of the more intriguing measures of success of HDACi compounds is their effects during the period imme-diately following nuclear transfer and during initial cell cycles when applied after nuclear transfer. Trichostatin A (TSA) increases histone acetylation and chromatin decon-densation, reverses the trend toward reduced histone H3–K4 dimethylation and increases H3–K4 trimethylation (thus imposing a more active global chromatin state), and increases transcriptional activity in two-cell stage cloned embryos (Bui et al., 2010). Scriptaid can overcome the variable delay in rRNA gene activation in individual blas-tomeres of two-cell stage mouse embryos (Bui et al., 2011) and also increases histone H3–K9 aceylation at the one-cell stage and mRNA synthesis during the two-cell stage (Van Thuan et al., 2009). Scriptaid corrects aberrantly regulated genes in pig blastocysts and improves histone acetylation pattern at the one-cell stage (Whitworth et al., 2011). These observations are intriguing because they reveal that the endogenous epigenome modifying machinery of the oocyte

and early embryo exerts undesirable effects on the incom-ing donor genome during the initial reprogramming period, and that a large portion of this undesirable effect is attrib-utable to histone deacetylation. Further study of the target genes that are susceptible to adverse epigenetic reprogram-ming with different donor cell types, their initial chromatin states, and their abilities to be acted upon by other tran-scriptional modulators will allow the cloning technology to be used to probe the specific chromatin regulatory events that contribute to cellular differentiation in different donor cell types as well as the characteristics of donor genome, oocyte, and early embryo that determine the limits and pace of reprogramming.

It is also important to bear in mind that some of the beneficial effects of HDACi compounds could reside at the level of other protein targets, even non-nuclear tar-gets. Modifying the activities of some of these other pro-teins could compensate for other deficiencies that arise in cloned constructs as a result of aberrant gene transcription. Interestingly, the optimum dose of HDACi compounds may vary with individual nucleus donor cell line (Akagi et al., 2011). Hence, cloning may provide a novel approach for probing epigenetic differences between cells, the stability of those differences, and how those differences can affect early development. TSA (Hai et al., 2011) and Scriptaid (Wang et al., 2011) improve activation of pluripotency markers and increase the number of ICM cells in cloned mouse embryos. Valproate is reported to have more benefi-cial effect on inner cell mass number (Kim et al., 2011).

These observations indicate that the different drugs either affect the acetylation levels of histones at different target genes, or have different effects on other arrays of proteins inside cells. Thus, further study with an expanded range of HDACi compounds in the context of cloning, cou-pled with detailed biochemical studies in other cell systems, has the potential to reveal the identities of other proteins and pathways that affect early development.

Procedural Modifications

A second area in which improvement in cloning outcome has been sought is in modifying the methods employed for removing the spindle–chromosome complex, inhibit-ing cytoskeleton function to permit this, or procedures used for microinjection or cell fusion to accomplish the nuclear transfer. In zebrafish, an improved method of depleting the oocyte genetic material by laser ablation enabled success-ful cloning (Siripattarapravat et al., 2009). For cloning in mice, incorporation of the piezo pipette driver (Wakayama et al., 1998) enabled nuclear transfer into oocytes by micro-injection. The same instrument facilitates penetration of the zona pellucida with a blunt pipette for the SCC removal. Early concerns that important SCC-associated molecules might be removed in this manner, as well as the laborious


and invasive aspects of physical SCC removal and diffi-culty in visualizing the spindle in some species, prompted the development of a “chemical enucleation” or “induced enucleation” method using a combination of etoposide and cycloheximide treatments (Fulka and Moor, 1993) to bind chromosomes together whilst inducing polar body extru-sion, ethanol-mediated oocyte activation in the presence of demecolcine (Ibanez et al., 2003) to elicit extrusion of the entire genome in a fraction of oocytes, or “chemically assisted enucleation” using activation in the presence of demecolcine and sucrose to allow a SCC-containing protru-sion to form followed by physical removal of the protrusion (Yin et al., 2002). Although adverse effects of the treat-ments can arise, these efforts reveal interesting aspects of oocyte and spindle biology.

