Recent advancements in cloning by somatic cell nuclear transfer

Atsuo Ogura, Kimiko Inoue, Teruhiko Wakayama

Abstract

Somatic cell nuclear transfer (SCNT) cloning is the sole reproductive engineering technology that endows the somatic cell genome with totipotency. Since the first report on the birth of a cloned sheep from adult somatic cells in 1997, many technical improvements in SCNT have been made by using different epigenetic approaches, including enhancement of the levels of histone acetylation in the chromatin of the reconstructed embryos. Although it will take a considerable time before we fully understand the nature of genomic programming and totipotency, we may expect that somatic cell cloning technology will soon become broadly applicable to practical purposes, including medicine, pharmaceutical manufacturing and agriculture. Here we review recent progress in somatic cell cloning, with a special emphasis on epigenetic studies using the laboratory mouse as a model.

1. Introduction

Somatic cell nuclear transfer (SCNT) in mammals is an assisted reproductive technique used to produce an animal from a single cell nucleus using an enucleated oocyte as a recipient. As somatic cells can be proliferated and gene-modified in vitro, this technique has been expected to contribute extensively to the farm animal production industry, drug production, regenerative medicine and conservation of invaluable genetic resources [1,2]. Besides its broad practical applications, SCNT can provide unique and interesting experimental systems for genomic research, especially in epigenetics, to learn how the somatic cell genome is reprogrammed into a state equivalent to that of the fertilized oocyte: the so-called totipotent state [3]. Although the somatic cell genome can be reprogrammed (or dedifferentiated) by other methods, including the introduction of transcription factors [4], incubation of permeabilized cells with cell extracts [5] and cell–cell fusion [6], the resultant reprogrammed state of this genome is ultimately a pluripotent state equivalent to that of embryonic stem (ES) cells (figure 1). Therefore, SCNT is the sole technique to date that can endow the somatic cell genome with totipotency.

Figure 1.

Two types of reprogramming of the somatic cell genome. The genome of differentiated somatic cells can be reprogrammed to the pluripotent state by introduction of the so-called Yamanaka factors [4], incubation of permeabilized cells with cell extracts [5] or cell fusion [6]. However, the resultant genomic state even with the best reprogramming is that of embryonic stem (ES) cells, which are epigenetically distinct from the inner cell mass (ICM) cells of blastocysts (see the text). It can be assumed that there is a somatic cell–embryo epigenetic barrier between preimplantation embryos and postimplantation embryonic tissues. Only somatic cell nuclear transfer (SCNT) using enucleated oocytes can overcome this barrier to endow the somatic genome with totipotency, the epigenetic state equivalent to fertilized oocytes (zygotes). Experimentally, SCNT bypasses the normal path of germ cell development, during which somatic epigenetic marks are erased and germ cell epigenetic marks are imposed. Conceivably, this might cause reprogramming errors in the somatic cell genome and the associated abnormal phenotypes following SCNT. By contrast, nuclear transfer (NT) using embryonic blastomeres is more efficient. The epigenetic barrier proposed here might be more stringent in mice and humans than in ungulates because the latter species have a non-attached preimplantation phase before the placenta is established in the uterus, after embryonic differentiation. iPS, induced pluripotent stem cell.

ES cells, especially those of mice and rats at their most ground state, have full pluripotency and can differentiate into all cell lineages comprising the body [7,8] and even into placental cells under specific culture conditions [9]. Their open hyperdynamic chromatin structure [10] and global transcriptional hyperactivity [11] sets them apart from differentiated somatic cells [12]. However, in a broad sense, ES cells seem to be a kind of somatic cell because they share some important characteristics common to other somatic cells or to postimplantation embryos, such as their DNA methylation patterns in promoter and centric/pericentric regions and complete loss of the germline-specific X-chromosome inactivation memory [9,1316]. It has also been demonstrated that the process of ES cell derivation causes robust changes in methylation of histone H3 at lysines 4 (H3K4me3) and 27 (H3K27me3) [17]. Similarly, many differences in transcriptional programmes between ES cells and inner cell mass (ICM) cell outgrowths have been detected by RNA-seq profiling [18,19]. Taken together, during the derivation of ES cells, they accumulate somatic cell-type epigenetic characters, faithfully reflecting the phenomena occurring during implantation in vivo. In accordance with this, it is known that cloned mice derived from ES cells show SCNT-specific hyperplastic placentas, which are about twice as large as those cloned from ICM cells [20,21]. By contrast, nuclear transfer (NT) from blastomeres of preimplantation mouse embryos is more efficient in terms of the birth rate per number of embryos transferred and causes very few abnormalities in the resulting clones [21,22]. As far as has been tested in mammals, there might be a gap between blastomere NT cloning and SCNT in terms of the birth rates and the normality of cloned offspring [2023]. Therefore, it is reasonable to assume that there is an ‘epigenetic barrier’ between preimplantation embryos and postimplantation somatic cells. This barrier might be more stringent in mice and humans than in ungulates because the latter species have a non-attached preimplantation phase before the placenta is established in the uterus, after embryonic differentiation. Some epigenetic marks are probably imposed on the somatic cell genome at the time of implantation, and remain in the genome throughout the subsequent life cycle. These somatic cell marks are erased only through gonadal germ cell development in a step-by-step manner, although the mechanism is still unclear [24]. SCNT forces the somatic cell genome to be reprogrammed directly to a totipotent state by bypassing these erasing steps, and this might make the technique prone to epigenetic errors and cause frequent death and loss of embryos. In addition, SCNT should also erase the cell-type-specific memory that had been imposed during differentiation. This differentiation-associated memory might be easier to reprogramme than the former putative somatic cell marks because during induced pluripotent stem (iPS) cell generation, most if not all of the epigenetic characters of original donor cells are erased successfully [25].

