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Chapter IV: Epigenetic Reprogramming: Implications for Clones and their Progeny

The previous Chapters of this Risk Assessment have introduced the concept that incomplete or inappropriate epigenetic reprogramming appears to be one of the primary underlying causes for the relatively low success rate of cloning, and the source of potential subtle hazards for the consumption of food from animal clones. Although a complete discussion of the rapidly emerging field of epigenetics is beyond the scope of this risk assessment, readers are directed to a series of excellent reviews for more details (Reik and Walter 2001b; Surani 2001; Bird 2002; Li E 2002; Davidson et al. 2003; Kelly and Trasler 2004; Santos and Dean 2004; Tian 2004; Allegrucci et al. 2005; Holliday 2005; Morgan et al. 2005; Eilertsen et al 2007; Reik 2007). An overview of the topic, however, is useful to put the issue of the source of potential subtle hazards in clones into context.

Briefly, epigenetics has been defined as the study of stable alterations in gene expression potentials that arise during development and cell proliferation (Jaenisch and Bird 2003), or alterations in DNA function without alterations in DNA sequence (Jones and Takai 2001). The central idea behind the concept of epigenetics is that although the DNA sequence of almost all the nucleated somatic cells in the body of an adult mammal is identical (except some very specialized cells whose development requires DNA rearrangements), the phenotypes of those cells can be quite different because alternate subsets of genes are expressed at different times in development and during cellular differentiation. In other words, each cell type in an organism has its own epigenetic profile or signature (Morgan et al. 2005).

Epigenetic changes have been implicated as the source of the anomalies noted in clones and other ARTs. The primary biological assumption is that as no exogenous genes are being introduced into the genome being copied and expressed (as in the case of clones) or being expressed following the union of two gametes (as in the case of the other ARTs), alterations in gene expression are responsible for the adverse outcomes noted in the resulting animals. Although much of the focus of the ongoing research in this rapidly expanding field is directed towards gathering and understanding observations of epigenetic changes in early development, epigenetic changes also occur later in life. They are part of the normal and necessary way that organisms adapt to their environments.

For example, Fraga et al. (2005) have demonstrated that monozygotic or “identical” human twins begin life with very similar epigenetic patterns. Over time, however, they accumulate epigenetic differences so that their epigenetic profiles become quite different. Smoking, diet, and other life experiences are proposed as exerting influence of the epigenetic differences observed between genetically identical twins, with more differences in life experiences correlated with more different epigenetic profiles later in life. Epigenetic changes have also been associated with various disease states that arise from the dysregulation of normal genes (reviewed in Egger et al. 2004, Jiang et al. 2004).

Epigenetic differences are also noted in conventional animals, and may reflect the status of the uterine environment. Cooney et al. (2002) investigated the effects of maternal methyl food supplements²⁵ prior to and during pregnancy on the epigenetic control of various health outcomes using an experimental system based on the expression of an epigenetically-regulated mouse coat color. The genome of mouse strain employed in this study includes an endogenous retrovirus containing viral genes and promoter enhancer sequences referred to as long terminal repeats (LTRs), which can drive the expression of retroviral genes and murine genes in their vicinity. When the LTR is active (relatively demethylated), it overpowers the endogenous mouse promoters, and allows the constant transcription of the genes giving rise to the “yellow” phenotype. This phenotype exhibits a solid yellow coat color, obesity, predisposition to cancer, diabetes, and a relatively short lifespan. When the LTR is suppressed (relatively methylated), the agouti²⁶ gene locus is regulated by its own promoters, and is expressed cyclically and only in hair follicles. The phenotype of these mice is lean, healthy animals of normal lifespan and agouti-patterned coats. In this study, pregnant dams were fed diets containing three levels of dietary 1-carbon sources or cofactors. The lowest consisted of typical laboratory mouse chow; the intermediate level was supplemented with choline, betaine, folic acid and vitamin B-12, while the highest supplementation included three times the supplement level of the intermediate diet plus methionine and zinc. In addition to evaluating coat color, Cooney et al. (2002) also determined the relative degree of methylation of the LTRs driving the agouti gene locus. They observed that as the level of methyl donors in the diet increased, phenotypes of progeny animals shifted towards the agouti phenotype. Corresponding changes were observed in the methylation status of the LTR, with increasing methylation in the animals whose dams received higher levels of methyl donors in their diets.

Another example of altered phenotype dependent on methylation status has been identified in inbred sexually-derived mouse strains. In this case, mice that carry a particular allele (specifically axin-fused which is referred to as Axin^Fu) variably express a “kinked tail” phenotype rather than a normal tail morphology. The variability in heritability of the phenotype at this locus has also been shown to be a result of epigenetic inheritance (Rakyan et al 2003). Similar to the variable coat color phenotype described by Cooney et al. (2002, above) the variable expression of the kinked tail phenotype has been shown to correlate with differences in methylation status of a particular type of transposable element at the locus (intracisternal A particle). Additionally, the variability of expression is influenced by the parent that contributed the Axin^Fu allele.  The authors hypothesize that the variability in the phenotype is related to resistance of this type of retrotransposon to epigenetic reprogramming and further influenced by differing levels of reprogramming of the genome contributed by the male or female parent. The two models are not mutually exclusive in that the retrotransposons at the loci are similar and reprogramming may be influenced by maternal methyl donor status and different for maternally and paternally derived alleles (Cropley et al 2006).

Because the field is relatively new, and the scientific community has not identified all of the mechanisms involved in epigenetic remodeling, with few exceptions (e.g., X-chromosome inactivation), the direct links between any one mechanism (or a series of mechanisms) and the health outcomes in live animals are not clear. Animals produced by non-SCNT ARTs, including natural mating, may have different epigenetic profiles, and even exhibit developmental abnormalities, but are not considered to pose unique food consumption risks.

A. Overview of Epigenetic Reprogramming in Early Embryonic Development

In conventional breeding, a new diploid genome is created by the fusion of two haploid genomes; one each from the sperm and the egg. The subsequent expression of that newly formed diploid genome to generate a functional multicellular organism is governed by a “program.” This term was first used by the genetic pioneers Jacob and Monod, who in 1961 proposed that “…the genome contains not only a series of blueprints, but a coordinated program…and a means of controlling its execution.” More than a half-century later, researchers are still trying to understand how that control is exerted.

Multiple mechanisms respond to the cell’s developmental stage or its environment by acting as positive (more transcription ²⁷) or negative (less transcription) control elements. Transcriptionally active regions of DNA (or heterochromatin) may be considered to be “open” so that various molecules involved in DNA processing can gain access to certain regions, whereas “euchromatin” is physically tightly condensed, or “closed” with respect to access by other molecules, and transcriptionally silent. The picture emerging through current research (see citations above) suggests that the overall system is extremely complex, with many degrees of “openness” existing.

One of the examples of this complexity is manifested via the extent and variety of modifications that can occur to DNA itself and its associated histones (positively charged proteins that are responsible for maintaining chromosome structure). These modifications include DNA and histone methylation at a number of positions, acetylation, phosphorylation, and ubiquitination of histones (Kanka 2003; Quivy et al. 2004; Cheung and Lau 2005; Fuks 2005; Verschure et al. 2005). Although histone modification seems to be important for fully appreciating the complete range and stability of regulation possible as well as the subtleties of the system, the methylation state of the DNA is central to the epigenetic regulation of gene expression. DNA methylation has been the subject of considerable research (reviewed by Holliday 2005, Scarano et al. 2005), as scientists begin to understand its role in gene regulation. The bulk of the discussion of epigenetics in this chapter centers on DNA methylation primarily because, to date, most of the studies of epigenetic changes in animal clones examine changes in methylation states.  

DNA methylation refers to the addition of methyl groups to the 5 position of cytosine, a non-coding portion of the nucleoside, to regulate the appropriate expression of genes (see Figure IV-1). Methylation tends to occur in areas of the chromosome that are rich in sequences that contain stretches of repeating cytosine-guanosine residues (CpG islands), which tend to be positioned at the 5’ ends of genes.²⁸ Most of these regions are unmethylated regardless of developmental stage, tissue type, or gene expression level. DNA methylation in somatic cells is generally faithfully restored at each replication cycle (for dividing cells), although changes in methylation levels are often associated with aging, or occur in abnormal cells (Bird 2002, Jaensich 2004). Methylation may affect gene transcription by physically impeding the access of cellular transcriptional machinery to coding regions, or by attracting proteins that bind specifically to the modified CpG sequences, thus impeding the transcriptional machinery (Cezar 2003).

Figure IV-1: Cytosine and 5-methyl cytosine Addition of a methyl group at the 5 position of the base is shown.

Mammalian embryos experience major epigenetic reprogramming primarily at two times in their development, both of which have significant implications for cloning. One of these takes place soon after fertilization, and is referred to as preimplantation reprogramming; the other occurs during gametogenesis (the development of cells that ultimately become the sperm and egg). Because preimplantation reprogramming occurs after fertilization, and in the case of nuclear transfer, after fusion of the donor nucleus with the oöplast, it is the most immediately impacted by the cloning process, and may be most directly implicated in the anomalous development of clones with defects. Gametogenic reprogramming may also be involved in the anomalies noted in clones, but it likely has more far-reaching implications for progeny, because it generates the gametes used for the sexual reproduction of clones, (although, by definition, the absence of gametogenic reprogramming in the somatic cell donors used for SCNT poses a high biological burden for the preimplantation reprogramming (Jaenisch et al 2004)). Most of the literature on epigenetic dysregulation in clones and animals produced using other ARTs addresses preimplantation reprogramming; the literature on gametogenic reprogramming often evaluates endpoints related to the sexual reproduction of clones (Yamazaki et al. 2003).

