Preimplantation Development of Somatic Cell Cloned Embryos in the Common Marmoset ( Callithrix jacchus )

Abstract

The somatic cell nuclear transfer technique has been applied to various mammals to produce cloned animals; however, a standardized method is not applicable to all species. We aimed here to develop optimum procedures for somatic cell cloning in nonhuman primates, using common marmosets. First, we confirmed that parthenogenetic activation of in vitro matured oocytes was successfully induced by electrical stimulation (three cycles of 150 V/mm, 50 μsec × 2, 20 min intervals), and this condition was applied to the egg activation procedure in the subsequent experiments. Next, nuclear transfer to recipient enucleated oocytes was performed 1 h before, immediately after, or 1 h after egg activation treatment. The highest developmental rate was observed when nuclear transfer was performed 1 h before activation, but none of the cloned embryos developed beyond the eight-cell stage. To investigate the causes of the low developmental potential of cloned embryos, a study was performed to determine whether the presence of metaphase II (MII) chromosome in recipient ooplasm has an effect on developmental potential. As a result, only tetraploid cloned embryos produced by transferring a donor cell into a recipient bearing the MII chromosome developed into blastocysts (66.7%). In contrast, neither parthenogenetic embryos nor cloned embryos (whether diploid or tetraploid) produced using enucleated oocytes developed past the eight-cell stage. These results suggest that MII chromosome, or cytoplasm proximal to the MII chromosome, plays a major role in the development of cloned embryos in common marmosets.

Introduction

Somatic cell nuclear transfer (SCNT) techniques are powerful tools in the fields of basic biology concerned with elucidating the mechanisms of early stage development and nuclear reprogramming. They have been especially well developed as a productive method for producing hereditary identical animals, so-called cloning techniques (Willadsen, 1986; Wilmut et al., 1997). Application to the production of superior domestic animals, maintenance of mutant animals with low fertility, and preservation of endangered species is also predicted in the fields of stock raising and animal experimentation.

Recently, cloning techniques have also been applied to various life science research fields such as biomedical science, and not simply for the manufacture of copy animals. For example, in animal experimentation studies, cloning techniques have been used in gene manipulation of donor cells (Cibelli et al., 1998; Rideout et al., 2000; Schnieke et al., 1997). Generally, knock-in/-out mice are produced using chimeric mice with embryonic stem (ES) cells through homologous recombination (Bradley et al., 1984; Robertson et al., 1986; Thomas and Capecchi, 1987). However, because mice (Bradley et al., 1984) and rats (Li et al., 2008) are the only animals for which ES cells that contribute to germ line cells have so far been confirmed, gene manipulation of other animal species in a similar manner remains difficult. In the field of medical science, cloning techniques are also expected to be applicable to regenerative medicine and gene therapy through the establishment of embryonic stem cells from cloned embryos (Byrne et al., 2007; Rideout et al., 2002).

As laboratory animals, mice and rats have been highly developed regarding developmental and genetic characteristics biotechnologically, contributing significantly to medical research as representatives of human disease models. However, because rodents are phylogenically distant from Homo sapiens, knowledge provided by such animal experiments cannot always be extrapolated directly to humans. In contrast, the common marmoset, a small primate belonging to the suborder Haplorhini, is a favored laboratory animal compared with other primates due to its high breeding rate. In addition, similarity of cytokines and hormones, and drug metabolism with humans has also been shown (Hibino et al., 1999; Mansfield, 2003). The marmoset is a potential model animal for such gene therapies, and if it were to be introduced as a preclinical model, the above-mentioned application of cloning techniques could also be adapted for primates. Recently, marmoset ES cell lines have been established and used in preclinical studies for regenerative medicine (Kurita et al., 2006; Sasaki et al., 2005; Thomson et al., 1996). To elucidate the pathogenic mechanisms of various diseases or the safety and efficacy of ES cell therapies, genetically manipulated human disease animal models using nonhuman primates are required. However, genetically manipulated nonhuman primate models for human disease have not yet been established, except in the recent report of transgene-mediated overexpression of polyglutamine-expanded human huntingtin in the rhesus macaque as a model for human Huntington's disease (Yang et al., 2008). Accordingly, if application of developmental and genetic biotechnological procedures such as genetic manipulation were to be achieved, it is expected that marmosets could increase the utility of primates as a human model animal.