In sum, the SCNT technique should somehow overcome these two epigenetic hurdles: somatic cell marking and cell-type-specific differentiation memory. Each hurdle might cause specific reprogramming errors and clone-associated abnormalities. We expect that while we seek to improve the efficiency of SCNT, such relationships between the epigenetic hurdles and their specific reprogramming errors will become clearer, leading to more precise understanding of epigenetic control mechanisms functioning during implantation and cell differentiation. The mouse probably provides the best experimental model for this purpose.

2. What determines genomic reprogrammability? Lessons from mice

Since the birth of Dolly the sheep in 1996, many attempts have been made to clone animals of different species using different somatic cell types [26]. Based on the accumulated information on the various levels of efficiency in those cloning experiments, it is broadly accepted that the efficiency of cloning in terms of the birth rates of offspring can be affected by a number of biological and technical factors. However, in cloning farm animals, there are inevitably considerable individual differences in the quality of recipient oocytes, donor cells and recipient females, so it is usually very difficult to determine the decisive factors statistically [2]. Therefore, attempts to determine the best experimental conditions for cloning farm animals can be compromised, and sometimes become issues of controversy. By contrast, laboratory mice offer more reproducible experimental systems because of the availability of defined genetic backgrounds [27] and well-established protocols for superovulation, embryo culture and embryo transfer. Since the first report of successful mouse cloning in 1998 [28], cumulus cells with a B6D2F1 genetic background have been the standard nuclear donor source and have been used as controls for the assessment of other donor cells for their ‘clonability’ [29]. A large-scale mouse cloning experiment using two different cell types and six different genotypes of donor cells revealed that the birth rates of clones were determined by the combination of these two factors [30]. In this analysis, the birth rate using B6D2F1 cumulus cells was 2.2 per cent while that of (B6 × 129) F1 neonatal Sertoli cell clones was 10.8 per cent. Overall, SCNT with neonatal Sertoli cells resulted in better efficiencies than with cumulus cells. This has been true in more recent studies [31,32] and is in accordance with the results of global gene expression analysis of blastocysts cloned from cumulus or Sertoli cells [33]. ES cells have also been used in cloning studies in mice, especially in the second step of a serial cloning protocol where NT-derived ES cells were generated as an intervening step between the first and second rounds of SCNT [34] (figure 2). The efficiency of cloning mice using ES cells is generally very high, provided their cell cycle is synchronized successfully with that of the ooplasm and genetic/epigenetic errors are avoided during in vitro culture. The birth rates per number of embryos transferred ranged from 12 per cent in the original report by Wakayama et al. [20] to 33 per cent in an experiment using F1 hybrid ES cells [37]. The high reprogrammability (the ability of the genome to be reprogrammed) of the ES cell genome might be attributed to the consecutive expression of Oct3/4, a key upstream factor required for normal early embryonic development [38]. Better cell cycle synchrony between the ES cell nucleus and the recipient ooplasm can be achieved by confluent culture [39] or through cell cycle arrest at the M-phase with nocodazole treatment [40].

Figure 2.

Two-step SCNT in mice. NT-derived embryonic stem (ntES) cells are generated by the first round of SCNT. These are then used to generate cloned mice by a second SCNT procedure or via an intervening stage using chimeric embryo production. This protocol has been used successfully for mouse cloning studies where conventional one-step SCNT is unsuccessful or when it is very difficult to generate mice; for example, when cloning lymphocytes [35] and when using somatic cells retrieved from frozen cadavers [36].