1. Preimplantation Reprogramming

a. Fusion and Cleavage

In sexual reproduction, mammals use cells of highly different morphology and function to deliver haploid genomes. Sperm are small relative to the oöcyte, and package their highly condensed DNA by tightly coiling the DNA around a set of proteins called protamines. The oöcyte’s genome is packaged more loosely around a different set of proteins called histones, also found in somatic cells (Cezar 2003). In order for the embryo to form a unique genome, the two chromatin structures must be resolved into one that is capable of coordinated gene expression. A number of factors present in the oöplasm of the ovum, only a few of which have been identified, are thought to aid in this remodeling (Kang et al. 2003a; Chen et al. 2006; Fulka and Fulka, 2007). In the first hour after fertilization, the sperm head swells, the nuclear envelope of the sperm breaks down, and protamines are replaced with histones (Santos and Dean 2004). The chromatin then decondenses, and the male pronucleus²⁹ forms (Mann and Bartolomei 2002). The female genome completes its second meiotic division, expels the resulting polar body, and then forms the maternal pronucleus. Both the male and female pronuclei begin to replicate DNA, and depending on the species, some transcription may ensue. In mice, transcription occurs in the male pronucleus in the first cell cycle, followed by a larger burst in the second cell cycle (Aoki et al. 1997), while in bovine embryos, transcription is delayed (Mann and Bartolomei 2002).

During SCNT, however, different initial events must take place. SCNT begins with the removal of the nucleus of the oöcyte that contains the meiotic metaphase II chromosome-spindle complex, followed by microinjecting or fusion of the donor cell or nucleus into the enucleated oöplast. The presence of oöcyte factors probably causes the breakdown of the nuclear envelope of the donor cell (similar to swelling and breakdown of the sperm head). Following oöcyte activation (usually by electrical stimulation), the chromatin from the donor nucleus decondenses, and a pseudopronucleus is formed. If successful, DNA replication and cellular division follow.

Figure IV-2: Epigenetic Reprogramming and Embryonic Development

 [D]

Legend to Figure IV-2. The top of the diagram illustrates the terminally differentiated sperm and egg (oöcyte). These gametes fuse to form the fertilized egg or zygote and begin preimplantation reprogramming (emphasized by the bracket at the left). Following the steps counter-clockwise around the figure sequential cell divisions are illustrated with corresponding differentiation from totipotent through pluripotent to differentiated. The right half of the figure represents gametogenic reprogramming (emphasized by the bracket at the right), the epigenetic marking of the primordial germ cells that will become the sperm or eggs of this new individual at sexual maturity and setting up another cycle.

Information from either terminally differentiated gametes (fertilization-derived zygotes) or a terminally differentiated somatic cell (in SCNT) must be reprogrammed so that the resulting zygote is totipotent (capable of developing into any cell type). Totipotency appears to be lost early in development and almost certainly after the blastocyst is formed, when the trophectoderm and inner cell mass begin to separate (see Figure IV-2). At this point in development, the cells are pluripotent—no longer capable of being any cell type, but retaining the ability to become many cell types. The end process is referred to as “terminal differentiation,” in which cells acquire a set of characteristics that allows them to perform a specific function (e.g., muscle cells contract, neurons transmit electrical pulses, and gametes serve as genome donors for subsequent generations). One of the ways that this overall process is accomplished is by resetting the epigenetic marks of the gametes. At this time, the signals that determine “lineage allocation” are not clear. Fujimori et al. (2003) have noted that each of the two cells in the early blastomere is completely totipotent; some lineage bias is observed when the developmental potency of four-cell stage blastomeres is evaluated. Cells inside the eight and 16 cell stage of the morula appear to be more likely to become committed to the inner cell mass lineage (which becomes the embryo), while those outside appear to be directed to the trophectoderm and the development of placental tissues (Morgan et al. 2005).

The next sections provide an overview of events as they are understood in the development of fertilization-derived embryos, followed by examples of observations noted in clones and, when available, other ARTs with significant in vitro culturing components. The examples are intended to be illustrative and not comprehensive, as an encyclopedic review is beyond the scope of this discussion. The important points to be made are that

• Mechanisms of epigenetic reprogramming are complex and not fully understood, even in “normal” fertilization-derived embryos;

• SCNT-derived embryos often do not develop normally, and all the available evidence indicates that this is due to incomplete or inappropriate epigenetic reprogramming;

• Genomes are “plastic” and can accommodate some errors in epigenetic reprogramming, regardless of whether those embryos are derived via fertilization or nuclear transfer; and that 

• Some SCNT-derived embryos go onto full gestation and delivery; clones that are born can range from those exhibiting some epigenetic dysregulation to no detectable differences.

Some have suggested (Wilmut 2002b, Jaenisch 2004) that no clone is completely “normal” with respect to its epigenetic profile. Although this is an important point for assessing the overall safety of the cloning process for any particular species, and for determining risk to animals involved in the cloning process, the relevance of “epigenetic normality” to food consumption risks is unclear. This is particularly true when considering the degree to which epigenetic changes are observed in other ARTs with a significant in vitro culturing component, and the accumulation of epigenetic changes expected during the aging process. The most compelling conclusions that can be made about food consumption risks are drawn from assessments of the health status of the animals and the composition of food products derived from them, and not from gene expression studies.
 
b. Demethylation and Remethylation in Early Embryos

Dean et al. (2001) and Morgan et al. (2005) have outlined how the process of demethylation and epigenetic resetting occur in various mammals. Hours after fertilization, but prior to DNA replication and cleavage, the paternal genome of mice, rats, pigs, cattle, and humans, but not sheep, is actively stripped of the epigenetic methylation markers by mechanisms not fully understood, but that likely require the activity of a demethylase enzyme present in the oöcyte (Morgan et al. 2005). This genome-wide methylation erasure appears to be conserved among cattle, swine, and rats, but is not observed in sheep (Wilmut et al. 2002; Beaujean et al. 2004; Young and Beaujean 2004). In mice and cattle, the maternal genome retains its methylation markers during this period, and does not undergo demethylation until the zygote undergoes the first cleavage to yield the two-celled embryo. Demethylation of the maternal genome is thought to be passive, that is, diluted by the lack of remethylation on newly replicated DNA (Cezar 2003). In two to eight cell bovine embryos, Dean et al. (2001) observed a further reduction in methylation, consistent with the passive demethylation occurring during DNA replication seen in the mouse. In contrast, mouse 16-cell embryos continued to remain demethylated, and genome-wide de novo methylation did not occur until approximately four cell divisions later, and appeared to occur preferentially in the inner cell mass (ICM). Thus, although the overall process of demethylation and de novo methylation appears to be conserved in the species evaluated, the timing of these phenomena may differ among species (Morgan et al. 2005). The more important observation, however, is that the first differentiation event in mammalian embryos (the differentiation of the trophectoderm and ICM and the resulting loss of totipotency of the ICM cells) is accompanied by genome-wide de novo methylation.

Fertilization-derived bovine embryos begin to demonstrate global genomic de novo methylation in the eight- to 16-cell stage, what is often referred to as the maternal to embryonic transition (MET). During this time, the developmental program that is initially directed by components within the egg (maternal) is replaced by a new program directed by the expression of new genes (Wrenzycki et al. 2005), and is accompanied by different rates of demethylation of maternally and paternally derived genes to give rise to a new methylation pattern for the embryo.

Although the early demethylation described above is global (occurring over the entire genome in general), methylation marks on imprinted single copy genes tend to be protected from demethylation so that parental imprints are preserved in the resulting somatic cells of the developing mammal (Li E 2002). It is unknown whether the extensive global demethylation of the genome during pre-implantation development is essential for normal development.

DNA-methylation patterns unique to the developing mammal are established in the embryo after its implantation in the uterus through lineage-specific de novo methylation that begins in the inner cell mass. DNA methylation increases rapidly in the primitive ectoderm, which gives rise to the entire embryo. Conversely, methylation is either inhibited or not maintained in the trophoblast and the primitive endoderm, from which the placenta and yolk-sac membranes develop, respectively (Li E 2002). The net effect is that extra-embryonic tissues appear to have a lower methylation state than embryonic tissues. These global differences in methylation status between the embryonic and extra-embryonic tissues appear to be conserved in mice, cattle, sheep, and rabbits (Morgan et al. 2005).

Reprogramming the donor nucleus in SCNT or the nucleus of the early fertilized embryo has been the subject of considerable investigation over the past few years. Much of this research has been summarized in reviews by Rideout et al. 2001; Jaenisch et al. 2002; Mann and Bartolomei 2002; Cezar 2003; Han et al. 2003; Jouneau and Renard 2003; Smith and Murphy 2004; Young and Beaujean 2004; Wrenzycki et al. 2005; Armstrong et al. 2006; and Eilertsen et al. 2007.

These reviews and the studies contained in them have come to approximately the same conclusions: although some clones may develop into healthy animals, the low success rate of SCNT is likely associated with the inability of clones to reprogram the somatic nucleus of the donor to the state of a fertilized zygote. Similarly, the rates of successful embryo formation resulting in term gestation and live births in ARTs that have a high degree of in vitro culturing are likely also due to difficulties in reprogramming (Gardner and Lane 2005, Wrenzycki et al. 2005). The sources of the stresses on the embryos that cause these difficulties may be a reflection of the intrinsic biological differences between fertilization- and nuclear transfer-derived embryos (e.g., the need to fully reprogram a differentiated nuclear donor), or technological (e.g., the in vitro environment in which early embryos are cultured prior to introduction into the uterus). The following discussion briefly outlines the current state of knowledge of how this is accomplished in fertilization- or nuclear transfer-derived embryos.

In embryos derived via nuclear transfer, epigenetic modification, such as the waves of demethylation and de novo methylation observed following fertilization must also occur, but may be hampered by both the nature of the donor DNA and the partially depleted oöplasm. There are reports of both aberrant and “normal” demethylation and remethylation in clones and fertilization-derived embryos. Differences may be reflections of different methodologies, source cells, species differences, or may reflect unexplained phenomena. The following discussion summarizes the key observations that contribute to the body of knowledge regarding epigenetic remodeling in SCNT- and other ART-derived embryos.

Some species-specific responses in the degree of methylation reprogramming have been observed, although in general, the overall processes appear to be relatively conserved among the clones of different species. Dean et al. (2001) found that somatic nuclei of mouse, rat, pig, and bovine embryos undergo the genome-wide reprogramming described previously, but that reprogramming occurred aberrantly in many cloned preimplantation embryos. Bourc’his et al. (2001), using a similar method, did not observe active demethylation in bovine SCNT zygotes, although they did observe that the somatic pattern of methylation from donor nuclei was preserved through the four cell stage.

