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Production of Cloned Pigs from Adult Somatic Cells by Chemically Assisted Removal of Maternal Chromosomes¹

^a Laboratory of Animal Reproduction, College of Agriculture, Kinki University, Nara 631-8505, Japan ^b Reproduction Section, Tottori Swine and Poultry Experiment Station, Tottori 683-03, Japan

	ABSTRACT

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The present study demonstrated that brief treatment of in vitro-matured porcine oocytes with demecolcine results in a membrane protrusion that contains a condensed chromosome mass, which can be easily removed by aspiration. This simple, chemically assisted method for removing maternal chromosomes enabled the production of a large number of nuclear-transferred porcine eggs. The development of eggs whose chromosomes were removed by this procedure following transfer of somatic cell nuclei to the blastocyst stage was not significantly different among groups activated using different procedures (6% to 11%) and was also not different among donor cells of different origins (3% to 9%), except for cumulus cells (0.4%). After transfer of 180 to 341 nuclear-transferred eggs that received somatic cells to 6 recipients, 2 of the recipients produced 8 healthy cloned piglets from the heart cells of a female pig. The chemically assisted method for removing maternal chromosomes was also effective for bovine and rabbit eggs.

developmental biology, embryo, gamete biology, oocyte development

	INTRODUCTION

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Production of cloned mammals by nuclear transfer of somatic cells into eggs in which the chromosomes were removed at the second metaphase stage (MII) is now feasible [1]. The technology has been extended to apply to the genetic improvement of farm animals, rescue of endangered species, and production of transgenic animals for medical use and organ transplantation. The nuclear transfer technique is currently unreliable, however, because the production efficiency of normal offspring is low [2]. The removal of chromosomes from recipient eggs is one of the key factors affecting cloning efficiency. The chromosomes of mouse and rat eggs can be observed and removed under an inverted microscope, but the chromosomes of rabbit, sheep, goat, bovine, and pig eggs are difficult to observe without DNA staining. Thus, the chromosomes in these species are removed mainly by aspirating or pushing out a large volume of cytoplasm underlying the first polar body with or without Hoechst staining [2]. Although cloned sheep have been obtained after fusion of a blastomere from a preimplantation embryo with a bisected egg with half the volume of normal cytoplasm [3], the developmental potential of nuclear-transferred eggs is low. The removal of chromosomes from activated eggs at the telophase stage is also effective [4], but decreased maturation promoting factor activity might decrease the viability of nuclear-transferred eggs with somatic cells [5]. Nocodazole treatment of rat MII eggs reportedly stabilizes the metaphase plate as a single and slightly protruding mass [6], which enables the mechanical removal of the metaphase cone [7]. The effectiveness of nocodazole treatment for removal of the maternal chromosomes and cloning, however, has not been demonstrated. These observations led us to attempt chemically assisted enucleation for the production of somatic cell cloned pigs.

Successful production of somatic cell cloned pigs has been reported by four laboratories [8–11]. The success rates, however, were low; recipients gave birth 1% to 7% transferred cloned embryos. Cloned piglets were obtained after transfer of nuclear-transferred eggs, which were recovered from superovulated females receiving somatic cells by serial nuclear transfer (5 of 72, 7%) [8], or by single nuclear transfer (2 of 49, 4%) [10]. In another study, cloned piglets were obtained following transfer of a large number of in vitro-matured eggs receiving fetal fibroblast cells, but the production of live piglets was low (4 of 307, 1.3%) [11].

In the present study, cloned piglets were produced after transfer of eggs that were matured in vitro and received nuclear transfer of adult somatic cells whose chromosomes were removed by chemically assisted procedures.