Cloning techniques have been examined in various mammals for the provision of cloned animals, but suitable methods vary depending on the species. For example, the method and timing of activation of the recipient cytoplasm and transplantation of donor nuclei varies with respect to the optimal conditions of reprogramming. In addition, especially in rats, maturation-promoting factor (MPF) activity, which is related to the reprogramming of donor nuclei, is decreased immediately in the recipient cytoplasm after removal of the female genome (Hirabayashi et al., 2003; Ito et al., 2005). So it should be necessary possible to investigate whether the presence of the oocyte genome in recipient ooplasm has an effect on the developmental potential, by creating polyploid SCNT or parthenogenetic embryos using the intact oocytes. To date, whole animal cloning of a monkey species has yet to be reported. In addition, there are few reports concerned on the reproductive engineering of marmosets (Gilchrist et al., 1997; Lopata et al., 1988; Marshall et al., 1998; Nayudu et al., 2003; Wilton et al., 1993). As a preliminary experiment to establish ES cell lines and produce cloned individuals from somatic cell cloned embryos in the present study, we examined the optimum procedure for SCNT and the in vitro development of SCNT embryos in common marmosets.

Materials and methods

Animals

We used common marmosets obtained from CLEA Japan (EDM: C. Marmoset (Jic), CLEA Japan, Inc.) and maintained the animals at the Central Institute for Experimental Animals. To obtain oocytes, 60 female marmosets, which were older than 2 years of age, were used for the experimental measures, and some of them were used repeatedly. All experiments were carried out after obtaining permission from the Institutional Animal Care and Use Committee at the Central Institute for Experimental Animals and the Committee of Animal Experimentation at Hiroshima University.

Preparation of matured oocytes

To collect germinal vesicle-stage (GV) oocytes, female marmosets were subjected to ovarian stimulation and oocyte collection procedures, with reference to previous reports (Marshall et al., 1998, 2003; Wilton et al., 1993) and our preliminary experiments. The estrous cycle was assessed by measuring serum concentration of progesterone using an automated immunoassay system (AIA360, Tosoh Corp., Tokyo, Japan). The females at the diestrus stage were treated by intramuscular injection of 50 IU FSH at 10:00 for 11 consecutive days. At 17:30 on the day following the final FSH injection, 75 IU hCG was administered by intramuscular injection. At 9:30 on the day following the hCG administration, animals were anesthetized with an intramuscular injection of 0.025 mg atropine sulfate (atropine sulfate injection 0.5 mg; Tanabe Seiyaku Co., Ltd., Osaka, Japan) and 70 mg/kg of ketamine hydrochloride (veterinary Ketalar 50; Sankyo Lifetech Co., Ltd., Tokyo, Japan) for immobilization. Immobilized animals were inhalation anesthetized using isoflurane (Forane; Abbott Japan, Tokyo, Japan), and the ovaries were exteriorized by midline laparotomy. Then, cumulus cell–oocyte complexes (COCs) were surgically collected from the ovarian follicles using a disposable syringe with a 23-G needle. The COCs were transported from the Central Institute for Experimental Animals to Hiroshima University by cargo service. During the 22- to 24-h period of transportation, they were kept in maturation medium at 38°C using a portable oven (Cell Transporter; Fujihira Industry Co., Ltd., Tokyo, Japan). The maturation medium was Waymouth's MB 752/1 Medium (Gibco, 11220-035, Carlsbad, CA) supplemented with 20% fetal bovine serum (Gibco, 16141-079), 1 μg/mL estradiol, 0.5 mM sodium pyruvate, 10 mM sodium lactate, 4 mM hypotaurine, and 1 mM glutamine. Upon arrival, the COCs were placed in PBI medium containing 300 units/mL hyaluronidase and cumulus cells were removed by pipetting. Oocytes with a polar body were confirmed as matured at metaphase II (MII) and used for subsequent experiments.

Parthenogenetic activation

Activation stimulation of the in vitro mature oocytes was examined with electric stimulation or strontium chloride. For activation by electric stimulation, two electrical pulses of 150 V/mm, lasting for 50 μsec each, were applied for three cycles to the oocytes every 20 min in Zimmerman's cell fusion medium (Wolfe and Kraemer, 1992). Next, the oocytes were cultured in modified Whitten's medium containing 5 g/mL cytochalasin B (CB) or modified Whitten's medium including 5 g/mL CB and 2 mM 6-dimethylaminopurine (DMAP) for 4 h. For activation by strontium chloride, the oocytes were cultured in calcium-free modified Whitten's medium containing 5 g/mL CB and 10 mM strontium chloride for 6 h. Six and 24 h later, the effects of these activation treatments were evaluated by pronuclear formation and cleavage of the eggs, respectively.