Given that the best donor cell types in terms of cloning efficiency are ES cells, followed by neonatal Sertoli cells and adult cumulus cells [28,30,34,41], this led us to hypothesize that the degree of differentiation might be correlated inversely with cloning efficiency. One of the strategies to test this assumption was to clone differentiated cells within the same cell lineage. So far as has been examined to date, neuronal lineage cells seem to confirm this assumption. When neural cells were collected from foetuses at 15.5–17.5 days postcoitus (dpc), normal cloned offspring were born at a relatively high rate (5.5%) [42]. By contrast, no live offspring were born from the neural cells of neonatal mice (days 0–4 after birth) because of embryonic death at around 10.5 dpc [43]. Adult neuronal cells also contributed to the reconstruction of cloned embryos, but supported their development only up to 6–7 dpc [28]. Neural stem cells derived from foetal or neonatal brains could be cloned to produce normal offspring by SCNT [31,44]. These results suggest that neural lineage cells lose their reprogrammability as they differentiate. However, another line of evidence using haematopoietic lineage cells suggested that this scenario is not always the case. Cloning T or B cells was extremely difficult while cloning natural killer T (NKT) cells was relatively easy, even though they are all terminally differentiated lymphocytes carrying rearranged DNA [35,45]. So far as has been tested to date, the generation of mice from T- or B-lymphocytes by direct SCNT has been unsuccessful and, therefore, a two-step round of NT—a technique involving ES cell generation and tetraploid complementation—is necessary [35] (figure 2). In this, the ES cell stage allows for extra reprogramming time, and tetraploid cell lines might contribute to most extra-embryonic tissues, which are commonly the more adversely affected components in cloned animals [46]. By contrast, we found that NKT cells with specific differentiated markers were suitable donors for the generation of cloned offspring and NT-derived embryonic stem (ntES) cell lines by direct SCNT [45]. We then further examined whether haematopoietic stem cells (HSCs) with innate differential plasticity could be cloned by SCNT. Unexpectedly, only 6 per cent of reconstructed embryos reached the morula or blastocyst stage in vitro (versus 46% for cumulus cell-derived clones) and at best only 0.7 per cent per embryo transferred reached full term [47]. No offspring were obtained when a standard B6D2F1 genetic background was used [47]. Sung et al. [48] also confirmed the unsuitability of HSCs for SCNT by comparing the birth rate of clones with that of granulocyte clones. The poor development of HSC-derived cloned embryos was consistent with their gene expression pattern at the 2-cell stage when major zygotic gene activation (ZGA) occurs in the mouse [49]. The HSC clones failed to activate five out of six important ZGA genes examined, including Hdac1 encoding histone deacetylase 1, a key regulator of ZGA [50,51]. As a result, only 34 per cent of the HSC clones reached the 4-cell stage (p < 0.05 versus 65–78% of other cloned embryos). This finding seems to be contradictory to the finding that HDAC inhibitors actually improve the development of cloned embryos (see below), but treatment with these drugs is usually restricted to the very early stage of development (less than 10 h after oocyte activation) to avoid their inhibitory effects on ZGA at a later stage [50]. We also confirmed that the expression of Hdac1 mRNA in HSCs was lower than in other somatic cells, probably reflecting an open chromatin structure that enables the easy access of transcriptional factors [52,53]. Taken together, we postulate that genomic reprogrammability is biologically distinct from the degree of genomic plasticity based on its differentiation status (or its ‘stemness’, in reverse). Rather, it might have a close correlation with the gene expression pattern or chromatin structure specific to each donor cell type. Oback [54] reviewed the relationships between the genomic reprogrammability of donors and their differentiation status in mice and other species in detail in 2009, and postulated that the differentiation status of the donor genome and its reprogrammability to totipotency might be unrelated.

According to Eminli et al. [55], HSCs and progenitor haematopoietic cells provided better efficiencies of iPS cell generation than did B or T cells. This is not surprising because genomic reprogramming to the pluripotent state does not need ZGA or activation of genes for early embryonic development. For example, the suppression of HDAC1 in HSCs might facilitate iPS generation, but might also hamper ZGA and subsequent embryonic development. Thus, the prerequisites for acquisition of pluripotency and totipotency are epigenetically different, although there might be a common machinery to reprogramme the chromatin structure and nuclear architecture.