	MATERIALS AND METHODS

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Isolation and Culture of Porcine Somatic Cells

Adult cells were obtained from liver, heart, kidney, muscle, ear, oviduct, and cumulus cells from one female Landrace pig (4 yr of age) and cultured as previously reported for bovine somatic cells [12, 13]. Fetal cells were also obtained from a fetus (7.5 cm in length) obtained from a slaughterhouse; the age and breed of the fetus were unknown. The cells were cultured and passaged 2–4 times in Dulbecco modified Eagle medium, modified for mouse embryonic stem cell culture, and supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin for at least 3 days. More than 80% of the cells were quiescent and used for nuclear transfer [12, 13]. The characteristics of porcine liver, muscle, oviduct, cumulus, kidney, ear, and heart cells were determined by labeling with antibodies against cytokeratin, vimentin, and desmine.

Removal of Chromosomes From In Vitro-Matured Eggs

Modified NCSU-37 containing 0.6 mmol/L cysteine and 4 mg/ml BSA with 100 IU/ml penicillin and 50 µg/ml streptomycin was used for in vitro culture of immature oocytes, micromanipulation, and in vitro culture of nuclear-transferred eggs [14]. Immature oocytes recovered from ovarian follicles were cultured in medium supplemented with 10% porcine follicular fluid, 0.6 mmol/L cysteine, 1 mmol/L dibutyryl cyclic adenosine monophosphate (dbcAMP, Sigma), and 0.1 IU/ml human menopausal gonadotropin (hMG, Teikokuzoki, Tokyo, Japan) for 20 h and then cultured without dbcAMP and hMG for another 18–24 h as previously reported [14]. As shown in Figure 1, matured eggs with the first polar body were cultured in medium supplemented with 0.4 µg/ml demecolcine (Sigma) and 0.05 mol/l sucrose for 1 h. Sucrose was used to enlarge the perivitelline space of the eggs. Treated eggs with a protruding membrane were moved to medium supplemented with 5 µg/ml cytochalasin B (CB) and 0.4 µg/ml demecolcine and then the protrusion was removed with a beveled pipette. A single donor cell was injected into the perivitelline space of each egg and electrically fused using two direct current pulses of 150 V/mm for 50 µsec in 0.28 mol/L mannitol supplemented with 0.1 mM MgSO₄ and 0.01% polyvinyl alcohol (PVA, Sigma). Fused eggs were cultured in medium with 0.4 µg/ml demecolcine for 1 h before parthenogenetic activation.

View larger version (15K): [in this window] [in a new window]   FIG. 1. Procedure of nuclear transfer. See Removal of Chromosomes from In Vitro-Matured Eggs in Materials and Methods

Activation of Nuclear-Transferred Eggs

Fused eggs were activated using 5 different procedures. One group received 2 direct current pulses of 100 V/mm for 20 µsec in 0.28 mol/L mannitol supplemented with 0.1 mmol/L MgSO₄, 0.05 mmol/L CaCl₂, and 0.01% PVA, and then cultured in 5 µg/ml of CB-supplemented medium for 4 h. The eggs in the other 3 groups were subjected to 2 direct current pulses and then cultured in media supplemented with either 50 µM butyrolactone [15] (Funakoshi, Tokyo, Japan), 2 mmol/L 6-dimethylaminopurine (6-DMAP, Sigma) [16], or 10 µg/ml cyclohexamide (Sigma) for 4 h [17]. Eggs in the fifth group were treated with 15 µmol/L calcium ionomycin (Sigma) for 20 min followed by 2 mmol/L 6-DMAP for 4 h [11]. Activated eggs were cultured in the medium for 6 days in an atmosphere of 5% CO₂ and 95% air at 39°C.

Embryo Transfer

To minimize the possibility of infection, ovaries were obtained from a slaughterhouse close to the experimental station where the nuclear-transferred eggs were transferred to recipients. Ovaries were transported in saline with 100 U/ml penicillin and 100 µg/ml streptomycin at 25°C to a laboratory located within 6 h by car, and nuclear-transferred eggs that were activated with electric pulses and cultured for 1 or 2 days were then transported to the experimental station in modified NCSU-37 with antibiotics. Nuclear-transferred eggs were surgically transferred into oviducts of noncycling pubertal gilts that were the offspring of a Landrace x Large White female mated with a Large White male, and synchronized with injections of 1000 IU eCG and 500 IU hCG.

