JSAR Outstanding Research Award Production of Cloned ... - J-Stage

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Oct 2, 2006 - When the giant oocytes were activated ... and after embryo transfer, some of these giant ..... Nagashima H, Baba T, Ogura A. Cytoplasmic asters.
Journal of Reproduction and Development, Vol. 53, No. 1, 2007

JSAR Outstanding Research Award Production of Cloned Mice and ES Cells from Adult Somatic Cells by Nuclear Transfer: How to Improve Cloning Efficiency? Teruhiko WAKAYAMA1) 1)

Center for Developmental Biology, RIKEN Kobe, 2–2–3 Minatojima-minamimachi, Kobe 650-0047, Japan Abstract. Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), most cloned embryos usually undergo developmental arrest prior to or soon after implantation, and the success rate for producing live offspring by cloning remains below 5%. The low success rate is believed to be associated with epigenetic errors, including abnormal DNA hypermethylation, but the mechanism of “reprogramming” is unclear. We have been able to develop a stable NT method in the mouse in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Especially in the mouse, only a few laboratories can make clones from adult somatic cells, and cloned mice are never successfully produced from most mouse strains. However, this technique promises to be an important tool for future research in basic biology. For example, NT can be used to generate embryonic stem (NT-ES) cell lines from a patient’s own somatic cells. We have shown that NT-ES cells are equivalent to ES cells derived from fertilized embryos and that they can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. In general, NTES cell techniques are expected to be applied to regenerative medicine; however, this technique can also be applied to the preservation of genetic resources of mouse strain instead of embryos, oocytes and spermatozoa. This review describes how to improve cloning efficiency and NT-ES cell establishment and further applications.

Key words: Clone, Nuclear transfer, Reprogramming, NT-ES cell, Trichostatin A (J. Reprod. Dev. 53: 13–26, 2007)

y the end of the 1980s, only the nuclei of early stage mammalian embryos were proven to enable development of full-term embryos following NT into enucleated oocytes, zygotes or 2cell embryo blastomeres [1–3]. Subsequently, NT techniques for domestic animals developed quickly, and the first adult somatic cell cloned animal, Dolly the sheep, was reported in 1997 [4]. However, notwithstanding the relative ease of mouse embryo culture and manipulation, cloning proved more difficult for the mouse than for most other species. The first cloned mice were born Accepted for publication: October 2, 2006 T. Wakayama (e-mail: [email protected])

following the fusion of enucleated oocytes with the blastomeres of late 2-cell embryos in 1991 [5]. Not until 1998 was it shown to be possible to clone mice by serial transfer of nuclei from morulae or blastocyst inner cell mass (ICM) cells, first into enucleated oocytes and then into the blastomeres of 2-cell embryos [6, 7]. Most of those experiments were done using cell fusion, which is used widely for most animal experiments. The alternative method, using a donor cell nucleus injected into the oocyte directly, skips several NT procedures, such as zona cutting, donor cell insertion into perivitelline space, movement of cells on the electro-fusion machine, and fusion and confirmation of cell fusion 30 min

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later; however, this method has proven to be much more technically difficult. Mouse oocytes in p a r t ic u la r a r e s o f r a g i l e t h a t c o n v e n t i o n a l microinjection through pipettes wider than 1 µm at their tips is impracticable because it leads to lysis. This was eventually overcome with the application of piezo-actuated micromanipulation [8], which permitted the use of larger microinjection pipettes for intracytoplasmic sperm injection (ICSI) and led to the realization that they could be utilized in NT experiments [9]. By 1998, we had developed a mouse somatic cell NT method in which donor nuclei are injected into enucleated oocytes using a piezo unit and succeeded in producing the first cloned mouse Cumulina [10]. However, cloning of animals has been inefficient since the first cloned animal Dolly was born in 1997 [4]. Although we have tried may new methods, such as activation [11] and enucleation timing [12], inhibition of cytokinesis [13], and treatment with dimethyl sulfoxide (DMSO) [13], most of the method had no effect. Moreover, abnormalities in mice cloned from somatic cells have been reported, such as abnormal gene expression in embryos [14, 15], abnormal placentas [16, 17], obesity [18] and early death [19]. Such abnormalities notwithstanding, success in generating cloned offspring has opened new avenues for study of complex processes, such as genomic reprogramming, imprinting, and embryonic development. Thus, there is an urgent need to improve cloning efficiency for further understanding. Very recently, one of my laboratory staff members, Dr. Kishigami, found that the efficiency of mouse cloning can be enhanced by up to six-fold through the addition of the histone deacetylation inhibitor trichostatin A (TSA) into the oocyte activation medium [20], even though high concentrations of TSA are toxic to embryonic development [21]. This result suggested the possibility that nuclear reprogramming can be enhanced by artificial chemical treatment. On the other hand, it became possible to create new types of embryonic stem (ES) cell lines via nuclear transfer (NT-ES cell) from adult somatic cells [22, 23]. We have previously shown that such NT-ES cell lines are capable of differentiating into all three germ layers in vitro or into spermatozoa and oocytes in chimeric mice [22]. We have also shown that they have the same developmental potential as ES cells from fertilized blastocysts,

even if NT is repeated several times [24]. Moreover, cloned mice can be obtained from these NT-ES cell lines using a second NT [22], which can be used as a backup of the donor cell genome and can help increase the overall success rate of mouse cloning [25]. NT-ES cell techniques have now been applied not only to preliminary medical research, including therapeutic cloning [26], but also to basic biological research. For example, such studies have shown irrevers ibl e chang es in the DN A of differentiated adult lymphocytes but not in that of olfactory neurons [27–29]. These techniques are also applicable to preservation of the genetic resources of any mouse strain instead of preserving them as embryos or gametes [30, 31]. At present, this technique is the only one available for preservation of valuable genetic resources from infertile mutant mice without the use of germ cells [30]. This review attempts to describe the efficiency of nuclear cloning and features of NT-ES cell technology for basic study, including the preservation of genetic resources.

Abnormalities Found in Cloned Mice Successful NT, whether for the purpose of reproductive cloning or for derivation of NT-ES cells, depends on the reprogramming of an adult somatic nucleus to an embryonic state. The current NT method, however, causes numerous abnormalities in cloned mice that are probably related to the efficiency of successful cloning. We review these clone-specific abnormalities briefly below before considering how we can improve the efficiency of cloning.

Early events of the donor nucleus following NT In primate cloning, it has been reported that removal of the meiotic spindle depletes the ooplasm of nuclear mitotic apparatus protein 1 (NuMA) and the kinesin family member C1 protein, which are both vital for mitotic spindle pole formation [32]. Although our previous experiments show that cloning efficiency could not be improved even if donor nuclei were injected into intact oocytes [12], enucleation of MII oocytes might result in the loss of some important factors such as asters [33]. We attempted to analyze in more detail such factors as the location of gamma tubulin and NuMA after enucleation and found

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that the level of gamma-tubulin in the poles and around the metaphase II spindle declines significantly, whereas only 10% of NuMA is removed during enucleation. Interestingly, retaining the donor cell centrosome establishes a monopolar spindle, whereas prior removal of the centrosome by a narrow micropipette leads to bipolar spindle formation [34]. At the same time, histone H3 phosphorylation and acetylation of injected somatic cell nuclei took place in the oocytes under regulation by the oocytes cytoplasm [35].