After a series of SCNT experiments using different donor cell types with a common male (B6 × 129) F1 genotype, we found a high correlation (r = 0.92, p = 9.1 × 10–5) between the rates of embryos that developed beyond the 2-cell stage and the rates of birth after embryo transfer (figure 3). This finding suggests that the degree of ZGA has a strong effect on embryonic development to term. The data also clearly indicate that there was no relationship between genomic reprogrammability and the undifferentiated status of the genome (figure 3).

Figure 3.

Correlation between the rates of development beyond the 2-cell stage and full-term development in cloned embryos. There is a close relationship between these parameters while the degree of ‘stemness’ or undifferentiated status of the donor cells has no association with these cloning efficiencies. All experiments were undertaken using male donor cells with a (B6×129) F1 genetic background, except for female primordial germ cells (PGCs). This is based on data both published [30,44,45,47,56] and unpublished (A. Ogura, K. Inoue, unpublished data). The birth rates were calculated, including placenta-only conceptuses. Results using donor cells with incomplete genomic imprinting such as early PGCs [57] or with abnormal chromosomal constitutions such as long-cultured mesenchymal stem cells [44] or cancer cells [58] have been omitted here. TSA, trichostatin A.

As mentioned above, the genotype of donor cells can also affect cloning efficiency. In one study, Japanese Black calves were born following SCNT at a rate as high as 80 per cent (8/10 per embryos transferred) using cumulus and oviductal epithelial cells [59]. This efficiency was much higher than that of bovine cloning performed during the same period (about 10%) [60]. Since then, high pregnancy rates have been reported for bovine SCNT using the Japanese Black breed [61,62]. Sheep also showed breed-specific variability in terms of the success of cloned embryo development [63]. However, there are no directly comparable data for cattle and sheep clones that can allow precise evaluation of the effects of genetic backgrounds. By contrast, mouse cloning experiments have allowed us to assess the effect of genotype on the development of clones in a more precise manner thanks to the availability of genetically defined strains of mice [27]. According to Van Thuan et al. [64], cloned mice could be obtained from all the inbred strains so far tested [64]. The best birth rate was obtained with the 129 strain, followed by the DBA/2 strain (figure 4). Based on these studies, the presence of the genome from the 129 strain is expected to increase the reprogramming efficiency of the donor genome following SCNT [30]. Historically, the plasticity of this mouse genome has been demonstrated by the relatively easy establishment of ES cells from this strain [65] and by the high incidence of testicular carcinoma [66]. Interestingly, the placentas of clones derived from the 129 strain showed nearly normal morphology, unlike those from other strains, which showed placentomegaly [67]. Therefore, there might be certain factors within the 129 strain genome that ensure its high genomic plasticity. At present, we have no idea of the identity of these factors, but if we could identify them they would contribute greatly to the safe and efficient development of new technologies in regenerative medicine and pharmacy. SCNT experiments using a set of recombinant inbred strains based on crosses between the 129 strain and other strains should help in the execution of this strategy by the so-called ‘forward genetics’.

Figure 4.

Effects of trichostatin A (TSA) and scriptaid treatment on inbred mouse cloning. Without such histone deacetylase inhibitor (HDACi) treatment (black bars), cloned mice could be obtained from hybrid and 129/Sv strains, but with a low success rate. Only one cloned mouse was obtained from the DBA/2 strain, but this animal never reproduced. When TSA (light grey bars) was used, the success rates for hybrid and outbred strains were increased but we have never succeeded in producing full-term cloned mice from inbred strains. However, when scriptaid (dark grey bars) was used, the overall success rate was increased even from inbred strains.

3. Technical improvements based on histone modifications

As mentioned above, the prevention of epigenetic errors during nuclear reprogramming is expected to improve the success rate of animal cloning. Enright et al. [68] have tried to alter the epigenetic status of donor nuclei before NT by using two chemicals: 5-azacytidine (an inhibitor of DNA methylation) and trichostatin A (TSA; a histone deacetylase inhibitor (HDACi)). The in vitro developmental potential of bovine cloned embryos was improved slightly. However, these drugs that affect epigenetics are very toxic [69,70] and each drug must be tested pharmacologically for its appropriate exposure, timing, concentration and duration. Thus, Kishigami et al. [71] discovered by trial and error the optimum concentration, timing and period of TSA treatment for cloning mouse embryos. Eventually, this method led to a greater than fivefold increase in the success rate of mouse cloning, except for cloning ES cells (figure 5). Unlike the situation in mouse cloning, the effects of TSA on cloning efficiency are controversial for bovine [72,73], pig [74,75], rabbit [76,77] and rat [78] models. Moreover, some groups have reported that TSA treatment had detrimental effects on the in vitro and in vivo development of SCNT embryos [73,76]. To our knowledge, the effects of TSA treatment on full-term development have not been determined in any species other than the mouse.