As mentioned above, diploid parthenogenetic embryos were derived from the activated oocytes, preventing a second polar body emission by CB. When tetraploid parthenogenetic embryos were produced, each blastomere of the two-cell stage parthenogenetic embryos was fused by stimulation of two electrical pulses of 150 V/mm, lasting for 50 μsec.

Preparation of donor cells

As the nuclear donor, marmoset bone marrow mononuclear cells (MBMMNCs) from male adult and embryonic fibroblast cells from a female fetus at around 60–70 days of gestation were used. MBMMNCs were isolated from the femoral bone marrow by Ficoll-Paque density gradient centrifugation (Hibino et al., 1999). Collected cells were cultured in DMEM (Gibco, 11885-084) supplemented with 10% FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin. Culture medium was changed every 3 days, and floating cells were removed while changing the culture medium. About 2 weeks later, adhesive MBMMNCs were split to new plates. For the preparation of fetal fibroblasts, small pieces of fetus depleted of the internal organs, head, feet, and arms were placed in the DMEM supplemented as mentioned above, and outgrowth cells were transplanted to new plates. To confirm the contribution of the donor nuclei to in vitro development of clone embryos, MBMMNCs were used as the nuclear donor, where the EGFP gene driven by CAG promoter was transduced using self-inactivating (SIN) lentiviral vectors based on the human immunodeficiency virus type 1 (HIV-1) vector (Miyoshi et al., 1998). An EGFP-expressing cell line was established by fluorescence-activated cell sorting (FACS) and subcloning.

The nuclear donors were introduced into the nuclear transfer procedure without treatment for cell cycle regulation. About 1 h before nuclear transfer, the cells from the Second to eighth passage were dispersed by treatment with 0.5% trypsin–5.3 mM EDTA solution and then kept in M2 medium at 4°C until use. The chromosome number and karyotype of the donor cells were analyzed as reported previously (Nesbitt and Francke, 1973; Sugawara et al., 2006).

Removal of chromosomes from recipient oocytes

The zona pellucida of the oocytes was slit with a glass needle along 10–20% of its circumference, close to the position of the first polar body. The MII chromosomes and spindle were located in the cortex of the oocyte near the first polar body and identified with differential interference microscopy without any staining. The oocytes were placed in PBI medium containing 5 g/mL CB and a small amount of cytoplasm containing the MII chromosomes was aspirated with an enucleation pipette. When confirmation of the removal operation was necessary, aspirated cytoplasm was stained with 10 μg/mL Hoechst 33342 and chromosomes were observed using a fluorescent microscope.

Nuclear transfer

After chromosome removal, the cytoplasms of the in vitro matured oocytes were used as recipients for nuclear transfer. Transplantation of donor nuclei into the recipient cytoplasms was performed through cell membrane fusion by electrical stimulation or sensitization of inactivated Sendai virus (HVJ, hemagglutinating virus of Japan). Using electrical stimulation, two electrical pulses of 150 V/mm, lasting 50 μsec each, were applied to Zimmerman's cell fusion medium after injection of donor cells into the perivitelline space of the recipient egg. In the case of inactivated Sendai virus, a donor cell was introduced into the perivitelline space with three to five times the volume of the virus, which was prepared at 2700 hemagglutinating units (HAU)/mL.

To construct diploid and tetraploid SCNT (NT-dip(o) and NT-tetra(dd)) embryos, single and two donor cell(s) were transferred to the oocyte cytoplasm after chromosome removal, respectively. Tetraploid SCNT [NT-tetra(od)] embryos with both oocyte- and donor-cell-derived nuclei were constructed by transferring a single donor cell to the oocyte cytoplasm without chromosome removal. After parthenogenetic activation, because the emission of a second polar body was suppressed by CB treatment, it follows that oocyte-derived haploid genomes within the constructed embryos formed diploidy. Therefore, NT-tetra(od) embryos formed tetraploidy, by combining the donor cell-derived diploid genomes with the oocyte-derived diploid genomes.