First differentiation in cloned embryo Most cloned embryos die during the pre- and peri-implantation period [36]. This early stage lethality may represent a potential problem when establishin g eithe r or both em bryonic an d extraembryonic lineages within the developing embryo. Loss of and reduced pluripotency in the inner cell mass (ICM) lineage of cloned blastocysts has previously been reported [15]. We have previously examined the trophectoderm (TE) and ICM lineages using Cdx2 and Oct4, key molecules for the specification of TE and ICM fate, respectively. Oct4 expressing blastocysts were as low as 50%, as previously reported [15]; however, regardless of Oct4 expression, the majority of the cloned blastocysts (>90%) normally expressed Cdx2. This result suggests that even though somatic cloned embryos have a reduced potential to produce the inner cell mass lineage, the TE lineage can be established and maintained [37].

Epigenetic abnormality in offspring and the placenta In general, abnormal phenotypes, such as placental hyperplasia in cloned mice [16, 17], are caused entirely by disruption of the mouse’s epigenetic state, as cloned mice are genetically identical to their donors, and the phenotypes are limited to the generation that is derived from cloning [38]. These abnormalities presumably reflect a dysregulation of gene function in cells of the trophectodermal lineage, which may be accounted for by aberrant reprogramming-the failure to completely reset the differentiative state of the host cell [39]. Conversely, we found that abnormal growth of cloned mice is associated with a failure to re-establish a tissue-specific DNA methylation pattern [40] and identified the Spaltlike gene3 (Sall3) locus as a hypermethylated region in the placental genome of cloned mice [41].

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Large placentas were more heavily methylated at the Sall3 locus in cloned mice, and this epigenetic error was found in all cloned mice examined regardless of sex, strain or cell type; the control placenta did not show such hypermethylation. We concluded that the Sall3 locus is an area with frequent epigenetic errors in cloned mice [41]. Abnormalities have also been reported in cloned offspring. When an inbred mouse strain was used as the donor, most of the cloned embryos died just after implantation or soon after birth as a results of respiratory catastrophe [16, 36, 42, 43], while those from a hybrid mouse strain did not [10]. However, with significant variance across strains, the postpubertal body mass of clones becomes significantly greater than that of non-cloned controls from approximately 8–10 weeks of age [18], and the clones also have elevated plasma leptin and insulin levels [38]. The increase in body mass is not a consequence of low activity or increased dietary intake, as behavioral assays do not show any significant difference between the clones and the controls. Hence, the difference in body mass may be due to lower basal energy expenditure in clones [38]. This data suggests that there is at least one genetic locus that is highly susceptible to epigenetic error caused by NT. This error in DNA methylation in cloned mice allowed us to attempt to further investigate X-inactivation. The ratio of cells containing inactivated maternal X chromosomes to cells containing inactivated paternal X chromosomes is approximately 1:1. Under some circumstances, however, Xinactivation is skewed. We have examined the Xinactivation ratio in adult B6C3F1 clones, in which CpG islands on the X chromosome between C57Bl/ 6 and C3H/He mouse strains were distinguished by a difference in a single nucleotide polymorphism (SNP). As a result, skewed Xinactivation was observed to various degrees in different tissues of different individuals, suggesting that skewed X-inactivation in cloned mice is the result of secondary cell selection in combination with stochastic distortion of primary choice [44]. These findings indicate that a cloned mouse is not a perfect copy of the original mouse in terms of placental development, body weight, or the methylation status of its genomic DNA [40]. Ogura et al. have reported that many cloned mice die prematurely due to liver necrosis, tumors, and pneumonia [19]. However, some clones survive as

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long as non-clones, and the first cloned mouse, Cumulina, died at the age of 2 years and 7 months, which is slightly longer than the lifespan of an average mouse (news in brief. Nature 2000, 405:268). This indicates that more research is needed on the life expectancy of cloned animals.

activated in the most natural manner, by spermatozoa, the success rates were virtually the same (1–2%) [11]. This result suggests that the choice of activation method is not critical for mouse cloning (Fig. 1D).

Effect of timing of the removal of the oocyte spindle New Attempts to Improve the Efficiency of Mouse Cloning Why is the success rate of cloning so low? Is it because only a few percent of any cell population is “cloning-competent” or is it simply because of technical problems? Probably many technical factors, such as the oocyte activation method and chemical agents, affect cloned embryo development. Here, I will introduce our attempt to improve mouse cloning.

Timing of oocytes activation The timing of oocyte activation is very important. When cumulus cell nuclei were microinjected into enucleated zygotes or enucleated pre-activated oocytes, highly fragmented chromosomes and poor development were observed [45], whereas when cumulus cells were injected into enucleated oocytes and then activated immediately or within a few hours, more than half of the cloned embryos developed to blastocysts [10, 13] (Fig. 1A and B).

Effect of activation method Reconstructed oocytes must be activated artificially for their development to progress. We have tried and compared four different oocyte activation methods (strontium, ethanol, electrostimulation, and spermatozoa injection) for mouse cloning. However, even when cloned oocytes were

Fig. 1.

Donor nuclei were injected into intact oocytes, and then oocyte chromosomes were removed. This was done because it is not yet known whether enucleation of oocytes results in developmental impairment or whether it removes the factors necessary to reprogram the incoming nucleus [12]. Of these, 1% of the embryos developed to full term, an efficiency that is comparable to that obtained for the controls (1–2%) in which enucleation preceded NT (Fig. 1E). This method suggests that the timing of the removal of oocyte chromosomes before or after injection of the somatic nucleus has no effect on the development of cloned embryos. These findings imply that neither oocyte chromosome depletion, per se, nor the potential removal of “reprogramming” factors during enucleation can explain the low efficiency of NT cloning [12].

Effect of cytokinesis inhibitor In our protocol, reconstructed mouse oocytes are activated with cytochalasin to prevent the loss of somatic cell chromosomes in the pseudo-polar body [10]. The most commonly used reagents are cytochalasin B and D [46–48], which disrupt actin filaments. Another commonly used agent is nocodazole [49], which interferes with microtubule assembly. When reconstructed oocytes were treated with Sr2+ in the presence of cytochalasin B or cytochalasin D, the reconstructed zygotes had 2 or 3 pseudo-pronuclei. On the other hand, when reconstructed oocytes were activated wit h

Diagrammatic representation of six mouse NT protocols. (A) Zygotes or pre-activated oocytes are enucleated and then a diploid (2n) donor nucleus are injected. This method results in embryo degeneration at the 2- to 4-cell stage or fragmentation soon after activation, respectively. (B) Original method. Metaphase II (MII) oocytes are enucleated, and 1– 3 h after nuclear transfer, reconstructed oocytes are activated with cytochalasin B. Of the resulting 1-cell embryos, 1–2% develop to term. (C) G2-/M-phase donor cell nuclei (4n) are injected into enucleated oocytes, with subsequent activation in the absence of a cytokinesis inhibitor. (D) MII oocytes are enucleated, and 1–3 h after nuclear transfer, reconstructed oocytes are activated by sperm injection with CB. Six hours after sperm injection, the male pronucleus is removed. (E) A donor nucleus is injected into intact MII oocytes and then oocyte chromosomes are removed 1–3 h after NT. After that, the oocytes are activated. (F) Newly improved method. MII oocytes are enucleated, and 1–3 hours after nuclear transfer, reconstructed oocytes are activated with CB and TSA. Of the resulting 1-cell embryos, 5–6% develop to term. With the exception of (C), all donor nuclei are diploid (2n). CB, cytochalasin B; MII, metaphase II; TSA, trichostatin A.