Another aspect of the cloning procedure has recently been modified in conjunction with the use of TSA. Using latrunculin A instead of cytochalasin to disrupt microfila-ments, in combination with the application of TSA, enabled an enhanced rate of SCNT success along with procedural simplification (Terashita et al., 2012). Latrunculin A and cytochalasin B differ in their effects, in that cytochalasin B can produce scattered microfilaments. The consequences of using of DMSO as a solvent in these studies were not examined. The apparently beneficial effects of latrunculin A indicate that the manner of microfilament dispersal may affect the health and physiology of the cloned embryo, and as a result suggests that further use of the two drugs may provide a useful approach to studying the roles of micro-filaments in oocyte and cell architecture and the ability of these structures to reform after different modes of depo-lymerization. Procedural modification in SCNT, such as co-transfer of parthenogenetic/fertilized embryos along with SCNT embryos, also improved post-implantation develop-ment in the mouse (Meng et al., 2008; Tsuji et al., 2012). However, the positive effects of procedural modifications are often confined to species with a short generation inter-val. While in vitro developmental efficiency is usually very high in mouse SCNT, post-implantation development is better in species with long gestation periods, such as cat-tle, horses, and camels. The early-onset embryonic genome activation, short gestation period, and rapid progression of events in fetal development in the mouse may limit the window of opportunity for compensating for deviations in temporal and spatial expression patterns of key genes, or deviation from their optimum expression levels.

Enhancing Oocyte Composition

It is feasible to address deficiencies in SCNT embryos by enhancing the protein composition of oocytes or SCNT constructs. Chromosome aneuploidies have been proposed to contribute to SCNT embryo demise (Nolen et al., 2005; Simerly et al., 2003; Mizutani et al., 2012). Proteomics

studies revealed clathrin heavy chain (CLTC) as a major deficient protein on spindle–chromosome complexes in mouse SCNT embryos, and augmenting the expres-sion of this protein by mRNA injection after SCNT led to improved chromosome congression at the first mitotic spin-dle in 40% of cloned embryos (Han et al., 2010). However, enhancing chromosome congression did not enhance over-all term development, indicating other barriers (e.g., limited reprogramming) (Han et al., 2010).

The ability to overcome chromosome congression defects in cloned constructs by augmenting the supply of clathrin heavy chain provides an example of how it is pos-sible to augment the molecular composition of oocytes to improve cloned embryo phenotype. Given that deficiencies in nuclear reprogramming likely remain, and that epige-netic drugs can enhance cloning outcome, it is reasonable to anticipate that cloning success rates could be substantially improved by creating “super-oocytes” that contain enhanced levels of key reprogramming factors, proteins to enhance the establishment of pluripotency, or proteins to modify the metabolism and physiology of the embryo. Future studies pursuing such initiatives could provide exciting new discov-eries concerning the biology of normal embryos.

MYSTERIES YET TO BE SOLVED

The foregoing discussion highlights the many interesting phenomena that have been observed during the course of cloning studies in a range of species. These observations tell us a great deal about processes that occur in normal embryos, and the consequences of disrupting those pro-cesses. While cloning outcomes are inefficient, and while this is often viewed in the light of limitations in cloning-specific processes (e.g., reprogramming), these limitations in fact inform us about restrictions that may apply to pro-cesses during normal embryogenesis. By concentrating attention on these phenomena and associated limitations, cloning technology should continue to provide new insights into normal development.

One area in which cloning should advance our knowl-edge of development is in understanding the dialog between the cytoplasm and the nucleus, or molecules/orga-nelles closely associated with it. One example of this is the intriguing mystery of how the origin of the nucleus affects spindle formation and composition. Another example will be understanding how the nucleus regulates maternal mRNA stability and degradation.

Another area that should prove interesting is how the initial epigenetic state of the donor genome affects its abil-ity to be reprogrammed. That initial epigenetic state may simultaneously direct the aberrant expression of gene products that influence the reprogramming process, while also limiting access of genes to be modified by ooplasmic factors.

PART | I Basics32

A third area of future discovery relates to what repro-gramming factors exist in the ooplasm and the understand-ing of their roles in normal embryogenesis. The advent of the induced pluripotency field, the ongoing characteriza-tion of factors that maintain developmental potential or undifferentiated cell states, and the identification of spe-cific proteins and protein complexes that modify chroma-tin structure have vastly expanded our knowledge of the array of proteins that regulate global epigenetic states. A challenge that remains is to decipher which of these pro-teins directs reprogramming processes during normal pre-implantation development, and whether other proteins exist that contribute stage-specific functions to the process. Further use of oocyte extracts and in vitro nuclear remod-eling/reprogramming systems will likely play an important role in this discovery.

ACKNOWLEDGEMENTSThe work in the author’s laboratory is supported in part by a grant from the National Institutes of Health, National Institute of Child Health and Development (R01 HD43092).

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Principles of Cloning || Nuclear Origins and Clone Phenotype

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