In vitro culture of embryos

The in vitro culture of constructed embryos was performed using ISM1 and ISM2 culture medium (Nosan, Tokyo, Japan, 10500010 and 10510010). The embryos were cultured in ISM1 medium for the first 48 h (from days 1 to 3) and then cocultured with inactivated marmoset embryonic fibroblasts in ISM2 medium supplemented with 10% FBS for 7 days. During culture in ISM2, 50% of the culture medium was exchanged every 48 h. The vapor phase conditions for culture were 38°C, 5% CO₂, and humidity saturation.

Analysis of the transplanted nuclei

Some constructed embryos were applied to whole-mount specimens 30, 60, 120, and 180 min after nuclear transfer. The status of the nuclei was classified into three groups: interphase (with a nuclear membrane), nuclear membrane breakdown (NMBD, with/without a slight nuclear membrane and without condensed chromosomes), and premature chromosome condensation (PCC, with condensed chromosomes).

Statistical assessment

Differences were analyzed using the chi-square test. Statistical significance was set at the p < 0.05 level.

Results

In vitro maturation of oocytes

For the present experiments, 1092 GV oocytes were collected from 104 marmosets. After 22–24 h incubation, 578 (52.9%) oocytes with a polar body were confirmed to be matured oocytes at metaphase II. Then, the matured oocytes were used in in vitro fertilization (IVF) to assess their viability. Of the IVF embryos, 5.3% (5/95) developed to the blastocyst stage, suggesting that the conditions of in vitro maturation may not have been optimal.

Oocyte activation

We conducted parthenogenetic stimulation using electrical pulses or strontium chloride to examine the activation procedure of in vitro matured oocytes in marmosets (Table 1). A large percentage of the oocytes could be effectively activated by electrical stimulation, with no need for supplementary treatment with DMAP. In contrast, oocyte activation did not occur with strontium treatment, even though this procedure has been successful in mice and rats. This finding suggests that the sensitivity of marmoset oocytes to chlorination strontium differs from that of mice and rats.

Table 1.

Activation of In Vitro Matured Marmoset Oocytes

	Number of oocytes (%)
Activation method ^a	Examined	Survived	Activated	Cleaved
EP	57	57	57 (100)	57 (100)
EP + DMAP	35	33	33 (100)	33 (100)
SrCl₂	14	14	0 (0)	0 (0)

EP = two electrical pulses of 150 V/mm, lasting 50 μsec each, were applied for 3 cycles to the oocytes every 20 min. DMAP: cultured in medium containg 2 mM DMAP for 6 h, SrCl₂ = cultured in medium containing 10 mM strontium chloride for 6 h.

Development of cloned embryos

To construct viable SCNT embryos, we examined the transfer of donor nuclei into recipient cytoplasm and the timing of activation treatment (Table 2). In this experiment, embryonic fibroblasts were used as the nuclear donors. The induction of cells was performed 1 h before, immediately after, or 1 h after activation using electrical pulses or inactivated Sendai virus. The results showed that 95 to 100% of the eggs in each group were successfully classified as pronuclear-stage eggs, while the induction rate was significantly high using HVJ. After in vitro culture of the constructed embryos, the highest developmental rate was observed when nuclear transfer was performed 1 h before activation. However, SCNT embryos in all groups and parthenogenetically activated oocytes did not develop beyond the eight-cell stage. These findings suggest that the most viable SCNT embryos were efficiently obtained by transferring donor cells into a nonactivated oocyte cytoplasm using HVJ.

Table 2.

Productive Efficiency and In Vitro Development of Cloned Embryos Produced by Different Procdures

		Number of eggs (%)			Number of cloned embryos developed to (%)***
Timing of NT	Fusion method*	Used	Fused	Activated**	Two-Cell	Eight-Cell	Morula	Blastocyst
After activation	EP	43	27 (62.8)^a	27 (100)	20 (74.1)^a	4 (14.8)^a	0 (0)	0 (0)
Immediately after activation	EP	91	54 (59.3)^a	54 (100)	52 (96.3)^b	13 (20.1)^a	0 (0)	0 (0)
Before activation	HVJ	45	45 (100)^b	43 (95.6)	34 (79.1)^a	21 (48.8)^b	0 (0)	0 (0)
Parthenogenetic control	—	—	—	86 (—)	78 (90.7)^b	16 (18.6)^a	0 (0)	0 (0)

In this experient, clone embryos were produced using embryonic fibroblasts as nuclear donor.

a,b

Different letters in same column indicate the significant differences (p < 0.05).