Figure 5.

Effects of HDACi treatment on mouse cloning using B6D2F1 cumulus cells as NT donors. Without HDACi treatment, cloned mice could be obtained but with a low success rate (Cont). When TSA, scriptaid, SAHA or oxamflatin were used, the success rates were increased significantly, but when APHA was used, no clones were obtained. All the HDACi agents listed here belong to the hydroxamic acid or hydroxamate chemical compound classes.

It must be emphasized that most cloned mice have only been generated from hybrid strains and have never been cloned from outbred or inbred strains [30,67]. However, when the drug scriptaid, which acts as an HDACi but is less toxic than TSA [79], was used for cloning, it could increase mouse cloning success rates not only in hybrid but also in supposedly ‘unclonable’ inbred strains of mice [64,80]. Similarly, when scriptaid was used instead of TSA, Zhao et al. [81] improved the success rate of pig cloning to full term. These results suggest that although the use of HDACi drugs can enhance reprogramming in cloned embryos, because of their toxicity the effects depend on the individual sensitivity of the donor strain or species. For this reason, we have tried to discover other useful HDACi drugs for mouse cloning, and we found that two other agents, suberoylanilide hydroxamic acid (SAHA) and oxamflatin, could also improve the full-term development of cloned mice significantly without leading to obvious abnormalities [82]. Another group found that m-carboxycinnamic acid bishydroxamide also improved the success rate of full-term mouse cloning [83]. However, although valproic acid was reported to increase the reprogramming efficiency of mouse fibroblasts by more than 100-fold to establish iPS cells [84], it had little [85] or no effect [82] on the success rate of mouse cloning. Another HDACi, aroyl pyrrolyl hydroxamide (APHA), also could not improve cloning efficiency [64]. Figure 4 summarizes the effect of HDACi agents on mouse cloning using BDF1 cumulus cells [64,71,82].

(a) How does HDACi treatment enhance reprogramming?

Although how HDACi treatment improves cloning efficiency remains unknown, it is thought that it can induce hyperacetylation of the core histones, resulting in structural changes in chromatin that permit transcription and enhanced DNA demethylation of the somatic cell-derived genome after SCNT [71]. This is a necessary part of genetic reprogramming [86]. In fact, several reports clearly showed that HDACi treatment during SCNT cloning improved histone acetylation [87], nascent mRNA production [64] and gene expression [88] in a manner similar to that in normally fertilized embryos. TSA treatment also improved the long-term consistency of genome-wide gene expression regulation: the total number of genes commonly exhibiting up- or downregulation in the TSA-treated clone pups decreased to half of the conventional SCNT pups and the total gene expression profile of the TSA clones came to resemble that of the intracytoplasmic sperm injection (ICSI) pups [89].

How histone methylation is modified in TSA-treated cloned embryos is not completely understood. Bui et al. [90] found that TSA treatment caused an increase in chromosome decondensation and nuclear volume in SCNT-generated embryos, similar to embryos produced by ICSI [90]. This was associated with a more effective formation of DNA replication complexes in treated embryos. Those embryos could overcome a failure in the timely onset of embryonic gene transcription by the activation of rRNA genes and promotion of nucleolar protein allocation during the early phase of ZGA [91]. Those results suggest that HDACi can enhance the reprogramming of the somatic nuclei in terms of chromatin remodelling, histone modification, DNA replication and transcriptional activity.

(b) Why do cloned embryos require HDACi treatment for better genomic reprogramming?

In nature, the ooplasm contains reprogramming mechanisms, such as histone acetylation or DNA demethylation, that convert the sperm and oocyte nuclei to a totipotent state [87,92,93]. Given that SCNT can result in the birth of viable animals, the oocyte's reprogramming machinery is sufficient to reprogramme a somatic cell nucleus, at least in some cases. However, the potential reprogramming machinery of the oocyte cytoplasm is prepared for the receipt of a haploid sperm nucleus, not a somatic cell nucleus. In general, it is considered that the incomplete reprogramming of somatic cell nuclei following SCNT arises from poor genomic reprogramming in the oocyte. However, we now think that the oocyte cytoplasm might reprogramme the somatic cell nucleus too strongly, or that the somatic cell nucleus is more sensitive to oocyte reprogramming factors than are sperm cell nuclei. Therefore, by inhibiting a particular HDAC or one of the reprogramming factors in ooplasm during reprogramming, the donor nuclei in our studies were possibly reprogrammed more correctly, resulting in a higher success rate for cloning [64,71].