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nocodazole, the reconstructed zygotes had many small pseudo-pronuclei. However, 1–3% of the reconstructed oocytes developed to full term irrespective of treatment, which suggests that the cytokinesis inhibitor has no harmful effect on development [13].

giant oocytes fertilized and developed normally, and after embryo transfer, some of these giant embryos developed to healthy and fertile offspring. However, giant oocytes or extra cytoplasm could not improve the development of cloned mice by NT (Wakayama S. et al., submitted).

Effect of in vitro culture period

Effect of DMSO

When reconstructed embryos were transferred to surrogate mothers at the 2-cell (one day of in vitro culture) or morula/blastocyst stage (4 days of culture), an average of 2% and 3% of the reconstructed embryos developed to full term, respectively. These results suggests that the in vitro culture period has no detrimental effect [13].

We accidentally discovered that 1% DMSO can significantly improve the frequency of development to the blastocyst stage in vitro; however, this improvement did not lead to a higher success rate of full term development [13]. It is not clear why DMSO enhanced the developmental potential of cloned embryos. DMSO is known to cause differentiation of ES cells into muscle [51, 52]. Recently, Iwatani et al. discovered that the DMSO affects the DNA methylation status at multiple loci, inducing hypomethylation as well as hypermethylation depending on the genomic loci [53]. It might be suggested that DMSO affects differentiation from ES cells to somatic cells, but for NT, DMSO might affect reprogramming of somatic cell nuclei after/during oocyte activation [13].

Effect of cell number of cloned embryos Boiani et al. reported that most cloned blastocysts show abnormal Oct4 expression [15] and that this could be improved by aggregation of the cloned embryos with each other [50]. This result suggests that the success rate of cloned offspring may be improved by increasing the number of normal ICM. Here, we hypothesize that the NT-ES cells may enhance the development of cloned offspring by injecting them into cloned embryos (NT-ES cell complementation method). In this study, cloned embryos were produced from NT-ES cell nuclei, and the same line’s NT-ES cells were inserted into the perivitelline space of 8-cell cloned embryos. As a result, the inserted NT-ES cells contributed to the ICM, and the number of Oct4 expression cells increased in the clone-chimera blastocyst (Mizutani et al., submitted).

Effect of the volume of oocyte cytoplasm It is not yet clear whether an oocyte’s cytoplasm is sufficient for reprogramming of one somatic cell nucleus. If this is not the case, cloning success rates might be improved by adding supplementary oocyte cytoplasm before the SCNT procedure. We reconstructed oocytes with two to nine times the normal volume by electrofusion or mechanical fusion using intact and enucleated oocytes. We examined their in vitro and in vivo developmental potential after parthenogenetic activation, intracytoplasmic sperm injection (ICSI), and somatic cell NT. When the giant oocytes were activated parthenogenetically, most developed to morulae or blastocysts, irrespective of their original size. Sperm heads injected by ICSI into diploid

Effect of trichostatin A Recent molecular analyses of cloned embryos reveal abnormal epigenetic modifications, such as DNA methylation and histone modification [40, 54– 56]. Therefore, prevention of epigenetic errors has been expected to lead to improvement of the success rate of animal cloning. Although there are a number of reports concerning 5-azacytidine, an inhibitor of DNA methylation, and Trichostatin A (TSA), an inhibitor of histone deacetylas e, treatments [57], none of them significantly improve the actual cloning efficiency. However, each drug must be examined for appropriate exposure timing, c o nc entr atio n and d ur atio n. Rec ently , we identified the optimal conditions for TSA in a study in which a 5–50 nM TSA-treatment for 10 h following oocyte activation led to more than a 5fold increase in the success rate of mouse cloning from cumulus cells without obvious abnormalities [20]. These results were independently and concurrently obtained by Tsunoda’s lab [58], which suggests that the effect of TSA are repeatable irrespective of the laboratory. Thus, our findings provide a new approach for practical improvement of mouse cloning techniques and insight into the reprogramming of somatic nuclei.

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Effect of Donor Cell Type Origin of somatic cell donor At the beginning, this technique [10] was difficult to reproduce for other laboratories. Several laboratories, such as Ogura’s lab, can now continuously reproduce cloned mice, and currently, cumulus cells [10], tail tip cells (probably fibroblasts) [16, 59], Sertoli cells [60], fetal cells [42, 61], and ES cells [62] are routinely used to produce cloned mice. NKT cells [63], primordial germ cells [64], hematopoietic stem cells [65], granulocyte [66], fetal neuronal cells [67], and neuronal stem cells [68] have also been used. Donor age and sex are not too important in mouse cloning because the success rate when using fetal cells is the same or lower than with adult somatic cell nuclei.

Effect of somatic stem cells In general, the success rate of cloned mice from ES cell nuclei is known to be higher than that of cloned mice from somatic cell nuclei [62, 69], suggesting that the undifferentiated state of donor cells, or somatic stem cells, may increase cloning efficiency. Previously, hematopoietic stem cells and mesenchymal stem cells had been examined for the efficiency of cloning in the mouse [65] and cow [70], respectively. Although both type of donor cell could support full-term development after NT, the efficiency was the same or lower than for differentiated somatic cell nuclei. We have also assessed the developmental ability of cloned embryos derived from cultured neural stem cell (NSC) nuclei and compared the success rate with other somatic and ES cells. Although we succeeded in producing cloned mice from cultured NSCs, the success rate of cloned mice (5 mice; 0.5%) was lower than for other cell types (2, 3 and 4% success rates from cumulus, Sertoli and ES cells, respectively). These results indicate that, at least for those particular somatic stem cells, the partially undifferentiated state of somatic cells does not enhance cloning efficiency in mice [68].

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recently, only hybrid mice and the 129 strain have been used successfully as sources of donor nuclei (either somatic or ES cell) or recipient oocytes. However, as mentioned above, we have established a new efficient cloning technique using TSA that leads to a 2–5 fold increase in the success rate for mouse cloning of B6D2F1 cumulus cells [20]. We attempted to clone an adult ICR mouse, an outbred strain, which has never been directly cloned before. We obtained both male fand female cloned mice from cumulus and fibroblast cells of adult ICR mice with a 4–5% success rates, which is comparable to the 6–7% success rate using B6D2F1 mice, but only when TSA was used. Thus, the TSA cloning technique allows us to successfully clone outbred mice, demonstrating for the first time that this technique not only improves the success rate of cloning from hybrid strains but also enables mouse cloning from normally “unclonable” strains [71].

Effect of cell cycle When Dolly, the first cloned animal, was reported, it was claimed that the donor cells had to be quiescent (G0) at the time of NT [4]. However, at least in the mouse, cloning efficiency is not markedly affected by the phase of the donor cell cycle [16]. Further experiments have suggested that donor cells at different cell cycle stages (G0, G1, G2 and M phase, see Fig. 1C) are cloning competent [62, 72–75].