*EP = two electrical pulses of 150 V/mm, lasting 50 μsec each. HVJ = inactivated Sendai virus applied at 2700 HAU/mL.

**All activated eggs were introduced to in vitro culture.

***Percentages were calculated from the number of activated eggs.

Chromosome number and karyotype of donor cells

To assess cytogenetic quality, we examined the chromosome number and karyotype of the donor cells used for nuclear transfer (after the second to eighth passage). In embryonic fibroblast and MBMMNC lines, at least 74.0% (37/50) and 72.0% (36/50) of the analyzed cells, respectively, were confirmed to have a normal diploid chromosome number of 46 with estimated sex chromosomes of XX (female) and XY (male). Because chromosome analysis is commonly accompanied by an artificial error, it is inferred that almost all of the cells might be cytogenetically normal.

Status of the transplanted nuclei

When the donor nuclei were transplanted to an activated recipient cytoplasm, NMBD was partially observed but not PCC (Fig. 1). NMBD and PCC were observed when the nuclei were transferred to a nonactivated recipient. All nuclei transferred to the recipient 1 h before activation underwent PCC 60 min after nuclear transfer. These observations suggest that MPF activity in the marmoset oocyte is reduced 2 to 3 h after activation stimulation.

FIG. 1.

Status of donor nuclei after transfer to recipient cytoplasm. Light-colored boxes show nuclei at interphase; gray boxes show nuclei that have undergone nuclear membrane breakdown; dark-colored boxes show nuclei that have undergone premature chromosome condensation. *Timing of nuclear transfer (into the recipient cytoplasm).

Effects of removal of oocyte genome and nuclear ploidy

To investigate the causes of low developmental potential of SCNT embryos, we determined whether the presence of the oocyte genome in recipient ooplasm and ploidy of constructed embryos affected the developmental potential, by creating diploid and tetraploid SCNT embryos or parthenogenetic embryos (Table 3). In this experiment, MBMMNCs and nonactivated oocytes were used as the nuclear donor and recipient cytoplasm, respectively, to construct SCNT embryos. As a result, only tetraploid NT-tetra(od) embryos produced by transferring a donor cell into a recipient bearing the MII chromosome (oocyte genome) developed into blastocysts (66.7%). In contrast, neither PG-dip/PG-tetra parthenogenetic embryos nor NT-dip(o)/NT-tetra(dd) embryos (whether diploid or tetraploid) produced using enucleated oocytes developed past the eight-cell stage.

Table 3.

In Vitro Development of Diploid and Tetraploid Cloned Embryos and Parthenogenetic embryos

		Genome			Number of embryos developed to (%)
Types of embryos	Ploidy	Oocyte	Donor cell	No. of embryos cultured	Two-Cell	Eight-Cell	Morula	Blastocyst
NT-dip(o)	2n	−	+	29	29 (100)	21 (72.4)^a	0 (0.0)	0 (0.0)
NT-tetra(od)	4n	+	+	21	21 (100)	19 (90.5)^b	14 (66.7)	14 (66.7)
NT-tetra(dd)	4n	−	++	21	20 (90.5)	12 (57.1)^a	0 (0.0)	0 (0.0)
PG-dip	2n	+	−	18	17 (94.4)	9 (50.0)^c	0 (0.0)	0 (0.0)
PG-tetra	4n	++	−	9	9 (100)	4 (44.4)^c	0 (0.0)	0 (0.0)

Nuclear transfer (fusion of donor cells) was performed using inactivated HVJ.

a,b,c

Different letters in same column indicate the significant differences compared with NT-dip(o) embryos (p < 0.05).

Moreover, to confirm the contribution of donor nuclei to embryo development, NT-tetra(od) embryos were constructed using EGFP gene-transfected MBMMNCs, and were examined under fluorescence microscopy. The fluorescence signal of EGFP was observed in the cytoplasm of the embryos at six- to eight-cell and blastocyst stages (Fig. 2), confirming that donor cells certainly contributed to the development of NT-tetra(od) embryos until blastocyst stage.

FIG. 2.

Tetraploid SCNT [NT-tetra(od)] embryos at the four- to six-cell stage on day 3 (A, B) and at the blastocyst stage on day 8 (C, D), under bright-field and fluorescence microscopy, respectively. NT-tetra(od) embryos were produced by transferring an EGFP-gene-transfected MBMMNCs cell into a recipient oocyte cytoplasm with the MII chromosome. Living embryos expressed gene derived from donor cells.