Ohgane et al. (2001) compared the methylation status of CpG islands (CG-rich sequences located at promoter regions) in placenta and skin cells of sexually reproduced mice to similar regions in normal-appearing mouse clones. Most of the methylated regions in fetal clones (99.5 percent in the placenta and 99.8 percent in the skin) were identical to those of the controls, but different methylation patterns were observed in the two different tissues. The sites of discordant methylation were located in regions responsible for expression of tissue-specific genes, despite the absence of grossly observable abnormalities. In bovine preimplantation embryos, however, Kang et al. (2001a) noted that bovine clone embryos failed to demethylate satellite regions of the genome (certain repetitive sequences), and instead maintained methylation levels similar to the donor cell. In a subsequent study, however, Kang et al. (2001b) were able to “rescue” the inefficient demethylation of bovine embryos by providing an additional “dose” of oöcyte factors to the early embryo. This work confirms the presence of an active element in the oöcyte for erasure of paternal epigenetic methylation, and implies that this component, which may be removed or diluted during the process of preparing an enucleated oöplast, is involved in the appropriate epigenetic modeling observed in zygotes and early embryos derived from fertilization. In a third study, Kang et al. (2001c) investigated demethylation in swine clone embryos relative to those derived by in vitro fertilization (IVF). They observed that, unlike mice and cattle, the sequences investigated (centromeric satellite DNA) were negligibly methylated in swine oöcytes, and hypermethylated in swine sperm. (Sperm satellite DNA sequences in cattle and mouse tend to be undermethylated.) The satellite sequences of the donor pig fibroblast cells were hypermethylated, and retained that status until the 4-8 cell stage. Demethylation began at that time, and the methylation status of the clone embryos decreased significantly in the blastocyst, just as it did in the blastocysts of in vitro or in vivo fertilization-derived embryos. Their finding thus indicated that satellite sequences of SCNT-derived pig embryos undergo preimplantation demethylation in a manner similar to fertilization-derived embryos. Analogous results were observed when another sequence, PRE-1 (from the euchromatin) was evaluated. These results are comparable to the pattern observed in mouse embryos by Dean et al. 2001.

Whether the results obtained from these two DNA sequences studied by Kang et al. can be extrapolated to global DNA methylation or other single-gene sequences in the pig remains unknown. Additionally, the reasons for the interspecies differences between mice and pigs on one hand, and cattle on the other, also remain unknown. Nonetheless, one of the key implications of these observations is that global demethylation soon after fertilization appears to be a prerequisite for successful reprogramming later in embryonic development, and possibly for successful SCNT outcomes.

Kang et al. (2003) have also demonstrated that at least some SCNT-derived bovine embryos are capable of normal remethylation during early embryogenesis. They evaluated the methylation status of a 170 base pair fragment of single copy gene in IVF and SNCT-derived bovine embryos. This sequence is negligibly methylated in both sperm and oöcyte DNA, and moderately (approximately 37 percent) methylated in the fibroblasts that served as nuclear donors for SCNT. In single celled zygotes, as well as the 4-to 8-cell stage embryos derived via IVF, the low methylation levels of the sperm and oöcyte genomes were observed. No significant changes in methylation status of the IVF-derived embryos were observed at the 8-16 cell stage, but by the time a blastocyst had formed, de novo methylation appears to have taken place. In SCNT-derived embryos, the methylation pattern of the donor cell was nearly completely lost by the 4-8 cell stage, and demethylation appeared to be complete by the 4-8 cell stage. At the blastocyst stage, the methylation stage of the SCNT-derived embryo was exactly that of the IVF blastocyst, with the same CpG sites exclusively methylated in both sets of embryos. The authors claim that this study is “the most elaborate example of recapitulation of normal embryonic process[es] occurring in [SC]NT embryos.” Although this study clearly demonstrates the ability of somatic cells to be epigenetically reprogrammed in an accurate manner relative to an IVF comparator, and that these molecular results are consistent with observation of apparently healthy and normal animal clones being generated from somatic cell donors, the predictive value of this particular gene for other single copy genes, or the entire genome has not been demonstrated.
 
In another study showing differences in methylation states among species, Beaujean et al. (2004) evaluated the global methylation status of fertilized and SCNT-derived sheep embryos. They observed that unlike mice and cattle, sheep oöcytes do not appear to demethylate the sperm-derived pronucleus after fertilization. In vivo-derived sheep embryos demonstrated that a partial demethylation of the global genome occurred up to the 8-cell stage, with similar qualitative findings in SCNT-derived embryos (fibroblast cell nuclear donor), but to a lesser extent. Interestingly, between the 8-cell and blastocyst stages, both in vivo- and SCNT-derived embryos showed comparable overall levels of methylation, but the distribution of methylation patterns differed among the SCNT-derived embryos and between some of the SCNT-derived embryos and those derived from fertilization. The authors attributed these differences to differences in the overall high-order chromatin structure, rather than simply to changes in methylation. They suggested that many SCNT-derived embryos do not undergo the rapid reorganization of the DNA prior to first cleavage that successful in vivo-derived (and a small proportion of SCNT-derived) zygotes do. Further, they suggest that perturbations in methylation (and possibly remodeling) correlate with the lack of appropriate trophectodermal development and subsequent placental development in later embryos and that these alterations may contribute to the high observed levels of placental defects and embryonic loss during SCNT-pregnancies. Beaujean et al. conclude that although “DNA methylation appears to be marker of reprogramming in all mammalian species examined to date, it is not yet clear to what extent it is a determinant.”

In a recent attempt to more directly address the role of methylation status in reprogramming, Blelloch and colleagues (2007), used cells in a mouse model system containing an allele (variant) of a DNA methyltransferase (Dnmt1) that has reduced levels of activity. As a consequence of the mutation the cells have reduced DNA methylation. They found that fibroblasts containing the mutation could be reprogrammed into embryonic stem cell lines three times more efficiently than comparators whose DNA was relatively more methylated, consistent with the hypothesis that decreased global DNA methylation yields a less differentiated state. 

c. Epigenetic Reprogramming in Later Development

This summary covers studies of epigenetic reprogramming from the fetal through adult developmental nodes (as described in more detail in Chapter VI).

In a study of genome-wide epigenetic reprogramming in bovine clone embryos and adults, Cezar et al. (2003) measured the amount of 5-methyl cytosine in DNA from various tissues in fetuses and term pregnancies generated via SCNT and fertilization. Their results showed that the amount of methylation was lower in spontaneously aborted fetal clones, fetal clones sacrificed as part of the study, and tissues collected from pregnancies that had experienced hydroallantois relative to controls. These results are in contrast to others that have found hypermethylation in clones relative to fertilization-derived controls (Bourc’his et al. 2001; Dean et al. 2001; Kang et al. 2001b). Adult clones, however, had similar levels of DNA methylation as adults derived via fertilization. Cezar et al. (2003) concluded that there may be an epigenetic reprogramming threshold that is met by a subset of animal clones. They also proposed that clones surviving into adulthood have the ability to overcome epigenetic challenges determined by their somatic cell origin. These hypotheses are consistent with the observations by Chavatte-Palmer et al. (2002), Chapters V and VI, and the Cyagra dataset, described in Appendix E, in which early physiological instabilities appear to resolve as the clones mature.

Similar to Cezar’s observations, Chen et al (2005) also noted that aberrant methylation likely plays a role in the poor development noted in clones and other forms of assisted reproduction. In their study, the methylation status of aborted bovine clone fetuses, aborted fetuses generated by artificial insemination (AI), and adult cattle generated via AI or cloning was studied. Three genomic regions were evaluated: a repeated sequence and the promoter regions of two single copy genes (interleukin 3 and cytokeratin). All of the aborted fetuses (AI- and SCNT-derived) were females between 60 and 90 days of gestation; adult animals were all classified as “healthy” and between 18 and 24 months of age. The adult animals all had approximately the same level of methylation at all of the loci examined, regardless of method of production. The aborted AI-derived fetuses all had similar, but lower levels of methylation than the healthy adults, as well as different methylation patterns. For the single copy genes, methylation could be classified into two groups: one group had very low methylation patterns in the promoter regions, while the other group had methylation patterns similar to the aborted AI-derived fetuses. One of these fetuses also showed low methylation patterns in the satellite region. Although this study is not conclusive, it does provide evidence that at least for certain regions of the genome, appropriate methylation appear to be correlated with normal development.

Dindot et al. (2004) developed a unique bovine hybrid interspecies model (Bos gaurus x Bos taurus) to study epigenetic markings and imprinting in gestation day 40 female SCNT-derived fetuses and placentae (derived from cumulus cell donor cells) that were genetically identical to fetuses derived by fertilization. Previously, Hill et al. (2000b) had shown that more than 80 percent of bovine clone pregnancies were lost between days 30-60 of gestation, and attributed the losses to placental anomalies including a reduction in the number of expected cotyledons and a decrease in chorio-allantoic blood vessels. These observations were similar to those of Stice et al. (1996) who reported that no placentomes had developed in NT fetuses that died between gestation days 33-55. Mouse clone pregnancies have also shown increases in placental size (Tanaka et al. 2001). These abnormalities have been hypothesized to arise from anomalies in nuclear reprogramming of the trophectoderm, which gives rise to placental structures including the chorion. By using the hybrid Bos gaurus/Bos taurus model, Dindot et al. were able to discriminate between parental alleles by following single nucleotide polymorphisms (SNPs) (changes in the nucleotide sequence of the DNA at only one site that allow for the cleavage or the lack thereof by enzymes that recognize specific DNA sequences). In particular, three genes associated with epigenetic reprogramming were selected including IGF-2, Gene trap locus 2 (GTL2), and the X chromosome inactivation specific transcript (Xist). Clone fetuses and placental tissues were isolated from surrogate dams at gestation day 40; none of the clone placentae developed cotyledons, unlike the fertilization-derived fetuses, which had 4, 16, and 25 cotyledons per pregnancy. Although appropriate allelic expression of IGF2 and GTL2 relative to fertilization-derived fetuses was observed in both chorionic and fetal liver tissues of the clones, disruptions of genomic imprinting of the Xist locus was found in the chorion, but not the liver tissues of any of the clones. Further analysis of two other regions of the genome in the chorion of the clone fetuses, the satellite I repeat element and epidermal cytokeratin promoter, indicated that the trophectoderm-derived tissues of the clones had higher levels of methylation relative to fertilization-derived controls. No differences in methylation levels were observed in the livers of clones or fertilization-derived embryos. In this study, at least, there were differences in the degree of epigenetic reprogramming between ICM-derived tissues (the fetus proper) and those derived from the trophectoderm, consistent with the observation by Hill et al. (2000b), that clones with aberrant placental structures can survive gestation and be born alive and apparently healthy.