Effect of re-cloning We and others have demonstrated that cloned animals can be generated from the somatic cells of cloned animals [76, 77]. However, the overall trend of the cloning efficiency is downward; in the mouse, four to six generations is the upper limit, whereas in the cow, the second generation is the upper limit. It is unclear why subsequent cloning is not possible. At the cellular level, the telomere length of the peripheral blood lymphocytes of cloned mice did not reveal evidence of shortened telomeres [77].

Effect of mouse genotype The cloning success rate depends on the mouse strain [42, 43]. Usually, a hybrid strain is more suitable for mouse cloning than an inbred strain. C57BL/6 and C3H/He are popular mouse strains in mouse genetics but have never been used successfully to produce cloned mice [42]. Until

Other Possibilities to Improve the Cloning Contamination of somatic cell cytoplasm into the oocyte may causes abnormalities During the process of somatic cell NT, all or partial cytoplasm is introduced into the enucleated

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oocytes with the donor nucleus, regardless of whether the electrofusion method is used or the somatic nucleus is injected directly. However, there are no reports demonstrating that somatic cell cytoplasm is a cause of the abnormalities that occur in the development of cloned embryos or in fullt e r m d e v e l o p m e n t . R e c e n t l y , T h u a n et a l . discovered that when somatic cell cytoplasm was injected into oocytes before fertilization, the subsequent in vitro fertilized embryos had decreased in preimplantation development and there was placental overgrowth [78]. So far placental overgrowth phenotypes are only caused by interspecies hybridization of mouse, genetically modified mice or cloned animal. This study suggests that somatic cell cytoplasmic material is one cause of the low rate of full-term development in cloned mammals. Next, we must find a new method to transfer the donor nucleus to oocytes without contamination of the donor cytoplasm.

From abnormal cloned mice to normal healthy mice Cloned mice-specific abnormalities, such as obesity and an enlarged placenta are not heritable and are absent from the progeny of both clones mated with clones and clones mated with wild type mice [38, 79]. This implies that an epigenetic abnormality, such as an imprinting and/or reprogramming phenomenon, caused by NT is corrected during gametogenesis. Although this is good news for rescuing endangered species by cloning [80], cloned animal have fundamental problems, including weakness and susceptibility to illness, and these animals often die before growing to adulthood [81]. These animals are never able to produce offspring due to their sexually immature state. We attempted to produce offspring from mice that were ill or that died due to large offspring s y n d ro m e i m m e di a t el y a f t e r b i r t h u s i n g a combination of testicular transplantation and ICSI. Two to 3 months after transplantation, the grafted clone’s testicular tissue was observed to have grown, and spermatogenesis occurred in the host testis. Intracytoplasmic sperm injection demonstrated that the testicular sperm generated in the grafted clone’s tissue were able to support full-term development and that these offspring did not show any clone-specific abnormalities. This result clearly demonstrated that even if spermatogenesis was induced artificially, the epigenetic errors in the cloned mice were corrected

during germ cell differentiation [82].

Establishment of NT-ES Cell Lines There has been a great deal of interest in the possible application of human embryonic stem (ES) cells in regenerative medicine, as ES cells are envisioned to be potential sources for cell replacement therapies. However, as with any allogeneic material, ES cells derived from fertilized blastocysts and the progeny of such cells inevitably face the risk of immunorejection on transplantation. It has been proposed that ES cells derived from embryos cloned from the host patient’s own cell nuclei represent a potential solution to the problem of rejection, as any replacement cells would be genetically identical to the host’s own somatic cell nuclei [83–85]. The first successful production of ES-like cell lines from somatic cells via NT was reported in a bovine model [86] and was followed by production in mouse models [23, 87]. These ESlike cell lines are believed to possess the same capacities for unlimited differentiation and selfrenewal as those of conventional ES cell lines derived from normal embryos produced by fertilization. We have previously shown that NTES cell lines are capable of differentiating into all three germ layers in vitro or into spermatozoa and oocytes in chimeric mice [22]. This was the first demonstration that NT-ES cells have the same potential as ES cells derived from normally fertilized embryos. Moreover, cloned mice can be obtained from NT-ES cells lines by a second NT procedure [22, 25, 30]. These NT-ES cells lines can be used not only for basic research but may also have applications in tissue repair and transplantation medicine, a concept referred to as “therapeutic cloning” [26].

Characterization of NT-ES cells It is still unclear whether any individual mouse or cell type can be used to generate NT-ES cell lines. We have demonstrated that cumulus cell nuclei from F 1 mouse genotypes show a significantly higher cumulative establishment rate from reconstructed oocytes than from other cells; however, there no differences between the genotypes in the success rates from cloned blastocysts [31]. Overall success, therefore, depends on preimplantation development, and

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once embryos have reached the blastocyst stage, the genotype differences disappear. In the mouse genotypes that were tested, all demonstrated at least one cell line that subsequently contributed to germline transmission in chimeric mice, indicating that these cell lines clearly possess the same potential as embryonic stem cells derived from fertilized embryos. These results therefore suggest that NT-ES cells can be generated relatively easily from a variety of inbred mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly [31].

Equivalency of NT-ES cells to ES cells

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therapy for these diseases is currently limited by the availability and immunocompatibility of tissue transplants [83–85]. We have previously demonstrated that dopaminergic and serotonergic neurons can be generated from NT-ES cells derived from tail tip cells [22]. Rideout et al. reported that therapeutic cloning combined with gene therapy was able to treat a form of combined immune deficiency in mice [26]. In addition, Barberi et al. reported an interesting study that showed that mouse tail- or cumulus cell-derived NT-ES cells could be differentiated into neural cells at even higher efficiencies than fertilization-derived ES cells [90].

It still unclear whether NT-ES cells can be used in medicine, as many reports state that cloned animals produced by somatic cell NT possess gross abnormalities [16, 18, 40, 88] and NT-ES cell lines are generated by the same procedure. Moreover, the rate of establishment of NT-ES cells is much higher than that of animal cloning [22, 25, 31]. Most NT-ES cell lines are therefore established from cloned embryos with negligible reproductive potentials. For this reasons, we analyzed and compared NT-ES cells to ES cells derived from fertilized embryos. In the majority of experiments, such as karyotype, ES cell marker, donor cell aging and multi-NT, no significant differences were found between ES and NT-ES cell [24]. Although we found a few differences between NT-ES cells and ES cells and even between two NT-ES cell lines, s u c h a s in t h e D N A m e t h y la t i o n a n d g e n e expression profiles, the amount of difference is still far less than that found between different ES cell lines established from other laboratories. We are therefore able to conclude that NT-ES cells are almost the same as ES cells and that they have the potential to be used in regenerative medicine [24]. Brambrink et al. also demonstrated that mouse NTES cells closely resemble ES cells from naturally fertilized embryos using global gene expression profiles [89].