Discussion

Embryo cloning in mammals can roughly be divided into two types of procedure depending on the timing of donor nucleus transplant and activation of the recipient egg cytoplasm. The first type involves reprogramming of the donor nucleus while using an activated or immediately after-activated oocyte cytoplasm as the recipient, as observed in sheep and cows (Campbell et al., 1996; Kato et al., 1998; Wilmut et al., 1997). The marmosets employed in the present study belong to the second type, as employed for mice, rats, and rhesus monkeys (Ogura et al., 2000; Ono et al., 2001a; Wakayama et al., 1998; Zhou et al., 2003; 2006), whereby a SCNT embryo with the highest developmental ability to the eight-cell stage is constructed when the donor cells have been transferred into the recipient prior to activation. However, the developmental ability of marmoset SCNT embryos was found to be extremely limited. On the other hand, tetraploid SCNT embryos produced by transferring a donor cell into a recipient oocyte cytoplasm with the MII chromosome [NT-tetra(od)] developed to the blastocyst stage, but neither tetraploid parthenogenetic embryos nor tetraploid SCNT embryos produced by transferring two donor cells into an enucleated oocyte cytoplasm [NT-dip(o) and NT-tetra(dd)] did. These results suggest that the presence of MII chromosome or cytoplasm proximal to the MII chromosome, but not the genome constitution of tetraploidy, plays an important role in the development of SCNT embryos to the blastocyst stage. However, parthenogenetic embryos did not develop to blastocyst, showing that the intactness of in vitro mature oocytes cannot support preimplantation development alone. Based on these observations, although the obvious mechanism is still unclear, it is concluded that the synergistic effects of an intact oocyte cytoplasm/genome, and some factors from donor cells passed through a fertilization process are necessary to gain developmental viability.

Previous studies on embryo cloning have shown that MPF activity in the cytoplasm of a meiotic oocyte at metaphase II plays an important role in reprogramming donor nuclei (McGrath and Solter, 1984; Roble et al., 1986; Wakayama et al., 2000). With exposure to high MPF activity surroundings, chromosome condensation is induced in nuclei at interphase through NMBD, and the status of early-stage embryos is initiated (Barns et al., 1993; Campbell et al., 1993; Colls and Robl, 1991; Szollosi et al., 1986). In the present study, whole-mount analysis confirmed that chromosome condensation of donor nuclei occurred after the transfer of nonactivated oocytes into the cytoplasm, resulting in the production of cloned embryos with the highest developmental ability. A similar observation was also reported in the rhesus monkey (Zhou et al., 2006). On the other hand, condensation did not occur in the case of activated oocytes, and the embryos showed poor development. These findings reconfirmed that sufficient exposure of the nuclear genome to high MPF activity surroundings is a principal event for cloned embryos to gain the benefit of reprogramming factor in the recipient cytoplasm. Moreover, a recent topical report in a clone study, which shows that zygotic cytoplasm at metaphase is also actively involved in the reprogramming of differentiated nuclei at metaphase (Egli et al., 2007), could support this logic.

Cloned animals have been obtained using nuclei from various kinds of mammalian cells, including ES cells, although the selection of donor cells remains controversial with regard to cloning procedures. The chromosome number/karyotype is one of the most essential factors related to the construction of viable healthy cloned embryos; however, concerning gene manipulation with donor cells, proliferation activity (ideally with motility) during in vitro culture is also an important factor. Here, when the cell lines used as the nuclear donor were subsequently cultured, the proliferation of a skin-derived fibroblast cell line decreased after the 10th passage, but that of the MBMMNC line did not (data not shown). From this point of view, fibroblasts may not be suitable as donors, although they are easy to collect. The MBMMNCs used in this study have not been characterized by cell selective markers such as CD45 or differentiation potency to adipocytes, chondrocytes, and osteocytes. It is, however, likely that part of these MBMMNCs are mesenchymal stem cells because mesenchymal stem cell lines are established from MBMMNCs (Fuchs and Segre, 2000; Prockop, 1997). These findings suggest that MBMMNCs are convenient for advanced cloning procedures.