To understand epigenetic reprogramming over the lifetime of mice and to identify differences between the methylation status of animal clones and sexually derived comparators, Senda and colleagues (2007) have extended their previous studies (Ohgane et al. 2001) using a system that allows them to simultaneously examine ~2,000 methylation sites. In this study, they compared the methylation patterns in kidney tissues of newborn, adult (8-11 months old), and aged (23-27 months old) mouse clones with age matched comparators. The study also includes up to third generation sequential clones (animal clones that were generated with SCNT of nuclei from animals that are themselves clones).

This study had three main findings. In the first, the authors determine that the methylation patterns for newborn mouse clones differed from age matched comparators in only three of ~2,000 methylation sites, indicating that these clones did not differ markedly from comparator mice derived from IVF. The second finding indicated that the differences in the methylation pattern between clones and sexually reproduced comparators disappeard by 23-27 months of age. Clones at the intermediate time point measured (11 months) differed from their sexually reproduced counterparts in only one point of the methylation pattern. Finally, sequential cloning (that is, using a cell from a clone as a nuclear donor for a subsequent round of cloning), did not appear to either increase or perpetuate differences in methylation patterns in this study.

Although the extent to which these data from this mouse model system is a compelling indication that the epigenetic dysregulation assumed to be responsible for adverse outcomes in clones may indeed resolve with age. These results may also be useful for observation that although animal clones are at greater risk for health problems at birth, those that reach adulthood are often healthy (See Chapters V and VI).

d. Studies of Gene Expression and Development in Clones and Animals Produced by Other ARTs

The previous sections summarized studies of alterations in methylation associated with cloning; the following section summarizes reports of gene expression and phenotypic observations in similar clone populations. The overview is intended to be more illustrative than comprehensive as the literature on this subject is large and growing rapidly. The studies indicate that for non-viable clone embryos, fetuses, or neonates, key genes are inappropriately expressed. In some cases, viable clones have differences in expression compared to fertilization-derived counterparts, leading investigators to speculate that genomes are plastic and that a certain level of gene dysregulation can be tolerated. In other studies of healthy, live clones, no significant differences can be observed between the expression profiles of animals generated via SCNT or other fertilization-based ARTs. Finally, it should be noted that studies comparing embryos generated via various ARTs (including SCNT) with significant in vitro culturing components, appear to be sensitive to the culture environment, with developmental success often being a function of the culture medium used.

Most of the earliest studies of gene expression in clones were performed in mice. Boiani et al. (2002) and Bortvin et al. (2003) evaluated patterns of gene expression in mouse blastocysts derived from SCNT to identify which critical genes were involved in the inability of most of those blastocysts to develop further. In particular, they evaluated the expression of Oct4 and Oct4-related genes in these embryos. (Oct4 is a transcription factor specifically expressed in stem and primordial germ cells, and appears to be required for maintaining pluripotency and the self-renewal ability of stem cells.) Boiani et al. (2002) compared Oct4 expression in blastocysts cloned from somatic cell nuclei and germ cell nuclei to that observed in synchronous blastocysts produced by IVF and intracytoplasmic sperm injection (as the control groups independent of cloning but involving micromanipulation). Their results demonstrated that mouse blastocysts derived from clones had abnormal Oct4 expression, and that the failure of mouse clones embryos to develop beyond the blastocyst stage was related to incorrect lineage determination by the inappropriate expression of Oct4. Bortvin et al. (2003) identified 10 candidate genes with expression patterns similar to Oct4 and compared their expression in preimplantation embryos derived from fertilization to embryos whose SCNT donors were somatic cumulus and pluripotent embryonic stem cells. They demonstrated that successful reactivation of the full set of 10 genes correlated with the development of embryo clones, but also noted that almost 40 percent of the cumulus cell-derived blastocysts failed to reactivate these genes faithfully, even though the blastocysts were morphologically normal. Thus, some other factors were required to maintain the pluripotency of the inner cell mast cells. Marikawa et al. (2005) found that the DNA methylation status of the Oct4 regulatory element in mouse embryos directly influences the level of gene expression. They further noted that the methylation status of the Oct4 regulatory element was highly heterogeneous among alleles in a population of adult somatic cells, and hypothesized that that the degree to which Oct4 can be reactivated in SCNT may be a function of the methylation status of the donor cell(s).

Boiani et al. (2005) further evaluated Oct4 expression in early post-activation SCNT-derived zygotes, fertilization-derived early embryos and parthenotes in six different culture media. (Lack of expression of Oct4 precludes further development beyond the blastocyst). Among their first observations was that similar to fertilization-derived embryos, progression to blastocyst did not ensure further development of the embryos, and that some of the primary influences on whether such development occurred could be environmental in origin. They also noted that nuclear transfer embryos appeared to be more sensitive to environmental conditions than the other two types of embryos. They concluded that not only was the ability of mouse clone embryos to progress through development contingent on the nature of the donor nucleus and recipient oöplasm, but that culture conditions could have a significant impact on the expression of key genes required for reprogramming (and subsequent development), and the ability of the blastocyst to continue to develop successfully.

To study the correlation between gene expression, survival, and fetal overgrowth (e.g., LOS-type symptoms), Humpherys et al. (2001) examined imprinted gene expression in mice cloned by nuclear transfer and in the embryonic stem cell donor population from which they were derived. They determined that transcript levels of selected imprinted genes varied widely in placentae from animal clones relative to non-clones, although alterations in the expression of one imprinted gene did not correlate with abnormal expression of other imprinted genes. They also observed that changes in DNA methylation levels at one imprinted locus did not necessarily predict changes at other loci. Certain genes (e.g., H19 and Igf2) were largely silenced in the heart and kidney, and their expression reduced in the livers of animal clones relative to conventional animals. No correlations were observed between changes in gene expression and birth weights, placental weights, or neonatal mortality. Culturing the embryonic stem cells in vitro resulted in highly variable levels of gene expression; gene expression in the animals resulting from those cells was even more variable than in the cells in culture, implying that culturing early embryos may contribute to the degree of embryonic gene dysregulation. Furthermore, mice derived from the cells of the same cellular lineage differed in their expression of imprinted genes. Given that viable animals were generated with variable expression of imprinted genes, the authors concluded that “mammalian development may be rather tolerant to epigenetic abnormalities and that lethality may only result from the cumulative effects of a stochastic loss of normal gene regulation at multiple loci....even apparently healthy animal clones can have gene expression abnormalities that are not severe enough to impede development to birth but that may cause subtle physiological abnormalities which could be difficult to detect.” The degree to which such subtle abnormalities could exist within conventional populations of animals is not discussed.

Humpherys et al. (2002) evaluated expression of more than 10,000 genes in the placenta and liver tissues of mouse clones derived from embryonic stem cells and cumulus cells using microarray analyses. The expression of 286 genes was altered in clones derived from cumulus cells compared to the fertilization controls, with a similar level of altered expression detected in the embryonic stem cell-derived clones. The general concordance in the expression differences between the mouse clones from different donor cell types suggested to the investigators that most of the expression abnormalities were common to all placentae of mouse clones rather than specific to animals derived from one particular cell type. Consistent with their previous summary (2001), the authors concluded that differences in gene expression, even those that are highly variable, may be tolerated during differentiation and even in clones that survive. 

Several groups have recently extended this type of microarray technology to analysis of bovine embryos derived via nuclear transfer and other ARTs (Smith SL et al. 2005; Herath et al. 2006; Beyan et al, 2007a).  Although there are procedural differences among the studies, the results are complementary and consistent. They include the observations that the gene expression patterns in cell lines from which embryos were derived were strikingly different from the patterns observed in the resulting embryos. In addition, gene expression patterns in the embryos were relatively similar overall regardless of the cell type and methods used for derivation of the embryos. Finally, a small fraction of the overall number of messages examined was differentially expressed in embryos derived using different methodologies. Similar to the studies using mouse embryos discussed above, these studies show that significant reprogramming also occurs in bovine embryos. These studies are also consistent in that the reprogramming is currently imperfect and there are a number of messages that are differentially expressed in SCNT derived embryos compared with embryos derived using other ARTs. Additionally, numerous studies have shown that many SCNT embryos will not reach term.  The embryos used in these studies were early in gestation, so we do not know if the expression differences reflect significant abnormalities or tolerable variation. However, these studies are useful because they contribute to a better understanding of both development and technical approaches to improving SCNT technology.

Sebastiano et al. (2005) noted that in single cells derived from early preimplantation embryos of mice developed via SCNT and in vitro fertilization, a series of genes important to appropriate embryonic development began transcription at approximately the same time in both types of embryos. Different levels of expression, however, were found in the nuclear transfer-derived embryos, particularly as the embryos progressed through development. They concluded that reprogramming was initially quickly shifted towards embryonic development, but that reprogramming was incomplete and inaccurate, particularly in the latest stages of preimplantation.

Several studies have attempted to determine whether the expression of any particular gene(s) could be used as a marker to determine the developmental success of embryos produced via SCNT or other ARTs. Camargo et al. (2005) evaluated differences in gene expression in individual preimplantation bovine embryos produced via SCNT (same donor cell line), in vitro fertilization (IVF), or in vivo derived embryos obtained following superovulation, artificial insemination, and harvested, and cultured in vitro to reach the same degree of development as the nuclear transfer or IVF embryos. Using real time PCR, they studied a panel of 11 genes (including Oct4) preferentially activated at the maternal-embryo transition (~ the 8-12 cell stage in bovine embryos), during which demethylation of parental genes (or donor cell genes) largely has been accomplished and de novo methylation, in which transcription of embryonic genes becomes predominant. Also evaluated was the expression of a fibroblast gene expressed in the donor cells to determine whether cessation of expression of donor genes was also appropriate. The results indicated that the expression patterns of the 11 genes common to the IVF and SCNT-derived embryos were virtually indistinguishable. Further, the expression of the donor cell gene was appropriately turned off in the SCNT-derived embryos. Compared to expression levels in the in vivo derived embryos, however, all transcripts except one, lactate dehydrogenase, in both the IVF and SCNT-derived embryos were found at lower levels. They attributed the differences in expression between the in vivo- and in vitro-produced embryos to differences in culture conditions. To support this hypothesis, the investigators noted that the IVF and SCNT embryos exhibited similar variability in expression among individual embryos, but different from their in vivo counterparts.