Microsatellite Analysis

Parentage analysis was performed on the piglets obtained by nuclear transfer and the surrogate recipient females to confirm the identity of the donor cells used for nuclear transfer. DNA was extracted from ear punch or tail clipping obtained from each newborn piglet, the recipients, and donor cells. Five porcine DNA microsatellite markers (SW717, SW936, SW1311, SW1327, and SWR414) were used to confirm the genetic identity of the cloned piglets to the donor heart cells used for nuclear transfer.

	RESULTS

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When matured eggs with a first polar body were treated with demecolcine for 1 h, more than 70% of the eggs had a membrane protrusion (Fig. 2a) and the chromosome mass migrated to a cortical location (Fig. 2b). Enucleating the eggs with a small volume of cytoplasm was easy and the enucleation rate was high (109 of 117, 93%) compared with that by aspirating a portion of cytoplasm underlying the first polar body (79%, 445 of 564).

View larger version (68K): [in this window] [in a new window]   FIG. 2. MII eggs before (a and b) and after nuclear transfer (c and d). An egg with a membrane protrusion following 1 h of demecolcine treatment. Arrow shows the membrane protrusion, and arrowhead indicates the first polar body (a). The condensed chromosome mass can be seen in the protrusion under fluorescent field (b) after Hoechst stain. Nuclear-transferred eggs with fetal cells were cultured in medium with (c) or without (d) demecolcine for 1 h, and mounted and stained with aceto-orcein. Chromosomes were condensed (c), but then started to scatter into the cytoplasm (d)

Enucleated eggs fused with somatic cells were cultured in the presence of demecolcine for 1 h to facilitate the remodeling of donor nuclei before parthenogenetic activation and to prevent scattering of donor chromosomes that was observed in the absence of demecolcine (Fig. 2, c and d). As shown in Table 1, the proportions of nuclear-transferred eggs that developed into blastocysts were not significantly different among groups activated using different procedures. The potential of eggs receiving somatic cells to develop into blastocysts was not different among donor cells of different origins, except for cumulus cells (Table 2). Although the number of blastocysts examined was small, cell numbers of blastocysts that developed from nuclear-transferred eggs receiving heart and muscle cells were significantly higher than those of blastocysts derived from kidney and oviduct cells (Table 2). The blastocysts derived from heart cells also had a large number of cells compared with those from fetal cells (Table 1). Cells from all tissues were positive for vimentin, a marker of fibroblast cells, and negative for desmine, a marker of muscle cells. Some oviduct and kidney cells were positive for cytokeratin, a marker of epithelial cells.

Nuclear-transferred eggs at the 1- to 8-cell stage (n = 180–341) were surgically transferred to the oviducts of 6 recipient gilts (Table 3). All of the gilts had an extended next heat to Day 29 and three were confirmed to be pregnant as determined by ultrasound visualization of fetuses at Days 34 to 42 of gestation. Two pregnant recipients that received nuclear-transferred eggs with heart cells maintained the pregnancy to term. One produced 4 live piglets and 1 dead fetus (Fig. 3a), and the other produced 4 live piglets and 2 dead fetuses (Fig. 3b) via vaginal delivery. The other pregnant recipient that received nuclear-transferred eggs with kidney cells aborted on day 62 of gestation, but the fetuses were inadvertently not recovered. The average birth weight of the cloned live piglets was 1.0 kg (0.9–1.2 kg) in recipient 1 and 0.7 kg (0.5–1.3 kg) in recipient 2, which is lower than that of female piglets of the same breed under natural mating conditions (average 1.3 kg; range 1.0–2.0 kg). Results of microsatellite marker analysis verified that the donor cells were the source of the genetic material used to produce the newborn piglets (Fig. 4).