4. Technical improvements based on X-chromosome inactivation status

It is very probable that the effect of HDACi treatment on the chromatin remodelling of cloned embryos is genome wide rather than genome region-specific and leads to nearly normal reprogramming of the whole genome. However, the presence of many SCNT-specific phenotypes, such as placental abnormalities [21,94,95], obesity [96] and immunodeficiency [97], suggests that SCNT inevitably induces specific epigenetic errors into the donor genome. They might be non-random and definable characteristics, perhaps caused by the fundamental epigenetic nature imposed on somatic cell nuclei at the time of implantation described above. To examine this possibility, single cloned blastocysts were analysed for their global gene expression patterns by comparing them with genotype- and sex-matched controls produced by in vitro fertilization under the same environment. We noted that when the relative expression levels of all 41 233 genes in cloned embryos were plotted on the 20 chromosomes, many genes on the X chromosome were specifically downregulated [32]. This suppression of the X-linked gene was chromosome wide and was associated with elevated expression of the Xist gene, which is responsible for inactivation of one of the X chromosomes in female cells. RNA fluorescent in situ hybridization (FISH) analysis for Xist mRNA in blastomere nuclei revealed an excessive signal in male and female cloned embryos, indicating that Xist was expressed ectopically from the active X chromosome in both sexes. We then examined to what extent its ectopic expression was responsible for the low birth rates of clones, using knockout and knockdown strategies. As the X chromosome is only one of the 20 chromosomes in mice (except for the Y), we first anticipated that normalization of Xist expression would simply result in some slight improvements in the cloning efficiency. However, the results were much more remarkable than we expected. When donor cells containing an Xist-deficient X chromosome were used for NT, the birth rates increased 8- and 14-fold following cumulus and Sertoli cell cloning, respectively [32]. Similarly, knockdown of Xist by the injection of specific short interfering (si)RNA into reconstructed oocytes resulted in about a 10-fold increase in the birth rate [98]. Interestingly, normalization of the Xist expression level resulted in a decreased number of downregulated autosomal genes to 6 per cent and 25 per cent in female and male clones, respectively. These results indicate that the ectopic Xist expression in clones might have adversely affected the gene expression in cloned embryos in a genome-wide manner. Although this knockdown strategy is currently only applicable to male clones because of our inability to achieve precise quantitative control of Xist mRNA repression, this technology is more realistic for the future practical applications of SCNT because it is technically easy and does not alter the genomic constitution of the donor genome or of the cloned offspring. The XIST gene is upregulated in bovine SCNT embryos and has been implicated in the prenatal death of cloned pigs and calves [99101]. In domestic animal species such as bovines and pigs, embryonic implantation takes place long after embryo transfer. Therefore, we should carefully examine whether the effect of XIST knockdown can persist through to the critical developmental period for the cloned embryos to survive.

Two discrete groups of genes remained suppressed in Xist-deleted mouse clone blastocysts. They were the Magea and Xlr gene clusters, localized in chromosomal regions XqF3 and XqA7.2–7.3, respectively [32]. These regions are within the blocks enriched with the dimethylation of histone H3 at lysine 9 (H3K9me2), which is responsible for gene silencing providing a constitutive heterochromatin status. They are called ‘large organized chromatin K9 modifications’ [102] and their pattern of distribution matched exactly between ES cells [102] and donor cumulus cells [32]. We postulated that the repressive state of the Magea and Xlr regions mediated by the somatic type-H3K9me2 modification in donor cells was resistant to reprogramming by the putative ooplasmic factor(s) and consequently transmitted to cloned embryos. The Magea and Xlr genes were actively transcribed in normal fertilization-derived blastocysts, but became shut down in ES cells [32], suggesting that the H3K9me2 blocks might serve as a ‘somatic signature’ imposed during implantation. These could be the next targets for technical improvements in SCNT.