Miyazaki et al. (2005) compared the expression of a different set of genes from SCNT-and intracytoplasmic sperm injection (ICSI)-derived 2-4 cell and blastocyst stage porcine embryos. The genes selected have previously been suggested as candidates as markers for identifying embryos that would successfully develop (Daniels et al. 2000) included two genes from the fibroblast growth factor family, Xist (important in X-chromosome inactivation), genes encoding interleukin-6 and its receptor, and c-kit ligand (another gene important in early embryonic development). Donor cells for the SCNT-derived embryos came from two different cell lines, with different degrees of success at developing blastocysts. Additionally, SCNT-derived embryos were developed using two different activation protocols. Although the percentage of embryos in which expression of these genes was similar between the SCNT- and ICSI-derived embryos, actual levels of transcripts of two of the genes (FGFr72IIIb, one of the fibroblast growth factor genes, and interleukin 6 receptor gene) were lower and higher, respectively, in SCNT-derived versus ICSI-derived embryos in one of the SCNT-activation protocols, while FGFr72IIIb and Xist transcripts were lower than ICSI-derived embryos when evaluating the other method of SCNT activation. No significant differences in gene expression were noted at these early developmental stages between the two different SCNT donor cell sources. No comparisons were made to in vivo derived embryos. It is not clear whether the differences between the results observed by Miyazaki et al. and Camargo et al. are due to experimental design, species, or the genes assayed.

Both appropriate and inappropriate gene expression have been observed later in the development of fetuses, neonates, or more mature clones. Yang L et al. (2005) used real-time PCR³⁰ to compare the expression levels of three imprinted genes associated with growth regulation (Igf2r and Igf2) or imprinting regulation (H19) in eight tissues from deceased newborn calf clones, three tissue sources from apparently healthy, genetically identical adult bovine clones, and cattle obtained from a slaughterhouse. The deceased clones all exhibited signs of LOS, and exhibited abnormal and highly variable expression of the genes, despite being produced from one nuclear donor. The decreased levels of expression of Igfr2 (which inhibit fetal growth) in the deceased clones compared to controls were consistent with the decreased expression of the same gene noted by Young et al. (2001), in LOS sheep clones, but interestingly, these levels were not correlated with increased birth weights of the deceased clones. Expression of the three genes in the healthy clones was largely normal, except for Igf2 in the muscle tissue of adult clones, which was found to be highly variable, although lower than the reported levels for the newborn controls. These results are consistent with the idea that significant dysregulation of imprinted genes results in embryonic or neonatal death, but that those animal clones surviving to adulthood can be epigenetically similar to control animals.

A similar study, this time using real-time PCR to study the expression of IGF binding proteins and IGF receptors in Japanese Black beef bovine embryos derived using via SCNT, BNT, IVF, and in vivo, production was performed by Sawai et al. (2005) to determine whether the rates of developmental failure seen in nuclear transfer embryos could be related to changes in this complement of genes. The results indicated that the amounts of Igf-1 receptor mRNA did not differ significantly among the types of embryos; in contrast, the amounts of mRNA of the Igf-2r differed depending on how the embryos were derived. In general, the proportion of embryos exhibiting Igf-2r receptor mRNA was more variable in embryos derived via SCNT. This is in contrast to the observations of Wrenzycki et al (2001), who found that the levels did not differ significantly. There are multiple reasons to account for the differences including the source of donor cells, their cell-cycle, etc.  Heyman et al. (2002a) also reported that there were no significant differences in the proportion of embryos expressing IGF-2r transcripts among nuclear transfer, IVF and in vivo produced embryos. These apparently contradictory observations serve to imply that it is not some process associated with cloning per se (e.g., electrical pulses for cell fusion or other treatments) that adversely affect development, but rather provide further evidence that epigenetic dysregulation (i.e., failure of appropriate gene expression) that appears to be responsible for growth dysregulation.

Li S et al. 2005 also used real time PCR to compare expression levels of eight developmentally important genes in six organs from bovine clones that within 48 hours of birth relative to control animals produced by artificial insemination and also slaughtered within 48 hours of birth. Organs that were evaluated included the heart, liver, kidney, spleen, lung, and brain. Aberrant and highly variable gene expression in the clones occurred in a tissue-specific pattern, with the heart most (five of eight genes), and the kidney, least (two of eight genes) again indicating the role of gene expression in the ability of particular tissues and organs to develop appropriately in clones. They also noted that organ systems could be affected independently of others, implying a stochastic process at work. No mention was made of whether a similar study had been performed on live, healthy clone births in this report.

Finally, Archer et al. (2003a) have performed the most comprehensive study of the correlation between epigenetic reprogramming and live clone outcomes in a cohort of female swine clones. (More detailed discussions of the results of this study are found in Chapters V and VI). In addition to evaluating methylation in two different regions of the genomes of these animals and half-sibling comparators, the investigators studied the growth, clinical chemistry, and behavior (Archer et al. 2003b) of these animals. The overall degree of methylation between clones and their half-siblings was the same, with a small random variability in the PRE-1 SINE regions, and one CpG site in the centromeric satellite region. Further, the clones exhibited two patterns in specific phenotypic traits: one set of traits exhibited variability similar to the comparators, and another set showed less variability than the comparators. CpG methylation was measured in PRE-1 SINE (repeat sequence in a euchromatic region) and centromeric DNA (repeat sequence in a heterochromatic region) obtained from skin punch samples. Finally, the clones appeared to have grown and developed normally: no differences were observed between clones and their comparators with respect to growth rates, physiological measures of health, or behavior.

e. Studies of Technical Contributions to Epigenetic Variability in Clones and Other ARTs

In the previous discussion, we have repeatedly referred to the contribution of the methodology used to generate embryos (particularly the culture environment) as playing a critical role in developmental success for cloning and other ARTs.  The continuing reports of relatively low efficiency of SCNT have stimulated a wide interest in how a variety of factors affect the efficiency of SCNT in particularly but including various ARTs with an eye to improving the process (Oback and Wells 2007a, Collas and Taranger 2006) particularly, but not exclusively, by improving nuclear reprogramming. A number of factors affecting the status of the cells used for nuclear transfer seem to be important considerations for improving the efficiency of the process and recent reports identify the morphology, proliferative characteristics, chromosome stability (Giraldo et al. 2006; Mastromonaco et al. 2006), cell type, culture conditions (Bosch et al. 2006; Inoue et al. 2006; Beyhan et al. 2007b), and stage of the cell cycle (Bordignon and Smith 2006) as important considerations for improving efficiency of SCNT. 

Others reports are more directed at identifying the technical conditions that influence cloning efficiency with most focusing on improving reprogramming. Several investigators have examined the contribution of the oöcyte to reprogramming in SCNT (Chen et al. 2006, Fulka and Fulka 2007) in the hope of identifying oöcyte factors that increase reprogramming and consequently cloning efficiency.  Preparation of the oöcyte (Li GP et al. 2006) as well as timing (Sung et al. 2007) and method (Schurmann et al. 2006) of activation after fusion of the oöcyte and transferred nucleus have been examined for their contribution for the process.  Reprogramming may be improved by the addition of exogenous “remodeling factors” such as nucleoplasmin (Betthauser et al. 2006) or even caffeine (Lee and Campbell, 2006). Although all of these studies (which use a variety of species and most of which harvest the embryos before term) identify factors in the process that are important, the efficiency of SCNT remains low.

Two studies by Wolf and colleagues (Hiendleder et al. 2004a, 2006) also provide insight into the relationship between reproductive methodology, methylation status, and fetal characteristics. The 2004 study compares a number of anatomic parameters to the “global” DNA methylation status of a variety of tissues from embryos produced using AI, IVF, and SCNT. In general, they correlate increased DNA methylation in fetal tissues and increased fetal size. In this study, 80 day AI fetuses were smallest and contained the least methylation, IFV fetuses of the same age were intermediate for both traits and clone fetuses, also 80 days old, were largest and had the most DNA methylation. The 2006 study extends these observations by focusing on the culture conditions used for IVF. This latter study demonstrates that the authors can manipulate the culture conditions so that under one set of culture conditions the fetuses are physically comparable to in vivo fertilized comparators while other culture conditions result in consistently large offspring as reported for IVF and SCNT derived fetuses as reported in their 2004 study.  However, even in the “normal” size IVF fetuses, global methylation status is not identical to the comparators, indicating that further work needs to be performed to identify and characterize key parameters related to fetal overgrowth syndromes.

2. Gametogenic Reprogramming

The development mechanisms involved in gametogenic reprogramming were initially studied most extensively in the mouse; conservation of mechanisms involved in sexual reproduction are similar in all species examined to date, although the timing of events differs depending on the length of gestation.

Germ cells (those developmentally destined to become gametes) are first detected as founder population cells at about embryonic day (E) 6.5 in the mouse. By E 7.2, approximately 45 primordial germ cells can be counted in the mouse embryo (Hajkova et al. 2002). These cells begin migration into the genital ridge (the portion of the embryo destined to become the reproductive organs) about 10 days after embryo formation (Hajkova et al. 2002, Yamazaki et al. 2003) (See Figure IV-2). Their epigenetic methylation status at this point resembles that of the rest of the embryo: they contain genomic imprints from the maternal and paternal genomes, and one of the two X chromosomes in female gametes has been inactivated in the somatic tissues (Surani 2001). Once the primordial germ cells migrate into the genital ridge (the thickening near the kidneys of the embryo that gives rise to the ovaries and testes), however, profound changes in their methylation status occur. A period of rapid demethylation ensues, in effect “erasing” all of the epigenetic modifications that were present on the cells prior to their migration (Yamazaki et al. 2003, 2005). This demethylation appears to be selective by affecting single copy imprinted and non-imprinted genes (e.g., coding sequences), whereas the reprogramming of repetitive elements (whose function in the cell is not fully understood but is thought to be structural and regulatory) is more protected and incomplete.