The current success rate of generating cloned mice from adult somatic cell nuclei is very low, whereas the generation of cloned mice from ES cell nuclei is relatively high [10, 31]. We have examined whether the success rate of cloning from somatic cells could be improved through the use of NT-ES cells established from the somatic cell nuclei of the same individual [25]. Although we obtained many cloned mice, the overall success rate of cloning from NT-ES cell nuclei was no better than when using somatic cell nuclei. We were ultimately able to obtain cloned mice from six individuals out of seven using either somatic or NT-ES cells. So, although NT-ES cells as donor nuclei do not ensure a superior success rate for mouse cloning over that of somatic cells, we recommend the establishment of NT-ES cell lines at the same time to preserve and clone valuable individuals. These can be used as an unlimited source of donor nuclei for NT and can therefore complement conventional somatic cell NT cloning approaches [25]. Unfortunately, if difficulty is experienced in the production of cloned mice from a donor cell, difficulty is also experienced when attempting to produce cloned mice by a second NT from NT-ES cell nuclei [30].

Application of NT-ES Cell Techniques

Preservation of genes from infertile mice without the use of germ cells

NT-ES cells are genetically identical to the donor and are potentially useful for therapeutic application. Therefore, therapeutic cloning may improve the treatment of neurodegenerative diseases, blood disorders, and diabetes, since

The genetically modified mouse is a powerful tool for research in the fields of medicine and biology. However, infertility was listed as a phenotype in more than half of the mutants described in one large-scale ethyl-nitrosourea (ENU) mutagenesis study [91, 92]. This is a

Improving the mouse cloning success rate from somatic cell via NT-ES cells

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challenge worth taking, as the ability to maintain such types of mutant mice as genetic resources will offer numerous advantages to research on human infertility and the biology of reproduction. Unfortunately, the success rate of somatic cell cloning is very low [20]. Even in cases in which the cloning of a sterile mouse is successful, due to the non-reproductive nature of the phenotype, it is still necessary to clone all subsequent generations. This represents another significant potential barrier, as the success rate of cloning from cloned mice decreases for each successive generation after the first NT [77]. On the other hand, NT-ES technology can allow us to maintain such interesting genes from mouse strains even without the use of germ cells [22, 25, 30]. We were able to find an infertile mouse in our ICR mouse colony. This mouse showed a hermaphroditic phenotype, possessing both testes and ovaries but entirely lacking germ cells. Although we failed to generate a cloned mouse using tail-tip cells, we were able to establish several NT-ES cell lines from the tail. As a result of NT-ES cell examination, the original hermaphrodite mouse was found to be a male with an abnormal Y chromosome. We next tried to produce cloned mice from the NT-ES cell nuclei. Despite attempting more than 1000 NTs, we only managed to obtain placentae. We also tried to make chimeric mice using tetraploid or diploid embryos. When NT-ES cells were injected into the blastocoels of tetraploid embryos, 2 live clonal mice [30] were obtained in which most of the cells of the chimeric mice originated from NT-ES cells; they both inherited an abnormal Y chromosome. When NT-ES cells were injected into the blastocoels of diploid embryos, many chimeric mice were produced. Although tetraploid chimeric and highcolor-coat contribution diploid chimeric mice were infertile, one low-color-coat contribution chimeric mice demonstrated germline transmission of NTES cells [30]. These findings provide a proof-inprinciple demonstration that generation of ES cell lines via somatic NT offers a novel way of preserving mouse genetic resources and at present is the only technique capable of obtaining offspring from mutants lacking germline cells [30].

Improving the quality of ES cells by NT Although parthenogenetic embryos lack the potential to develop to full term [93, 94], they can be used to establish parthenogenetic embryonic stem (pES) cells for autologous cell therapy in females without needing to destroy normally competent embryos [95]. Unfortunately, the capacity for further differentiation of these pES cells in vivo is very poor [96, 97]. Recently, we succeeded in improving the potential of pES cells using an NT technique [98]. The donor nuclei from the original pES cell were transferred into enucleated oocytes, and the resulting cloned embryos were used to establish new NT-pES cell lines. All examined NTpES cell lines were positive for several ES cell markers and had a normal extent of karyotypes. The DNA methylation status of differentially methylated doma in H 19 a n d d i ff e r ent ia ll y methylated region IG did not change after NT. However, the in vivo and in vitro differentiation potentials of the NT-pES cells were significantly (2 to 5 times) better than the original pES cells, as judged by the production of chimeric mice and by in vitro differentiation into neuronal and mesodermal cell lines. Thus, NT could be used to improve the potential of pES cells and may enhance that of otherwise poor quality ES cells. It also offers a new tool for studying epigenetics.

Perspective The advent of mouse cloning from adult-derived cells in July 1998 marked a new departure in the study of key biological problems in cloning biology. However, even though we have successfully improved the cloning efficiency by TSA treatment, it is still greatly lower than the rate of fertilized embryos, and we must find better method for further application. On the other hand, abnormality of cloned animals is also big obstacle for application, but this may be resolved when the mechanisms of reprogramming are better understood. We believe that the mechanisms of reprogramming will be clear when cloning efficiency is improved though a technical approach.

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References 1. Willadsen SM. Nuclear transplantation in sheep embryos. Nature 1986; 320: 63–65. 2. Tsunoda Y, Yasui T, Shioda Y, Nakamura K, Uchida T, Sugie T. Full-term development of mouse blastomere nuclei transplanted into enucleated twocell embryos. J Exp Zool 1987; 242: 147–151. 3. McGrath J, Solter D. Nuclear transplantation in the mouse embryo by microsurgery and cell fusion. Science 1983; 220: 1300–1302. 4. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH. Viable offspring derived from fetal and adult mammalian cells. Nature 1997; 385: 810– 813. 5. Kono T, Tsunoda Y, Nakahara T. Production of identical twin and triplet mice by nuclear transplantation. J Exp Zool 1991; 257: 214–219. 6. Tsunoda Y, Kato Y. Full-term development after transfer of nuclei from 4-cell and compacted morula stage embryos to enucleated oocytes in the mouse. J Exp Zool 1997; 278: 250–254. 7. Tsunoda Y, Kato Y. Not only inner cell mass cell nuclei but also trophectoderm nuclei of mouse blastocysts have a developmental totipotency. J Reprod Fertil 1998; 113: 181–184. 8. Kimura Y, Yanagimachi R. Intracytoplasmic sperm injection in the mouse. Biol Reprod 1995; 52: 709–720. 9. Kishigami S, Wakayama S, Thuan NV, Ohta H, Mizutani E, Hikichi T, Bui HT, Balbach S, Ogura A, Boiani M, Wakayama T. Production of cloned mice by somatic cell nuclear transfer. Nat Prot 2006; 1: 125–138. 10. Wakayama T, Perry AC, Zuccotti M, Johnson KR, Yanagimachi R. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 1998; 394: 369–374. 11. Kishikawa H, Wakayama T, Yanagimachi R. Comparison of oocyte-activating agents for mouse cloning. Cloning 1999; 1: 153–159. 12. Wakayama S, Cibelli JB, Wakayama T. Effect of timing of the removal of oocyte chromosomes before or after injection of somatic nucleus on development of NT embryos. Cloning Stem Cells 2003; 5: 181–189. 13. Wakayama T, Yanagimachi R. Effect of cytokinesis inhibitors, DMSO and the timing of oocyte activation on mouse cloning using cumulus cell nuclei. Reproduction 2001; 122: 49–60. 14. Bortvin A, Eggan K, Skaletsky H, Akutsu H, Berry DL, Yanagimachi R, Page DC, Jaenisch R. Incomplete reactivation of Oct4-related genes in mouse embryos cloned from somatic nuclei. Development 2003; 130: 1673–1680. 15. Boiani M, Eckardt S, Scholer HR, McLaughlin KJ. Oct4 distribution and level in mouse clones: consequences for pluripotency. Genes Dev 2002; 16:

1209–1219. 16. Wakayama T, Yanagimachi R. Cloning of male mice from adult tail-tip cells. Nat Genet 1999; 22: 127–128. 17. Tanaka S, Oda M, Toyoshima Y, Wakayama T, Tanaka M, Yoshida N, Hattori N, Ohgane J, Yanagimach R, Shiota K. Placentomegaly in cloned mouse concepti caused by expansion of the spongiotrophoblast layer. Biol Reprod 2001; 65: 1813– 1821. 18. Tamashiro KL, Wakayama T, Blanchard RJ, Blanchard DC, Yanagimachi R. Postnatal growth and behavioral development of mice cloned from adult cumulus cells. Biol Reprod 2000; 63: 328–334. 19. Ogonuki N, Inoue K, Yamamoto Y, Noguchi Y, Tanemura K, Suzuki O, Nakayama H, Doi K, Ohtomo Y, Satoh M, Nishida A, Ogura, A. Early death of mice cloned from somatic cells. Nat Genet 2002; 30: 253–254. 20. Kishigami S, Mizutani E, Ohta H, Hikichi T, Thuan NV, Wakayama S, Bui HT, Wakayama T. Significant improvement of mouse cloning technique by treatment with trichostatin A after somatic nuclear transfer. Biochem Biophys Res Commun 2006; 340: 183–189. 21. Svensson K, Mattsson R, James TC, Wentzel P, Pilartz M, MacLaughlin J, Miller SJ, Olsson T, Eriksson UJ, Ohlsson R. The paternal allele of the H19 gene is progressively silenced during early mouse development: the acetylation status of histones may be involved in the generation of variegated expression patterns. Development 1998; 125: 61–69. 22. Wakayama T, Tabar V, Rodriguez I, Perry AC, Studer L, Mombaerts P. Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer. Science 2001; 292: 740–743. 23. Munsie MJ, Michalska AE, O’Brien CM, Trounson AO, Pera MF, Mountford PS. Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei. Curr Biol 2000; 10: 989–992. 24. Wakayama S, Jakt ML, Suzuki M, Araki R, Hikichi T, Kishigami S, Ohta H, Van Thuan N, Mizutani E, Sakaide Y, Senda S, Tanaka S, Okada M, Miyake M, Abe M, Nishikawa S, Shiota K, Wakayama T. Equivalency of nuclear transfer-derived embryonic stem cells to those derived from fertilized mouse blastocysts. Stem Cells 2006; 24: 2023–2033. 25. Wakayama S, Mizutani E, Kishigami S, Thuan NV, Ohta H, Hikichi T, Bui HT, Miyake M, Wakayama T. Mice cloned by nuclear transfer from somatic and ntES cells derived from the same individuals. J Reprod Dev 2005; 51: 765–772.

24

WAKAYAMA

26. Rideout WM. 3rd, Hochedlinger K, Kyba M, Daley GQ, Jaenisch R. Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy. Cell 2002; 109: 17–27. 27. Hochedlinger K, Jaenisch R. Monoclonal mice generated by nuclear transfer from mature B and T donor cells. Nature 2002; 415: 1035–1038. 28. Eggan K, Baldwin K, Tackett M, Osborne J, Gogos J, Chess A, Axel R, Jaenisch R. Mice cloned from olfactory sensory neurons. Nature 2004; 428: 44–49. 29. Li J, Ishii T, Feinstein P, Mombaerts P. Odorant receptor gene choice is reset by nuclear transfer from mouse olfactory sensory neurons. Nature 2004; 428: 393–399. 30. Wakayama S, Kishigami S, Van Thuan N, Ohta H, Hikichi T, Mizutani E, Yanagimachi R, Wakayama T. Propagation of an infertile hermaphrodite mouse lacking germ cells by using nuclear transfer and embryonic stem cell technology. Proc Natl Acad Sci USA 2005; 102: 29–33. 31. Wakayama S, Ohta H, Kishigami S, Thuan NV, Hikichi T, Mizutani E, Miyake M, Wakayama T. Establishment of male and female nuclear transfer embryonic stem cell lines from different mouse strains and tissues. Biol Reprod 2005; 72: 932–936. 32. Simerly C, Dominko T, Navara C, Payne C, Capuano S, Gosman G, Chong KY, Takahashi D, Chace C, Compton D, Hewitson L, Schatten G. Molecular correlates of primate nuclear transfer failures. Science 2003; 300: 297. 33. Miki H, Inoue K, Ogonuki N, Mochida K, Nagashima H, Baba T, Ogura A. Cytoplasmic asters are required for progression past the first cell cycle in cloned mouse embryos. Biol Reprod 2004; 71: 2022–2028. 34. Van Thuan N, Wakayama S, Kishigami S, Wakayama T. Donor centrosome regulation of initial spindle formation in mouse somatic cell nuclear transfer: roles of gamma-tubulin and nuclear mitotic apparatus protein 1. Biol Reprod 2006; 74: 777–787. 35. Bui HT, Van Thuan N, Wakayama T, Miyano T. Chromatin remodeling in somatic cells injected into mature pig oocytes. Reproduction 2006; 131: 1037– 1049. 36. Wakayama T, Yanagimachi R. Cloning the laboratory mouse. Semin Cell Dev Biol 1999; 10: 253– 258. 37. Kishigami S, Hikichi T, Van Thuan N, Ohta H, Wakayama S, Bui HT, Mizutani E, Wakayama T. Normal specification of the extraembryonic lineage after somatic nuclear transfer. FEBS Lett 2006; 580: 1801–1806. 38. Tamashiro KL, Wakayama T, Akutsu H, Yamazaki Y, Lachey JL, Wortman MD, Seeley RJ, D’Alessio DA, Woods SC, Yanagimachi R, Sakai RR. Cloned mice have an obese phenotype not transmitted to

