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a Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan
b Research Institute for the Functional Peptides, Yamagata 990-0823, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An alternative strategy has been to use ES cells for nuclear transfer and to produce an animal derived entirely from a cultured cell, thus bypassing the chimeric step. Recently, production of viable lambs from cells of an established embryonic cell line was achieved [10]. This result was all the more extraordinary because the cultured cells used were not ES cells but a more differentiated epithelial cell type. This raised the possibility that nuclei from other cell types can be similarly reprogrammed and can yield offspring. In a subsequent study, lambs were produced from fetal fibroblasts and adult mammary-derived cells [11]. In addition, this production system was applicable to other species such as cattle [12–14], the mouse [15, 16], and the goat [17].
Establishment of a similar system in the pig is useful for the production of transgenic animals in which organs do not contain antigens for xenotransplantation or for analysis of functions of isolated human genes. In the present study, the isolation of a porcine cell line from in vitro-produced blastocysts is reported. Furthermore, the cells were transferred into enucleated oocytes, and the viability and development of the reconstructed embryos were tested in vitro.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Blastocysts were produced using the method designated the new system in our previous study [18], except that embryos were transferred to 100 µl of BSA-free NCSU-23 [19] supplemented with 10% (v:v) fetal calf serum (FCS; ICN Biomedicals, Aurora, OH) covered with paraffin oil (Nacalai Tesque, Kyoto, Japan) at 120 h after insemination and cultured for an additional 48–72 h until hatching from the zona pellucida.
Establishment and Maintenance of an Embryonic Cell Line
Zona pellucida-free hatched blastocysts (3–6 embryos) were transferred to a well of a 4-well plate (Nunc, Roskilde, Denmark) containing a monolayer of mitomycin C-inactivated STO cells with 0.5 ml of medium. Thioguanine- and ouabain-resistant STO cells were derived from SIM mouse fibroblasts [20], and have been used as feeder cells for culture of ES cells. Embryos were cultured under 5% CO2 in air at 38.5°C, and the medium was changed every 2 days. Attachment of embryos on feeder cells and formation of colonies of attached embryo-derived cells was observed at 2 and 8 days after culture, respectively.
After 8 days of culture, the medium was aspirated from the well and replaced with 0.5 ml Dulbecco's PBS(-) composed of 136.9 mM NaCl, 2.68 mM KCl, 1.47 mM KH2PO4, and 8.1 mM Na2HPO4. Colonies of attached embryo-derived cells were freed from the STO cell layer with a fine pipette, transferred individually into 20 µl of PBS(-) supplemented with 0.25% (w:v) trypsin and 0.5 mM EDTA (T-E), and cultured for 5 min under 5% CO2 in air at 38.5°C. After culture, the colonies were partially dispersed by repeated passage through a fine pipette and individually reseeded onto new mitomycin C-inactivated STO feeder layers in 4-well plates; wells contained 0.5 ml of 60% (v:v) Buffalo rat liver (BRL) cell-conditioned medium [21] supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM minimal essential medium (MEM) nonessential amino acids, nucleosides (0.03 mM adenosine, 0.03 mM guanosine, 0.03 mM cytidine, 0.03 mM uridine, and 0.01 mM thymidine), 2000 IU/ml leukemia inhibitory factor (LIF; Gibco, Grand Island, NY), 10 ng/ml basic fibroblast growth factor (bFGF; Boehringer Mannheim Biochemica, Mannheim, Germany), and 20% FCS (cell culture solution). BRL cell-conditioned medium was obtained by incubating 10 ml of Dulbecco's modified Eagle's medium (DMEM; containing L-glutamine and 4.5 mg/ml glucose; Gibco) with 10% FCS per confluent 100-mm dish of BRL cells and harvesting every 3 days for up to 2 wk. Before use, conditioned medium was diluted to 60% with DMEM. After 6 days of seeding, formation of colonies with a morphology resembling that of mouse ES cells, in which it is difficult to discern the individual component cells because they are packed tightly together in small nests, was observed. The well was washed with PBS(-) and treated with T-E, and ES-like colonies were transferred to 35-mm gelatinized dishes containing 2 ml of the medium. After 6 days of culture, cells were trypsinized, resuspended in the medium, and transferred to 100-mm gelatinized dishes. Cells were subcultured at 6-day intervals, and the culture medium was changed every 48 h. Aliquots of cells from each passage were frozen in DMEM supplemented with 10% FCS and 10% (v:v) dimethyl sulfoxide (Wako Pure Chemical Industries, Tokyo, Japan).