In describing this phenomenon, Surani (2001) states that this “mechanism also erases any aberrant epigenetic modifications, so preventing the inheritance of epimutations, which consequently occurs very rarely.” The mechanism by which erasure of the epigenetic markings, including demethylation, in primordial germ cells is not yet understood. Other “resetting” mechanisms also occur in primordial germ cells, including the restoration of telomere length, and repair of lesions to the coding regions of the DNA (Surani 2001).

Random X inactivation in XX (female) germ cells also occurs during the migration phase of PGCs, coinciding with the timing of X inactivation in somatic tissues (reviewed by Avner and Heard 2001; Heard 2005). Inactivation of one X chromosome in female mammals is absolutely essential to compensate for the potential doubling of the “gene dosage” that a XX genotype would present. Although not fully understood, the process by which this occurs involves coating one of the X chromosomes by an RNA molecule itself encoded by a gene (Xist) on the X chromosome, followed by DNA methylation, and covalent modifications of the histones associated with the inactive chromosome. In mice, X inactivation first occurs in the placental trophoblast cells, where the paternal X tends to be inactivated by a mechanism thought to involve the expression of a maternal gene at the blastocyst stages that exclusively inactivates the paternal X chromosomes in the trophoblast cells. The end result is that the structure of the chromosome is altered from an active, relatively loosely coiled state to a highly condensed and transcriptionally silent DNA molecule (Avner and Heard 2001).

Restoration of epigenetic modification in primordial germ cells in mice appears to take place several days later when the male germ line appears to acquire methylation at 15-16 days after conception. Remethylation of the female germ line in mice does not appear to occur until after birth during the growth of the oöcytes, and probably continues until the first meiotic division (a stage in the maturation of the cells destined to become gametes in which the chromosome number is reduced from 2n to n) (Davis et al. 2000; Surani 2001). This overall process appears to be conserved in other mammals, although the exact timing may differ according to species.

Although the preceding discussion has focused on methylation as the primary marker of imprinting, it is important to remember that there are other modifications that may contribute to the retention of “epigenetic memory” in germ cells whose identity and mechanism remain to be characterized (Davis et al. 2000; Fazzari and Greally 2004).

3. Mitochondrial Heteroplasmy

In addition to incomplete or inappropriate epigenetic reprogramming, the relatively low success rate of cloning has been hypothesized to be related to changes in the pattern of mitochondrial DNA (mtDNA) transmission following SCNT (Hiendleder 2007; St. John et al. 2005; Spikings et al. 2006). Because sperm deposit very few of their own mitochondria³¹ during sexual reproduction, mtDNA in developing embryos tends to come almost exclusively from the oöcyte and tends to be maternally inherited. During the SCNT process, if intact donor cells are used as nuclear donors, following fusion with the enucleated oöcyte, the resulting embryo may have mtDNA from both the donor and recipient cells i.e., mitochondrial heteroplasmy. If the nuclear and mitochondrial DNA originate from different sources, the normal coordination of expression of nuclear and mtDNA may be altered, resulting in altered or impaired energy production in the cell or developing organism.

The extent to which mtDNA heteroplasmy is observed in animal clones is inconsistent (see reviews by St. John et al. 2005; Bowles et al. 2007; Hiendleder 2007). Bowles et al. (2007) reported that donor mtDNA is deleted by normal cellular regulation of mtDNA transmission during embryonic development in both NT embryos and clones themselves. Hiendleder (2007) observed that most of the sheep, cattle, and swine SCNT clones investigated appeared to be homoplasmic (have nuclear and mtDNA from the same source) or display only mild heteroplasmy. Hiendleder (2007) further noted that factors involved in the process of nuclear transfer (e.g., embryo culture conditions, the choice of donor cell types, and the quality of the oöcyte recipient) may affect the level of heteroplasmy,

Even if mtDNA heteroplasmy were present at a significant level in clones, the extent to which it could affect clone health or food consumption risk is difficult to determine. Clones may show little or no heteroplasmy, or may have considerable levels of mtDNA diversity but be phenotypically normal (Bowles et al. 2007). Heteroplasmy may result in impaired mitochondrial function and energy production, contributing to the poor success rate of cloning, but the empirical demonstration of that possibility has not yet been proven. Smith LC et al. (2005) noted that “To date, there is no clear indication that heteroplasmy caused by nuclear transfer procedures in farm animals is detrimental to development.”

4. Conclusions from Studies of Epigenetic Reprogramming

• Inappropriate or incomplete epigenetic reprogramming is the source of the frank adverse outcomes and subtle anomalies that pose animal health risks in animals developed by SCNT or other ARTs.

• SCNT-derived embryos must demethylate the differentiated and generally relatively highly methylated nuclear donors to restore totipotency. The high rate of failure to progress beyond the early stages of cleavage of SCNT-embryos may be a function of the inability to carry out that demethylation, and likely involves other mechanisms, some of which may involve higher-order chromatin remodeling.

• In studies evaluating the differential reprogramming of trophectoderm- and ICM-derived tissues, more dysregulation is observed in the trophectodermally-derived tissues (placental tissues) than in the somatic tissues derived from the ICM. Whether this disparity is a function of the more stringent requirement of appropriate reprogramming of the ICM-derived tissues for survival (embryos and fetuses with significantly altered epigenetic reprogramming simply do not survive) is not known.

• Live and apparently healthy clones can exhibit some level of epigenetic differences relative to fertilization-derived comparators. Many of these differences appear to resolve as the animals age, consistent with the adaptation observed in clone populations studied for physiological and growth parameters. It is not known whether these animals are tolerant of these differences, or whether a “threshold” of epigenetic differences exists that has not been exceeded in the live and apparently healthy animals.

B. Phenotypic Evidence for Gametogenic Reprogramming

The initial observations confirming the biological assumption that phenotypic expression of underlying inaccurate epigenetic reprogramming observed in clones disappear in the progeny due to gametogenic reprogramming come from the studies of Shimozawa et al. 2002a and Tamashiro et al. 2003, who demonstrated that a phenotype observed in mouse clones was not transmitted to their progeny. These studies have led to the conclusion that “Progeny of animal clones, on the other hand, are not anticipated to pose food safety concerns, as natural mating resulting from the production of new gametes by the clones is expected to reset epigenetic reprogramming errors that could persist in healthy, reproducing clones” (NAS 2002a). Or stating a similar conclusion “. . . epigenetic rather than genetic aberrations are the cause; epigenetic changes, in contrast to genetic changes, are reversible modifications of DNA or chromatin that are usually erased in the germ line” (Hochedlinger and Jaenisch 2002). This postulate can be further summarized as: “All epigenetic problems in the parents seem to be erased when cell nuclei go through the germ line” Yanagamichi (2002), and, “. . .the progeny of cloned animals will be normal” Fulka et al. (2004b).

In the following section, the studies that have led to these conclusions, as part of a summary of the utility of the mouse model for estimating risks in livestock clones are reviewed. It is organized by Developmental Nodes, as in the Critical Biological Systems Approach to evaluating the health status of livestock clones, although several nodes are combined to better reflect the existing mouse dataset.

1. Phenotypic Anomalies Observed in Mouse Clones

a. Utility of Mouse Model

Although the subject animals of this assessment are domestic livestock clones, the use of the mouse as a model system provides some insights into the underlying biology of the cloning process and its implications for food safety, particularly for understanding the role of sexual reproduction in resetting residual epigenetic reprogramming errors. SCNT in mice was first reported by Wakayama et al. (1998) using the “Honolulu technique” at approximately the same time as publication of the “Dolly” paper (Wilmut et al. 1997). Since that time, mice have been cloned from a range of cells from embryonic and adult sources (reviewed by Yanagimachi 2002). The mouse model is useful because of its well-characterized genotypes, small size, short generation period, and shorter life span than larger animals.

b. Pregnancy (Developmental Node 1)

The key measure of the success of SCNT is the normal development, maturation, and reproduction of the animal clones. As with livestock, the efficiency of this process in mice is very low, and in the same range as livestock: approximately 0.2-3.4 percent when calculated from the total number of reconstructed embryos resulting in live offspring (Yanagimachi 2002). In mice, the rate of embryo survival is most reduced early in development, particularly in the days immediately before and after implantation (Yanagimachi 2002). Yanagimachi (2002) also found that more than 90 percent of mouse embryos cloned with cumulus cells had normal chromosomal constitutions, indicating that the poor survival rates are not due to chromosomal problems, again pointing to epigenetic reprogramming as the determining factor in cloning efficiency.

Placental enlargement has been observed in almost all of the studies of mouse clones reported to date (Wakayama and Yanagamichi 1999; Humphreys et al. 2001; Ono et al. 2001b; Tanaka et al. 2001; Ogura et al. 2002; Yanagimachi 2002). Tanaka et al. (2001) performed histological examination of term placentae from mouse clones and evaluated the expression of a number of genes relevant to fetal development. Placentae from these animals were larger than from conventional controls, and exhibited histological changes in all three layers of the placenta (i.e., the trophoblastic giant cell, spongio-trophoblast, and labyrinth layers). Most of the anomalies appeared to be related to the expansion of the spongio-trophoblast layer, which exhibited an increased number of glycogen cells and enlarged spongio-trophoblast cells. Despite these morphological changes, there were no critical disturbances in regulation of gene expression in the placentae associated with term clone placentae. Unlike cattle and sheep, in which clone fetuses tended to be larger than comparators, the average weight of the mouse clone fetuses appeared to be lower than that of comparators, suggesting that a “latent negative effect from somatic cell cloning may occur on fetal growth, potentially due to incomplete placental function” (Tanaka et al. 2001). Despite the morphological changes observed in their study, Tanaka et al. (2001) noted that the placentae “could support full development of the fetus, suggesting that their functions are adequate for apparently normal fetal development” similar to the observation of Hill et al. (2000b) for cattle clones.