their offspring. Nat Med 2002; 8: 262–267. 39. Inoue K, Kohda T, Lee J, Ogonuki N, Mochida K, Noguchi Y, Tanemura K, Kaneko-Ishino T, Ishino F, Ogura A. Faithful expression of imprinted genes in cloned mice. Science 2002; 295: 297. 40. Ohgane J, Wakayama T, Kogo Y, Senda S, Hattori N, Tanaka S, Yanagimachi R, Shiota K. DNA methylation variation in cloned mice. Genesis 2001; 30: 45–50. 41. Ohgane J, Wakayama T, Senda S, Yamazaki Y, Inoue K, Ogura A, Marh J, Tanaka S, Yanagimachi R, Shiota K. The Sall3 locus is an epigenetic hotspot of aberrant DNA methylation associated with placentomegaly of cloned mice. Genes Cells 2004; 9: 253–260. 42. Wakayama T, Yanagimachi R. Mouse cloning with nucleus donor cells of different age and type. Mol Reprod Dev 2001; 58: 376–383. 43. Inoue K, Ogonuki N, Mochida K, Yamamoto Y, Takano K, Kohda T, Ishino F, Ogura A. Effects of donor cell type and genotype on the efficiency of mouse somatic cell cloning. Biol Reprod 2003; 69: 1394–1400. 44. Senda S, Wakayama T, Yamazaki Y, Ohgane J, Hattori N, Tanaka S, Yanagimachi R, Shiota K. Skewed X-inactivation in cloned mice. Biochem Biophys Res Commun 2004; 321: 38–44. 45. Wakayama T, Tateno H, Mombaerts P, Yanagimachi R. Nuclear transfer into mouse zygotes. Nat Genet 2000; 24: 108–109. 46. Snow MH. Tetraploid mouse embryos produced by cytochalasin B during cleavage. Nature 1973; 244: 513–515. 47. Siracusa G, Whittingham DG, De Felici M. The effect of microtubule- and microfilament-disrupting drugs on preimplantation mouse embryos. J Embryol Exp Morphol 1980; 60: 71–82. 48. Bos-Mikich A, Whittingham DG, Jones KT. Meiotic and mitotic Ca2+ oscillations affect cell composition in resulting blastocysts. Dev Biol 1997; 182: 172–179. 49. Johnson MH, Pickering SJ, Dhiman A, Radcliffe GS, Maro B. Cytocortical organization during natural and prolonged mitosis of mouse 8-cell blastomeres. Development 1988; 102: 143–158. 50. Boiani M, Eckardt S, Leu NA, Scholer HR, McLaughlin KJ. Pluripotency deficit in clones overcome by clone-clone aggregation: epigenetic complementation? Embo J 2003; 22: 5304–5312. 51. Vidricaire G, Jardine K, McBurney MW. Expression of the Brachyury gene during mesoderm development in differentiating embryonal carcinoma cell cultures. Development 1994; 120: 115– 122. 52. McBurney MW, Jones-Villeneuve EM, Edwards MK, Anderson PJ. Control of muscle and neuronal differentiation in a cultured embryonal carcinoma

HOW TO IMPROVE MOUSE CLONING EFFICIENCY?

cell line. Nature 1982; 299: 165–167. 53. Iwatani M, Ikegami K, Kremenska Y, Hattori, N, Tanaka S, Yagi S, Shiota K. Dimethyl sulfoxide (DMSO) increases expression of Dnmt3as and affects genome-wide DNA methylation profiles in mouse embryoid body. Stem Cells 2006 (in press). 54. Santos F, Zakhartchenko V, Stojkovic M, Peters A, Jenuwein T, Wolf E, Reik W, Dean W. Epigenetic marking correlates with developmental potential in cloned bovine preimplantation embryos. Curr Biol 2003; 13: 1116–1121. 55. Dean W, Santos F, Stojkovic M, Zakhartchenko V, Walter J, Wolf E, Reik W. Conservation of methylation reprogramming in mammalian development: aberrant reprogramming in cloned embryos. Proc Natl Acad Sci USA 2001; 98: 13734– 13738. 56. Kang YK, Koo DB, Park JS, Choi YH, Chung AS, Lee KK, Han YM. Aberrant methylation of donor genome in cloned bovine embryos. Nat Genet 2001; 28: 173–177. 57. Enright BP, Kubota C, Yang X, Tian XC. Epigenetic characteristics and development of embryos cloned from donor cells treated by trichostatin A or 5-aza2’-deoxycytidine. Biol Reprod 2003; 69: 896–901. 58. Rybouchkin A, Kato Y, Tsunoda Y. Role of histone acetylation in reprogramming of somatic nuclei following nuclear transfer. Biol Reprod 2006; 74: 1083–1089. 59. Ogura A, Inoue K, Takano K, Wakayama T, Yanagimachi R. Birth of mice after nuclear transfer by electrofusion using tail tip cells. Mol Reprod Dev 2000; 57: 55–59. 60. Ogura A, Inoue K, Ogonuki N, Noguchi A, Takano K, Nagano R, Suzuki O, Lee J, Ishino F, Matsuda J. Production of male cloned mice from fresh, cultured, and cryopreserved immature Sertoli cells. Biol Reprod 2000; 62: 1579–1584. 61. Ono Y, Shimozawa N, Ito M, Kono T. Cloned mice from fetal fibroblast cells arrested at metaphase by a serial nuclear transfer. Biol Reprod 2001; 64: 44–50. 62. Wakayama T, Rodriguez I, Perry AC, Yanagimachi R, Mombaerts P. Mice cloned from embryonic stem cells. Proc Natl Acad Sci USA 1999; 96: 14984–14989. 63. Inoue K, Wakao H, Ogonuki N, Miki H, Seino K, Nambu-Wakao R, Noda S, Miyoshi H, Koseki H, Taniguchi M, Ogura A. Generation of cloned mice by direct nuclear transfer from natural killer T cells. Curr Biol 2005; 15: 1114–1118. 64. Miki H, Inoue K, Kohda T, Honda A, Ogonuki N, Yuzuriha M, Mise N, Matsui Y, Baba T, Abe K, Ishino F, Ogura A. Birth of mice produced by germ cell nuclear transfer. Genesis 2005; 41: 81–86. 65. Inoue K, Ogonuki N, Miki H, Hirose M, Noda S, Kim JM, Aoki F, Miyoshi H, Ogura A. Inefficient reprogramming of the hematopoietic stem cell genome following nuclear transfer. J Cell Sci 2006;

25

119: 1985–1991. 66. Sung LY, Gao S, Shen H, Yu H, Song Y, Smith SL, Chang CC, Inoue K, Kuo L, Lian J, Li A, Tian XC, Tuck DP, Weissman SM, Yang X, Cheng T. Differentiated cells are more efficient than adult stem cells for cloning by somatic cell nuclear transfer. Nat Genet 2006; 38: 1323–1328. 67. Yamazaki Y, Makino H, Hamaguchi-Hamada K, Hamada S, Sugino H, Kawase E, Miyata T, Ogawa M, Yanagimachi R, Yagi T. Assessment of the developmental totipotency of neural cells in the cerebral cortex of mouse embryo by nuclear transfer. Proc Natl Acad Sci USA 2001; 98: 14022– 14026. 68. Mizutani E, Ohta H, Kishigami S, Van Thuan N, Hikichi T, Wakayama S, Kosaka M, Sato E, Wakayama T. Developmental ability of cloned embryos from neural stem cells. Reproduction 2007; 132: 849–857. 69. Rideout WM, 3rd, Wakayama T, Wutz A, Eggan K, Jackson-Grusby L, Dausman J, Yanagimachi R, Jaenisch R. Generation of mice from wild-type and targeted ES cells by nuclear cloning. Nat Genet 2000; 24: 109–110. 70. Kato Y, Imabayashi H, Mori T, Tani T, Taniguchi M, Higashi M, Matsumoto M, Umezawa A, Tsunoda Y. Nuclear transfer of adult bone marrow mesenchymal stem cells: developmental totipotency of tissue-specific stem cells from an adult mammal. Biol Reprod 2004; 70: 415–418. 71. Kishigami S, Bui HT, Wakayama S, Tokunaga K, Van Thuan N, Hikichi T, Mizutani E, Ohta H, Suetsugu R, Sata T, Wakayama T. Successful mouse cloning of an outbred strain by Trichostatin A treatment after somatic nuclear transfer. J Reprod Dev 2006; 53: 165–170. 72. Ono Y, Shimozawa N, Muguruma K, Kimoto S, Hioki K, Tachibana M, Shinkai Y, Ito M, Kono T. Production of cloned mice from embryonic stem cells arrested at metaphase. Reproduction 2001; 122: 731–736. 73. Amano T, Tani T, Kato Y, Tsunoda Y. Mouse cloned from embryonic stem (ES) cells synchronized in metaphase with nocodazole. J Exp Zool 2001; 289: 139–145. 74. Kasinathan P, Knott JG, Wang Z, Jerry DJ, Robl JM. Production of calves from G1 fibroblasts. Nat Biotechnol 2001; 19: 1176–1178. 75. Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce de Leon FA, Robl JM. Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 1998; 280: 1256–1258. 76. Kubota C, Tian XC, Yang X. Serial bull cloning by somatic cell nuclear transfer. Nat Biotechnol 2004; 22: 693–694. 77. Wakayama T, Shinkai Y, Tamashiro KL, Niida H, Blanchard DC, Blanchard RJ, Ogura A, Tanemura