5. Other factors that might affect the development of clones

(a) Placental abnormalities

Most SCNT-derived clone embryos arrest their development at some time during the early postimplantation period, depending on the species: before 6.5 days in mouse [103] and before 60 days in bovine embryos [104]. These periods are critical for early placentation. Indeed, SCNT-associated abnormal placental phenotypes have often been described in several of the species cloned to date [105]. Cloned mice are associated with hypoplastic placentas during early gestation [106,107] and with placental hyperplasia from mid-gestation to term [94,95,108]. Placentas of cloned calves have fewer but much larger placentomes, presumably to compensate for the reduced number of placental sites for maternal–foetal exchange [109]. Thus, it is possible that defects in the extra-embryonic cell lineage are one of the major causes of the low success rate of reproductive cloning [105]. At first, this assumption was thought to be consistent with the easy derivation of ntES cells from the inner mass cells of cloned blastocysts [110]. However, this scenario was found to be invalid because trophoblast stem (ntTS) cells were also efficiently established from SCNT embryos [111,112]. To determine how the extra-embryonic tissues are involved in poor development of clones and enlarged placenta phenotypes in mice, several laboratories have analysed chimeras generated by fusing cloned embryos and fertilized embryos. In addition, ntTS cells have also been characterized in vivo and in vitro for this purpose. As a result, some studies implied that disorganized interactions between embryonic and extra-embryonic tissues are responsible for inefficient cloning or for placental abnormalities [111,113,114], while others indicated the predominant involvement of the extra-embryonic tissues themselves [107,112]. Recently, Lin and co-workers [115] re-evaluated the developmental consequences of chimera embryos using isolated ICM cells, not whole blastocysts. By aggregating clone-derived ICM cells with tetraploid fertilized embryos, they successfully rescued abnormal placental phenotypes and increased the birth rate of clones up to 15.7 per cent, indicating that defects in the extra-embryonic lineage underlie the low success rate of SCNT cloning. The rescue of SCNT-derived embryos by Xist knockout or knockdown described above had no beneficial effect on the hyperplastic placental phenotype. It will be interesting to see whether the two strategies for rescuing SCNT embryos might have a synergistic effect.

(b) Maternal inheritance of the mitochondrial DNA

Since the early SCNT studies in the 1990s, whether the mitochondrial DNA (mtDNA) transmitted from the donor cells might cause poor development of cloned embryos has been an issue. This is a set of cytoplasmic genomes that encodes a subset of genes encoding oxidative phosphorylation and is transmitted to offspring by strict maternal inheritance [116,117]. Generally, a single oocyte contains more than 105 copies of mtDNA, whereas somatic cells contain only 102–103 copies [117,118]. Theoretically, cloned animals possess two types of mtDNAs derived from oocytes and donor cells; this condition is called heteroplasmy. Evans et al. [119] demonstrated that the first cloned mammal, Dolly, did not possess nuclear donor cell-derived mtDNA in its tissues, whereas Steinborn et al. [120] detected heteroplasmy in bovine embryos produced from three types of donor cells. Many attempts have been made to discriminate oocyte- from donor cell-derived mtDNA quantitatively. These have supported the presence of heteroplasmy in cloned animals, although its degree varied greatly among experiments [121124]. In mice, 24 of 25 cloned offspring carried the donor cell-derived mtDNA up to a ratio of 13.1 per cent of the total [125]. However, as far as we know, there is no experimental evidence that heteroplasmy might compromise the development of cloned embryos or the health status of offspring, at least in conventional SCNT. By contrast, the situation of interspecies SCNT (iSCNT) is very different. Incompatibility between the mtDNA genes and the nuclear genes encoding mitochondrial proteins is thought to be one of the major causes of developmental arrest among iSCNT embryos [126]. In iSCNT among the Canidae and Felidae, this might not be a critical issue because wolves and wild cats have been born using oocytes from domestic dogs and cats, respectively [127,128].

6. Future prospects

(a) The possibility of resurrecting an extinct animal

Cloning animals by SCNT provides an opportunity to preserve endangered mammalian species, provided viable cells can be collected. However, the ‘resurrection’ of extinct species from permafrost (such as the woolly mammoth) is thought to be impractical, because no live cells will be available. On the other hand, it is known that ‘dead’ spermatozoa from freeze-drying treatments [129] or from a whole frozen cadaver [130] still possess a complete haploid genome. When such spermatozoa were injected into oocytes, the resulting embryos could develop to full-term healthy offspring. Surprisingly, the toughness of DNA was demonstrated not only in the sperm head but also in somatic cells. We attempted to produce cloned mice from cadavers kept frozen at –20°C for up to 16 years without any cryoprotection. These conditions are similar to those of a frozen body recovered from permafrost, and the cells from all organs of the cadavers were completely disrupted. When we injected those cell nuclei into enucleated mouse oocytes, some of the embryos could develop to blastocysts. Although we could not produce cloned offspring from the somatic cells directly, several ES cell lines were established from the cloned embryos. Finally, healthy cloned mice were produced from these ES cells by a second round of NT (figure 2) [36,131]. Thus, these techniques could be used to resurrect animals or to maintain genome stocks from tissues that have been frozen for prolonged periods or even when no live cells are available. In such cases, all the anticipated clones would be the same gender as the donor and they could never propagate by natural breeding. In a recent cloning experiment, we obtained a female mouse from an immature Sertoli cell. This ‘male-derived female’ clone grew into a normal adult and produced offspring by natural mating [132]. Although this was an accidental phenomenon arising from a sex chromosomal error, the result unequivocally suggests the possibility of producing females from male donor animals if the techniques of sex chromosome manipulation are sufficiently well developed.