Both Ono et al. (2001) and Ogura et al. (2002) reported morphological changes in the placenta of mouse clones similar to those observed by Tanaka et al. (2001). Ono et al. (2001) observed that increased placental size was caused by proliferation of the trophoblastic cells, endometrial glycogen cells, and unusually large giant cells. They also found limited distribution of maternal blood vessels in the spongio-trophoblast layer and suppressed development of the labyrinth layer. They suggested that these abnormalities would greatly reduce the functional capacity of the placenta and could contribute to high rates of neonatal death in mouse pup clones derived from somatic cells. Ogura et al. (2002) compared the histological findings for mouse clone placentae with those of embryos derived from other micromanipulation techniques, such as microinsemination, aggregation chimera, and pronuclear exchange. Disruption of labyrinth layer morphology was common to placentae from cloning and other micromanipulation techniques, whereas disruption of the basal layer with marked proliferation of glycogen cells was the only phenotype unique to cloning.

The underlying mechanisms responsible for the observed placentomegaly are unknown, but Tanaka et al. (2001) cite their previous findings (Ohgane et al. 2001) of aberrant methylated genomic regions in placental tissues and suggest that slight disturbances in the expression of a number of genes, rather than a drastic change in the expression of a single gene, may impact on placental growth and function. Humpherys et al. (2002) reported that approximately 4 percent of the expressed genes in placentae from nuclear transfer-derived mouse clones differed dramatically in expression levels from those in controls. Placental size was not correlated with abnormal gene expression, indicating that the changes in cellular composition observed in Tanaka et al. (2001) are unlikely to account for the observed expression changes (i.e., changes in placental gene expression did not reflect changes in relative abundance of certain cell types). Ono et al. (2001) and Wakayama and Yanagimachi (2001) speculated that the observed placental abnormalities may be a function of disrupted patterns of expression of imprinted genes important for placental development. However, Inoue et al. (2002), using donor cells from a number of different sources, found that placentae of mouse clones at term were two to three times larger than those of controls, despite the developmentally appropriate expression of imprinted genes in both the placentae and fetuses of mouse clones. They concluded that placental genes were thus regulated by some upstream function that is independent of imprinting and is either dysregulated by nuclear transfer cloning itself, or by some other aspect of nuclear transfer.

More recently, Ohgane et al. (2004) investigated whether placental overgrowth was related to the existence of aberrant DNA methylation at certain loci (and subsequent abnormal gene expression) in mouse clones. They identified a tissue-dependent differentially methylated region within the Sall3 locus that is hypermethylated in the placenta of all mouse clones examined. Ohgane et al. concluded, given that the methylation rate of the Sall3 locus correlated with the occurrence of placentomegaly in mouse clones, this was an example of “a genomic locus highly susceptible to epigenetic error caused by nuclear transfer.”

c. Perinatal Period (Developmental Node 2)

As in the pregnancy and parturition developmental node, mouse clones have demonstrated some of the same abnormalities observed in the perinatal periods of larger mammalian clones, including reports of perinatal deaths from respiratory problems similar to that observed in cattle clones (Wakayama and Yanagimachi 1999; Eggan et al. 2001; Yanagimachi 2002). Interestingly, LOS, a relatively high frequency event in cattle cloning, was only evident in one mouse study (Eggan et al. 2001).

Eggan et al. (2001) investigated whether the phenotypic abnormalities noted in mouse clones, such as loss of neonatal growth control, respiratory failure, and high neonatal mortality, were due to the effects of nuclear transfer, or instead reflected some fundamental characteristic of the cell(s) used as donors. Using mouse embryonic stem cells with either inbred or hybrid (F1) genetic backgrounds, they compared the phenotypes of animals created by either tetraploid embryo complementation or nuclear cloning. After evaluating four endpoints (embryos transferred to surrogate dam, pups alive at term, pups respiring after Caesarian section, and pups surviving to adulthood) the authors concluded that genetic heterozygosity (i.e., hybrid vigor) was crucial for influencing the survival of mouse clones. They further concluded that difficulties with neonatal mouse clone survival and respiratory competence were a function of the genetic makeup of the donor cell nucleus, whereas neonatal overgrowth was more likely to be a consequence of the nuclear transfer procedure.

Ogura et al. (2002) reported that more than 90 percent of mouse clone fetuses that developed to term were mostly normal. Birth weights were not significantly different from controls (produced by IVF or spermatid injection), and fetal overgrowth was not observed. This is in contrast to the high incidence of placental enlargement observed in these studies (as discussed earlier in this Chapter). Of the 159 term pups, 12 had abnormalities: umbilical hernia (two cases), respiratory failure (six), developmental retardation (one), severe anemia (one), and intrauterine death shortly before birth (two).

d. Juvenile Period to Reproductive Maturity (Developmental Nodes 3 and 4)

The amount of information on the health status of mouse clones from postnatal development to reproductive maturity is limited. The finding of note within this period was postpubertal obesity in mouse clones reported by a single research group.

Tamashiro et al. (2000) evaluated the postnatal growth and behavioral development of mice cloned from adult cumulus cells relative to control mice specifically generated to eliminate confounding factors associated with the effects of embryo micromanipulation, in vitro embryo culture, embryo transfer, litter sizes, Caesarean delivery, and pup placement with lactating foster mothers. No physical abnormalities were noted at birth or through the course of the study. Body weight at birth was not statistically significantly different between clones and controls. Beginning at approximately 8-10 weeks, however, the body weights of the clone group were significantly higher than that of controls. The late onset of increased body weight in clones was distinguished by the authors from the LOS observed at birth in many mammalian clones. Although preweaning development of these mouse clones was similar to that of controls, there was a delay in first appearance of eye opening, ear twitch, and negative geotaxis (the ability of mice placed on a downward slope to turn and climb upwards). Subsequent tests of spatial learning, memory, and motor abilities in the same subjects did not show any deficits or long-term behavioral alterations. There was no significant difference in activity levels of clones compared to controls up to 180 days of age. The authors concluded that the cloning procedure did not adversely affect the overall postnatal behavior of mice.

Tamashiro et al. (2002) further investigated the obesity phenotype in mouse clones of two different background strains (B6C3F1 and B6D2F1). Comparisons were made relative to two groups: animals manipulated in vitro similar to SCNT-derived animals (in vitro embryo manipulated, or IVEM, mice), and stock (conventional) control mice. At birth, animals derived from in vitro manipulation (mouse clones and IVEM mice) were both heavier than stock control mice. Clones and IVEM mice gained about the same amount of weight over the next eight weeks, after which time the clones became significantly heavier than either IVEM or stock mice. Clones continued to weigh more than controls throughout their lives, unlike control animals whose body weight peaked at approximately 18 months of age. The increased body weight was independent of the strain of mouse used as the nuclear donor. Although mouse clones ate more than the IVEM mice, they consumed approximately the same amount of food as the stock mice. All animals lost the same percentage of baseline body weight when deprived of food, and all animals compensated by increasing consumption when it was returned. Carcass analysis showed that clones had more body fat than either the IVEM or stock mice. Mouse clones had increased plasma levels of leptin and insulin than either control group, whereas plasma corticosterone levels in mouse clones did not differ significantly from the control groups.

The authors concluded that mouse clones are truly obese and are not simply larger than controls. The process of in vitro culture appeared to be a factor in body weight, given that both the IVEM mice and clones were significantly heavier than controls. Further, the clones had more carcass fat than the IVEM mice, suggesting that some aspect of the somatic donor cell or the nuclear transfer technique may be a causative factor in the development of obesity. Faulty epigenetic programming was proposed as a possible mechanism responsible for the obesity phenotype observed in these clones.

Further study by Tamashiro et al. (2002) to determine whether a malfunctioning leptin-melanocortin system was involved in the observed obesity, involved administering melanocortin 4 receptor (MTII) and leptin, known suppressors of intake, to mouse clones and examining food intake. Inui (2003), who analyzed the results of Tamashiro et al. (2002) in context of knowledge gained of the leptin-melanocortin system from studies in rodent models of obesity and human obesity, agreed that the phenotype observed in mouse clones is unique, is not due to defects in the leptin-melanocortin system, and may be attributable in part to cloning procedures. Tamashiro et al. (2003) provides a more thorough discussion of the role body weight regulatory systems may play in this phenotype, but concludes that the mechanisms for the observed obesity remain to be elucidated. Inui (2003) proposed that inappropriate placentation may be at least partially responsible for the obese phenotype. This opinion is based on observations in other species, including humans, indicating that decreased intra-uterine nutrient levels can have significant repercussions on later human health. For example, diabetic human mothers have been observed to have births that result in large placentae, altered birth weights, respiratory distress syndromes, and subsequent obesity and diabetes in offspring (the “thrifty-phenotype” hypothesis) (Hales and Barker 2001).

To determine whether the obese phenotype was likely due to events in the cloning process, or the result of a genetic mutation, Tamashiro et al. (2002) mated male and female mouse clones and found that the offspring did not appear to be obese, nor did they have the enlarged placentae commonly found in mouse clones. Obesity was, therefore, not transmitted through the germline, indicating to the authors “that epigenetic modifications that occur during the cloning procedure are eliminated, or ‘corrected’ during gametogenesis.” The authors further proposed that “reproduction by natural mating may be recommended as soon as offspring with specific desired traits are produced by cloning.”

With respect to other possible health outcomes in this developmental period, Ogura et al. (2002) reported that more than 90 percent of mouse clones reached puberty when nursed by good foster mothers, a rate not significantly different from that of microinsemination-derived mice. In this study, cloning did not appear to have any adverse effects on reproductive performance. Of the 25 animals studied, no cases of complete sterility were observed; two female clones delivered only one litter and then became sterile for unknown reasons. No further details were provided.

e. Maturity and Aging (Developmental Node 5)³²

Given that one of the advantages of using the mouse model to study SCNT is the relatively short life span of mice compared to livestock animals, the impact of SCNT cloning on maturity and aging of animal clones has been examined in several reports. Ogonuki et al. (2002) followed weight gain, serum biochemical values, and lifespan in a group of 12 male mouse clones derived using immature Sertoli cells as donors, and compared them to the same values from male mice with the same genetic background derived from natural mating or spermatid injection. At one year after birth, weight gain of mouse clones did not differ from that of natural mating controls. Of the 16 serum biochemical values measured at 3 and 14 months of age, only lactate dehydrogenase (LDH), and ammonia (NH3) were significantly higher in clones than in control mice. Clone survival rate, however, was significantly different from the two control groups. The first death in the clone cohort occurred 311 days after birth, and 10 of the 12 animals died before day 800. Histopathological examination of necropsy samples of six of the mouse clones revealed severe pneumonia (6/6), extensive liver necrosis (4/6) and tumors (leukemia and lung cancer, 1/6 each). Elevated serum LDH and ammonium levels were consistent with liver damage.