View larger version (87K): [in this window] [in a new window]   FIG. 3. Piglets cloned by nuclear transfer of cultured heart cells from recipient 1 (a) and recipient 2 (b)

View larger version (13K): [in this window] [in a new window]   FIG. 4. Representative polymerase chain reaction analysis of microsatellite marker (SW936) in genomic DNA from recipient females (1 and 8), cloned piglets (2–6 and 9–14), and donor cells used in nuclear transfer (7 and 15)

	DISCUSSION

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Because mouse eggs have a small swelling in which the metaphase chromosomes are present, mechanical removal of chromosomes for nuclear transfer in mice is possible without treating the eggs [18]. Matured pig eggs do not have such a swelling, but more than 70% of eggs treated with demecolcine had a membrane protrusion in which the condensed chromosome mass was located (Fig. 2). Similar to rat meiotic chromosomes, which remain in a single mass within a protrusion overlaid by an actin-rich domain after nocodazole treatment [6], porcine meiotic chromosomes are also observed within a protrusion after demecolcine treatment. Although the mechanisms of action of demecolcine are not clear, the appearance of the protrusion might be related to the condensation of maternal chromosomes. When eggs were treated with demecolcine and sucrose for 1, 3, 6, 12, and 24 h, the proportions of eggs with condensed chromosomes were 100%, 97%, 87%, 96%, and 74%, respectively. All eggs with condensed chromosomes had a protruding membrane, but eggs with dispersed chromosomes did not. Such protrusions are observed more frequently in demecolcine-treated bovine (unpublished) and rabbit [19] eggs. This simple, chemically assisted method to remove maternal chromosomes makes it possible to produce a large number of nuclear-transferred eggs and to efficiently produce cloned piglets.

The present pregnancy rate (3 of 6, 50%) after transfer of eggs matured in vitro in which the chromosomes were removed using this chemically assisted method and that received adult somatic cells was higher than that in studies using in vivo-matured eggs and serial nuclear transfer (2 of 7, 29%) [8], in vivo-matured eggs receiving somatic cells from transgenic boar (2 of 5, 40%) [10], and in vitro-matured oocytes receiving fetal somatic cells (7 of 23, 30%) [11]. The proportion of live piglets from recipients that gave birth in the present study (8 of 467, 1.7%) was similar to that (4 of 307 1.3%) reported by Betthauser et al. [11], who used in vitro-matured eggs in which the absence of the metaphase plate after enucleation was verified by ultraviolet fluorescence.

Although the number of recipients was too small, considering the cell number of blastocysts derived from nuclear-transferred eggs, heart cells from a mature female pig seemed to be good donor cells for pig cloning. The reason for the low developmental potential of nuclear-transferred eggs receiving cultured cumulus cells, which are considered suitable donor cells for cloning in bovine [12], mice [20], and pigs [8], is not known.

The morphologic abnormalities that are frequently reported in bovine and mouse cloning of somatic and embryonic stem cells [2, 21] were not observed in the eight live piglets or three dead fetuses. Postnatal death of the young and excessive weight of the offspring, also typically reported for bovine and mouse [2] clones, were not observed. All eight cloned piglets, now 8 mo old, appear quite healthy. Because all piglets, except knockout pigs [22], thus far produced by different procedures from somatic cell nuclear transfer are apparently normal [8–11], species-specific differences might exist in the remodeling of somatic cell nuclei after nuclear transfer. The methylation patterns in pig nuclear-transferred embryos are the same as those in normal fertilized embryos [23], which is different from the methylation patterns in cloned bovine embryos [15].

Although normal cloned pigs were produced from adult somatic cells using a chemically assisted method to remove the maternal chromosomes, further studies are required to increase the successful development to term.

	ACKNOWLEDGMENTS

 
We thank Dr. H. Funahashi, Okayama University, for his kind suggestions on in vitro maturation of oocytes.

	FOOTNOTES

 
First decision: 17 December 2001.

¹ This work was supported in part by grant 12358014 from the Ministry of Education, Science, and Culture.

² Correspondence: Yukio Tsunoda, Laboratory of Animal Reproduction, College of Agriculture, Kinki University, 3327-204, Nakamachi, Nara 631-8505, Japan. FAX: 81 7 4243 1155; tsunoda{at}nara.kindai.ac.jp

³ These authors contributed equally to this work

Accepted: February 21, 2002.

Received: December 3, 2001.

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