(b) The possibility of selecting high-quality embryos before transfer

In addition to epigenetic alterations, genetic abnormalities arising during early cleavage stages, such as chromosomal abnormalities [105,133,134], might also be reasons for the low success rate of cloning. If so, it is probable that the SCNT-derived embryos could develop to term if such epigenetic reprogramming were to occur correctly leading to normal chromosomal segregation. Unfortunately, the morphology and/or rate of development to the blastocyst stage are not significant predictive markers for the full-term development of cloned embryos. However, we have recently succeeded in developing a ‘less-damage’ live-cell fluorescent imaging system optimized for preimplantation mouse embryos [135]. Using this system, we succeeded in selecting chromosomally normal cloned embryos and could improve the cloning success rate after embryo transfer [136]. If these selection and epigenetic modification methods could be combined, exploration of the mechanisms involved in genomic reprogramming would be accelerated and questions about the differences between embryonic cell-derived clones and SCNT-derived clones might be solvable. The cloning success rate per transferred embryo would be sufficient for applications in commercial animal breeding.

(c) Nuclear transfer techniques for analysing germ cell epigenetics

Pre-meiotic germ cells can also be used for constructing diploid embryos by NT, and this can work as a powerful tool to determine the dynamics of epigenetic changes during germ cell development. One important study in this category is the analysis of the genomic imprinting status of germ cells, especially primordial germ cells (PGCs) and gonocytes. Genomic imprinting involves an epigenetic ‘memory’ for parental allele-specific expression in about 100 genes in eutherian mammals [137,138]. Based on the principle that reprogramming in the mature ooplasm does not alter the genomic imprinting, cloned foetuses generated from germ cells are expected to reflect the donor's genomic imprinting status faithfully [139]. Therefore, analysis of foetuses and placentas reconstructed from germ cells might give invaluable information on their genomic imprinting status based on DNA methylation status and gene expression pattern. It is usually difficult to determine the gene expression profile of imprinted genes by conventional direct analysis of germ cells because most imprinted genes are primarily expressed in developing foetuses and placentas [137]. Furthermore, such direct analysis gives us only an averaged picture of a mixed population of germ cells. NT cloning of germ cells can overcome these biological and technical disadvantages. By employing the NT technique, we could analyse the dynamics of the imprinting erasure process in PGCs at 11.5 dpc and found the coordinated order of erasure specific for each imprinted gene [57]. This is consistent with the results from a comprehensive genomic analysis using PGCs at each developmental stage [140]. We identified the ‘default status’ of each imprinted gene either as a biallelic expression or as no expression, which were achieved in PGCs by 12.5 dpc. Similarly, this experimental system can also be applied to pre-meiotic male germ cells such as gonocytes and spermatogonia. According to the gene expression pattern of foetuses generated from these cells, the paternal genomic imprinting was thought to be imposed by 16.5 dpc for both the H19-DMR (differentially methylated region) and IG-DMR (A. Ogura, K. Inoue, unpublished data). Furthermore, by using germ cells at different developmental stages (PGCs, gonocytes, germline stem cells, round spermatids and parthenotes), we are now examining which genome ensures the correct Xist expression after NT. The results from RNA FISH and quantitative mRNA expression analysis imply that, for the Xist gene, transcription from the 4-cell stage onwards is the default pattern, and some unknown imprinting machinery, probably imposed during oogenesis, represses this transcription (A. Ogura, K. Inoue, unpublished data). This is consistent with the previous finding that the X chromosome derived from non-growing oocytes was inactivated whereas that from fully grown oocytes remained active [141]. Interestingly, the establishment of this Xist-repressing mechanism was independent of de novo DNA methylation [142].

We also expect that NT using germ cells will provide important clues in understanding the factors that ensure the normal process of genomic reprogramming for producing totipotency. Our recent findings on the phenotypes of cloned embryos and offspring derived from PGCs at 10.5 dpc suggested a somatic cell nature of their genome; they showed stage-specific developmental arrest and hyperplastic placentas as those derived from SCNT [56]. Therefore, at some time during germ cell development between 10.5 dpc and the mature gamete stage, the germ cell genome acquires the ability to be reprogrammed fully for the beginning of new life. Recently developed high-resolution and genome-wide epigenetic analysis of germ cells could be combined effectively to achieve this research goal [143].

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