26

78.

79.

80.

81.

82.

83. 84.

85. 86.

87.

88.

89.

90.

WAKAYAMA

K, Tachibana M, Perry AC, Colgan DF, Mombaerts P, Yanagimachi R. Cloning of mice to six generations. Nature 2000; 407: 318–319. Van Thuan N, Wakayama S, Kishigami S, Ohta H, Hikichi T, Mizutani E, Bui HT, Wakayama T. Injection of somatic cell cytoplasm into oocytes before intracytoplasmic sperm injection impairs full-term development and increases placental weight in mice. Biol Reprod 2006; 74: 865–873. Shimozawa N, Ono Y, Kimoto S, Hioki K, Araki Y, Shinkai Y, Kono T, Ito M. Abnormalities in cloned mice are not transmitted to the progeny. Genesis 2002; 34: 203–207. Lanza RP, Cibelli JB, Diaz F, Moraes CT, Farin PW, Farin CE, Hammer CJ, West MD, Damiani P. Cloning of an endangered species (Bos gaurus) using interspecies nuclear transfer. Cloning 2000; 2: 79–90. Cibelli JB, Campbell KH, Seidel GE, West MD, Lanza RP. The health profile of cloned animals. Nat Biotechnol 2002; 20: 13–14. Ohta H, Wakayama T. Generation of normal progeny by intracytoplasmic sperm injection following grafting of testicular tissue from cloned mice that died postnatally. Biol Reprod 2005; 73: 390– 395. Gurdon JB, Colman A. The future of cloning. Nature 1999; 402: 743–746. Mombaerts P. Therapeutic cloning in the mouse. Proc Natl Acad Sci USA 2003; 100 Suppl 1: 11924– 11925. Wakayama T. On the road to therapeutic cloning. Nat Biotechnol 2004; 22: 399–400. Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce de Leon FA, Robl JM. Transgenic bovine chimeric offspring produced from somatic cell-derived stem-like cells. Nat Biotechnol 1998; 16: 642–646. Kawase E, Yamazaki Y, Yagi T, Yanagimachi R, Pedersen RA. Mouse embryonic stem (ES) cell lines established from neuronal cell-derived cloned blastocysts. Genesis 2000; 28: 156–163. Kohda T, Inoue K, Ogonuki N, Miki H, Naruse M, Kaneko-Ishino T, Ogura A, Ishino F. Variation in gene expression and aberrantly regulated chromosome regions in cloned mice. Biol Reprod 2005; 73: 1302–1311. Brambrink T, Hochedlinger K, Bell G, Jaenisch R. ES cells derived from cloned and fertilized blastocysts are transcriptionally and functionally indistinguishable. Proc Natl Acad Sci USA 2006; 103: 933–938. Barberi T, Klivenyi P, Calingasan NY, Lee H, Kawamata H, Loonam K, Perrier AL, Bruses J, Rubio ME, Topf N, Tabar V, Harrison NL, Beal MF, Moore MA, Studer L. Neural subtype specification of fertilization and nuclear transfer

91.

92.

93.

94.

95.

96.

97.

98.

embryonic stem cells and application in parkinsonian mice. Nat Biotechnol 2003; 21: 1200– 1207. Nolan PM, Peters J, Strivens M, Rogers D, Hagan J, Spurr N, Gray IC, Vizor L, Brooker D, Whitehill E, Washbourne R, Hough T, Greenaway S, Hewitt M, Liu X, McCormack S, Pickford K, Selley R, Wells C, Tymowska-Lalanne Z, Roby P, Glenister P, Thornton C, Thaung C, Stevenson JA, Arkell R, Mburu P, Hardisty R, Kiernan A, Erven A, Steel KP, Voegeling S, Guenet JL, Nickols C, Sadri R, Nasse M, Isaacs A, Davies K, Browne M, Fisher EM, Martin J, Rastan S, Brown SD, Hunter J. A systematic, genome-wide, phenotype-driven mutagenesis programme for gene function studies in the mouse. Nat Genet 2000; 25: 440–443. Hrabe de Angelis MH, Flaswinkel H, Fuchs H, Rathkolb B, Soewarto D, Marschall S, Heffner S, Pargent W, Wuensch K, Jung M, Reis A, Richter T, Alessandrini F, Jakob T, Fuchs E, Kolb H, Kremmer E, Schaeble K, Rollinski B, Roscher A, Peters C, Meitinger T, Strom T, Steckler T, Holsboer F, Klopstock T, Gekeler F, Schindewolf C, Jung T, Avraham K, Behrendt H, Ring J, Zimmer A, Schughart K, Pfeffer K, Wolf E, Balling R. Genome-wide, large-scale production of mutant mice by ENU mutagenesis. Nat Genet 2000; 25: 444– 447. Surani MA, Barton SC, Norris ML. Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis. Nature 1984; 308: 548–550. McGrath J, Solter D. Completion of mouse embryogenesis requires both the maternal and paternal genomes. Cell 1984; 37: 179–183. Cibelli JB, Grant KA, Chapman KB, Cunniff K, Worst T, Green HL, Walker SJ, Gutin PH, Vilner L, Tabar V, Dominko T, Kane J, Wettstein PJ, Lanza RP, Studer L, Vrana KE, West MD. Parthenogenetic stem cells in nonhuman primates. Science 2002; 295: 819. Fundele RH, Norris ML, Barton SC, Fehlau M, Howlett SK, Mills WE, Surani MA. Temporal and spatial selection against parthenogenetic cells during development of fetal chimeras. Development 1990; 108: 203–211. Allen ND, Barton SC, Hilton K, Norris ML, Surani MA. A functional analysis of imprinting in parthenogenetic embryonic stem cells. Development 1994; 120: 1473–1482. Hikichi T, Wakayama S, Mizutani E, Takashima Y, Kishigami S, Van Thuan N, Ohta H, Bui HT, Nishikawa SI, Wakayama T. Differentiation potential of parthenogenetic embryonic stem cells is improved by nuclear transfer. Stem Cells 2007 (in press).