Immune function also was investigated by Ogonuki et al. (2002) in a different group of animals derived from Sertoli cells. In mouse clones as early as 4-5 months of age, antibody production following injection of live bacteria was significantly reduced relative to age- and genotype-matched controls. Phagocytic activity was also lower than controls, although the difference did not reach the level of statistical significance.

Ogura et al. (2002) provided additional information on the same mouse clones. The longest surviving clone died at 857 days of age, with the 50 percent survival point of the mouse clones at 550 days, relative to the 1,028 days for the naturally mated control animals. The average lifespans of the two control groups (natural mating vs. spermatid injection) were not significantly different. The authors suggested that the major cause of early death was related to dysfunction of the respiratory system. Necropsy results showed that all six examined clones had severe pneumonia that resulted in destruction of alveolar structures throughout the entire lobes. Given that the animals were maintained in a pathogen-free environment, and the observed reduced immunocompetence, the authors suggested that the respiratory effects were caused by chronic infection by opportunistic organisms that are usually asymptomatic in immunocompetent mice. Interestingly, the early pneumonia-associated death of mouse clones was restricted to mice of a specific genetic background (B6D2F1). Clones of other genotypes exhibited neither early death nor severe pneumonia.

In his overview of mouse cloning, Yanagimachi (2002) reported that in his laboratory’s experience, mice cloned with adult cumulus cells, tail-tip cells, and embryonic neural cells generally had normal life spans with no serious health problems before death except for the postpubertal obesity as described by Tamashiro et al. (2000, 2002). In reviewing the Ogonuki et al. (2002) data, Tamashiro et al. (2003) stressed the importance of considering the age and type of donor cell used in the animal clones, as they may influence the health status of the animal clone later in life. This is especially important in attempting to extrapolate data to other mouse clones, or other animal clones. Tamashiro et al. (2003) cite the immature Sertoli cells used by Ogonuki et al. (2002) as possibly harboring defects that would result in adverse effects such as the observed hepatic failure and immune incompetence. Tamashiro et al. (2003) summarized their own experience with mouse clones, observing that histopathology at the time of death of their cumulus cell clones indicated that most died of conditions associated with normal aging, and that the lifespan of their clones was comparable to animals followed by the National Institute of Aging.

2. Conclusions from Phenotypic Studies of Gametogenic Reprogramming in Mouse Clones and their Progeny for Reprogramming in Domestic Livestock Clones and their Progeny

• Mouse clones offer insights into physiological mechanisms that may be perturbed in animal clones, and provide evidence that certain epigenetic changes may lead to common anomalies in livestock clones. Placental enlargement, an outcome observed in cattle and sheep clone pregnancies, also has been observed in mouse clone pregnancies and appears to be linked to dysfunctional reprogramming of cells of trophectodermal origin. Fetal size, on the other hand, does not appear to be increased in the animals with placental enlargement, and in fact, appears to be decreased in mouse clones.

• The mouse literature also confirms that the genetic make-up of the donor cells is critical in the development and growth of the animal clone, and that cloning methodology (e.g., in vitro culture conditions, effects of micromanipulation, methods of oöcyte activation, technical skill) may also have a significant effect on cloning outcomes (see also Chapters V and VI).

• At this time, it is not possible to say whether the life span shortening observed in one strain of mouse clones will be observed in other species of clones. The shortened life-spans of mouse clones appear to be due to chronic alterations in metabolism, while the only observed early deaths of livestock clones appear to be due to more acute phenomena. Nonetheless, it is too early to make a definitive judgment on longevity, as most domestic livestock clones have not yet begun to approach even the midpoint of their natural life-spans (See Chapters V and VI).

• Clones are not the only animals that exhibit differences in epigenetic programming relative to their genetic antecedents. There are examples of fertilization-derived embryos responding to dietary levels of methyl donors in their dam’s diets resulting in offspring whose phenotypes differ significantly from their parents. Although the cited case provides a clear molecular correlation between the exposure and outcome, it is important to remember that epigenetic markers are reversible by “nature’s design,” and are intended to help provide organisms with multiple, interactive mechanisms with which they may adapt to environmental challenges.

The most important implication of the mouse clone literature for domestic livestock clones is the observation that anomalies noted in clones are not transmitted to their progeny. The obese phenotype, for example, is not transmitted to progeny of those clones, and progeny of mouse clones appear to be normal and healthy. This observation is consistent with the biological assumption that gametogenesis effectively “re-sets” epigenetic markings, and allows for the appropriate development of normal organisms (i.e., sexual reproduction). It is also consistent with the limited but consistent observations of healthy, fully functional progeny born to domestic livestock clones. Thus, the empirical evidence supports the assertion that “Progeny of animal clones, on the other hand, are not anticipated to pose food safety concerns, as natural mating resulting from the production of new gametes by the clones is expected to reset epigenetic reprogramming errors that could persist in healthy, reproducing clones” (NAS 2002a).

C. Implications of Epigenetic Reprogramming for Animal Health and Food Consumption Risks

The Center assumes that if clones were to pose food consumption risks, the only mechanism by which those risks could arise would be from inappropriate epigenetic reprogramming, similar to those observed for other ARTs. It is important to note that the genes that are being dysregulated are the “normal,” naturally present genes that comprise the animal’s genome, and have not been introduced via recombinant DNA techniques from other sources (i.e., these are not transgenic or genetically engineered animals).

• Anomalous epigenetic reprogramming is observed at the global genomic and individual gene level in clone embryos and fetuses, and in similar developmental stages of animals produced using ARTs with significant in vitro culturing components. Various factors influence the success rate of SCNT and these other ARTs, including the source of the donor cells and oöcytes, culture medium, and factors that have not yet been identified. Many of these anomalies are lethal, as demonstrated by the low success rate of IVF and the even lower success rate of SCNT.

• Because abnormalities arise from the dysregulation of intrinsic genes, adverse outcomes that would likely be expected in clones and animals derived via other ARTs are those that result from the inappropriate development of tissues and organs. For example, it would be reasonable to expect both overgrowth phenomena, and the poor development (aplasia or hypoplasia) of tissues and organs. Examples of outcomes that affect the health status of animal clones are presented in detail in Chapter V (Animal Health) and Appendix C, and those that may have an impact on food consumption risks are described in Chapter VI.
  

• The studies that have evaluated epigenetic reprogramming of live, healthy clones indicate that although there is some variability between clones and their fertilization-derived counterparts, clones are capable of carrying out sufficient methylation-based reprogramming (and other coordinated functions) to allow for survival. Molecular analyses reveal relatively small methylation differences, and either the animals are tolerant of such differences, or the epigenetic differences are below the threshold that poses observable adverse health outcomes.

• It may be, as many have suggested (Wilmut 2002b; Jaensich et al. 2004), that no clone is completely “normal” with respect to its epigenetic profile. Although this is an important point for assessing the overall safety of the cloning process for any particular species, the relevance of “epigenetic normality” to food consumption risks is unclear. Further, because similar abnormalities have been noted in animals produced using other ARTs, the issue of defining normality becomes significantly more complex. It may be that normality encompasses a range on a continuum, and that animals that are healthy, meet appropriate developmental and behavioral milestones, reproduce and bear healthy young are “normal,” regardless of their epigenetic status. The most compelling conclusions that can be made about food consumption risks, then, are drawn from assessments of the health status of the animals and the composition of food products derived from them, and not from gene expression studies.

• Progeny of animal clones, on the other hand, are not anticipated to pose food safety concerns, as natural mating resulting from the production of new gametes by the clones is expected to reset even those residual epigenetic reprogramming errors that could persist in healthy, reproducing clones (Tamashiro et al. 2002; Yanagimachi 2002; NAS 2002a, 2004, Fulka et al. 2004). Thus any anomalies present in clones are not expected to be transmitted to their progeny.

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²⁵ Methylation of DNA is performed by specific enzymes (methylases) that obtain methyl sources from either the diet (as folates or folic acid) or from endogenous one-carbon metabolism. The latter requires essential dietary components such as methionine, zinc, and vitamin B-12 to act as cofactors in the synthesis of intermediates that give rise to 1-carbon donors
²⁶  The agouti coat color is a continuous spectrum of variegated coat color patterns on a yellow background. In cats, this coat color pattern is referred to as “tortoiseshell.” 
²⁷  Information encoded in DNA is converted into RNA by a process referred to as transcription.  Those RNA molecules (messenger RNA) that encode information for protein synthesis are converted to proteins by the process of translation.
²⁸  The 5’ end of a gene is often considered to be at the start of the coding sequence on the DNA molecule. The nomenclature is derived from the position of a hydroxyl group in the deoxyribose sugar ring at the beginning of the strand of the DNA.
²⁹ The pronucleus is the structure that contains the haploid genome of the sperm or ovum after fertilization occurs, but before they fuse to make the nucleus of the zygote, or the single-celled diploid organism. Once the zygote has undergone the first division (or cleavage), it is referred to as an embryo.
³⁰ Real-time PCR is a technique that allows for the rapid and precise identification and quantification of genetic material (in this case, RNA) during the actual time that the reaction is running.
³¹ Mitochondria are the only organelles in animals that contain their own DNA, and are considered to be the “powerhouses” of eukaryotic cells. They are the site of the electron transport chain, which is the cell’s major source of energy. Proteins in the electron transport chain are encoded by chromosomal or mtDNA, necessitating tight coordination and regulation of expression between the nuclear and mtDNA. In sexual reproduction, these interactions, which govern mtDNA copy number, mitochondrial morphology, and the number of mitochondria per cell are tightly controlled to optimize energy production (St. John et al. 2005).
³² For a discussion of telomeres and their possible role in aging, see Chapter V.

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