In the SCNT procedure in mammals, transfer of donor nuclei into an egg cytoplasm is performed with membrane fusion of the whole cell/karyoplast (nuclei with surrounding cytoplasm) and egg, or direct injection of nuclei through the egg membrane (Kimura and Yanagimachi, 1995). In this study, nuclear transfer was attempted through membrane fusion using physical stimulation in the form of electrical pulses (Vienken and Zimmermann, 1982; Willadsen, 1986; Wolfe and Kraemer, 1992), or the hemagglutinating activity of HVJ (Sendai virus) (McGrath and Solter, 1983). With the electrical pulses, nuclear transfer was successfully achieved, but the efficiency was evidently improved with HVJ. HVJ has a wide host range, with mice, rats, including cotton rats, hamsters, guinea pigs, rabbits, ferrets, pikas, pigs, and marmosets, all showing sensitivity. Especially in mice, HVJ is often used for nuclear transfer; however, the fusion activity of cell membranes in other species remains unclear. Here, HVJ was also shown to be an effective tool for nuclear transfer in marmosets.

In the present study, parthenogenetic development of marmoset embryos was not observed beyond the eight-cell stage. As reported previously, in the monkey Macaca mulatta (Mitalipov et al., 2001; 2002), in mammals such as mice (Graham, 1970; Kaufman, 1973), rats (Jiang, et al., 2002; Krivokharchenko et al., 2003), cows (Campbell et al., 2000; Liu et al., 1998), pigs (Grupen et al., 1999; Wang et al., 1999), and so on, in which in vitro culture of preimplantation fertilized eggs has been established, it has been confirmed that parthenogenetically activated oocytes can develop to the blastocyst stage in vitro. Also, in marmosets (Marshall et al., 1998), parthenogenetic embryos can undergo implantation after transfer to a recipient female, in which the parthenogenetic embryos were derived from in vivo mature oocytes and transferred at the four-cell stage. Thus, as our study showed poor development of the parthenogenetic embryos, there might be room for the improvement in in vitro maturation/culture conditions of marmoset oocytes/embryos.

In the marmoset, the optimal procedure for viable SCNT embryo has not yet been established. Meanwhile, considering the necessary characteristics of laboratory primates, marmosets are considered as a model animal for preclinical experiments, as already shown for spinal cord injuries (Iwanami et al., 2005a, 2005b). This study also indicated the possibility of advanced applications using the marmoset, similar to those that have been established in mice, and perhaps the production of cloned individuals aimed at gene manipulation (Rideout et al., 2000) and the establishment of somatic cell nuclear transfer-derived ES cells aimed at regenerative medicine (Kishigami et al., 2006; Rideout et al., 2002). However, although there is a gradual progress in the gene expression analysis of cloned embryos or fetuses (Blelloch et al., 2006; Hiiragi and Solter, 2005; Humpherys et al., 2001; Inoue et al., 2002; Kang et al., 2001; Ogawa et al., 2003; Suemizu et al., 2003), the cause of developmental abnormality, and thus, the low success rate (Eggan et al., 2001; Hill et al., 1999; Ono et al., 2001a, 2001b; Renard et al., 1999; Shimozawa et al., 2002, 2003, 2006; Wakayama and Yanagimachi, 1999; Tamashiro et al., 2000, 2002) and the precise mechanism of nuclear reprogramming remain unclear. Prior to the introduction of cloning techniques through SCNT for therapeutic application to human patients, these problems need to be addressed, and accordingly, simultaneous progress in clone studies regarding basic biological mechanisms and preclinical application is expected.

Footnotes

Acknowledgments

This study was supported by a grant-in-aid for Scientific Research (18500336) to E.S. from the Japan Society for the Promotion of Science, a grant from Japan Science and Technology Agency (SORST) to H.O., and a Grant-in-aid for 21st Century and Global COE program to Keio University from The Ministry of Education, Culture, Sports, Science, and Technology (MEXT). A part of this study is the result of “Highly creative animal model development for brain sciences” carried out under the Strategic Research Program for Brain Sciences by the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Dr. T. Kono (Tokyo University of Agriculture, JP) and Dr. N. Maeda (Hiroshima University, JP) for helpful discussion. We also thank Ms. M. Kamioka, Ms. F. Toyota and Ms. S. Oba (JAC Inc., JP) for their professional animal cares. Lenti viral vector was kindly provided by Dr. H. Miyoshi at RIKEN BRC.

Author Disclosure Statement

The authors declare that no conflicting financial interests exist.

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