Genetic Transformation of Hatched Blastocyst-Derived Cells
A neomycin resistance gene containing a phosphoglycerate kinase promoter was inserted into an enhanced green fluorescent protein (EGFP)-containing vector, pCX-EGFP [22] (pCX-EGFP/Neo, Fig. 1). Hatched blastocyst-derived cells at passage 9 were trypsinized and resuspended in PBS(-) to give a concentration of 4.0 x 106 cells/ml. Then 500 µl of the cell suspension and 20 µg of linearized pCX-EGFP/Neo (SalI was used for linearization) were transferred into a cuvette with electrodes 4 mm apart and electroporated under a voltage of 400 V and a capacitance of 500 µFa. After electroporation, cells were resuspended in 10 ml of the cell culture solution, transferred to a 100-mm gelatinized dish, and cultured under 5% CO2 in air at 38.5°C. Selection in 500 µg/ml Geneticin (Gibco) was initiated 24 h after electroporation and continued for 12 days thereafter. After selection, surviving cells continued to be maintained in the cell culture solution, and a green fluorescent protein (GFP)-expressing cell line was established. Aliquots cells from each passage cells were frozen as described above.
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Nuclear Transfer
In vitro-matured oocytes produced as described above were washed once in the activation solution, which consisted of 0.3 M mannitol, 0.1 mM CaCl2, 0.1 mM MgSO4, and 2 mg/ml fatty acid-free BSA (Sigma Chemical Co., St. Louis, MO); they were then placed between 2 wire electrodes (1 mm apart) of the fusion chamber slide with 10 ml of activation solution. Direct current (D.C.) pulses of 120 V/mm were applied twice to the oocytes for a duration of 60 µsec at intervals of 5 sec.
The oocytes were transferred to 100 µl of PBS(-) supplemented with 5.55 mM glucose, 1.0 mM glutamine, 7.0 mM taurine, 5.0 mM hypotaurine, and 10% FCS (manipulation solution); and zonae pellucidae were cut (10–20% of their circumference) with a fine glass needle. After cutting, the oocytes were enucleated by pushing out the first polar body and the metaphase II plate in a small amount of surrounding cytoplasm with a glass pipette. The oocytes had been previously stained in the manipulation solution supplemented with 5 µg/ml Hoechst 33342 (Sigma) and 7.5 µg/ml cytochalasin B (Sigma) for 20 min. Confirmation of successful enucleation was achieved by visualizing the cytoplast and removed cytoplasm under ultraviolet light. After enucleation, the cytoplasts were washed in the manipulation solution, transferred to the same medium, and kept in a CO2 incubator until injection of donor cells.
Donor cells were used for nuclear transfer between passages 9 and 30 of culture. Immediately prior to injection, a single-cell suspension of the donor cells was prepared by standard trypsinization. The cells were pelleted and resuspended in the manipulation solution.
Recipient cytoplasts and donor cells were transferred to 100 µl of the manipulation solution on a microscope stage, and a cell was inserted into the perivitelline space of each enucleated oocyte using a glass pipette.
Cell-oocyte complexes were cultured in NCSU-23 until fusion. Fusion of cells and oocytes was induced at 6 h after activation [23]. The chamber for fusion was a 100-mm dish filled with 10 ml of the activation solution. Two stainless steel wires (100-µm diameter) were used for electrodes and were attached to micromanipulators. The single cell-oocyte complex was sandwiched between the electrodes and oriented with the contact surface between the cytoplast and the donor cell perpendicular to the electrodes. The distance between the electrodes was about 100 µm. Membrane fusion was induced by applying a single D.C. pulse with a pre- and postpulse alternating current field of 5 V, 1 MHz for 5 sec each. Following the fusion pulse, the complexes were washed in NCSU-23 and cultured for a period of 1 h in 100 µl of the same medium. Fusion was then determined by microscopic examination. Fused embryos (5–15 embryos) were transferred to 100 µl of NCSU-23, cultured for an additional 7 days, and evaluated for blastocyst formation. At 2 days after culture, the embryos were examined for cleavage.
Experimental Studies
The cell culture solution used in the present study was suitable for culture of mouse ES cells and porcine oviductal epithelial cells but did not support the isolation of cell lines from porcine blastocysts (data not shown). In experiment 1, therefore, to evaluate various media in relation to proliferation of hatched blastocyst-derived cells, the blastocysts were cultured in DMEM, a basic medium of the cell culture solution, or BSA-free NCSU-23, which is suitable for culture of porcine embryos. Both media were supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM MEM nonessential amino acids, nucleosides (0.03 mM adenosine, 0.03 mM guanosine, 0.03 mM cytidine, 0.03 mM uridine, and 0.01 mM thymidine), 2000 IU/ml LIF, 10 ng/ml bFGF, and 20% FCS.
To produce reconstructed embryos, fusion of hatched blastocyst-derived cells and enucleated oocytes by electric stimulation is necessary. Therefore, experiment 2 was an examination of the effect of electric field strengths on the fusion of donor cells (passages 24–30) and recipient oocytes. A single D.C. pulse of 100, 150, 200, 250, or 300 V/mm was applied to the cell-oocyte complexes for a duration of 20 µsec.
Although 5–15 reconstructed embryos are cultured together in a drop, it is believed that only one or none of them can develop to the blastocyst stage. It is known that blastocyst formation of embryos cultured individually is poor. In experiment 3, therefore, to improve the developmental ability of the reconstructed embryos, half of them were cocultured with oocytes activated as described above. Donor cells (passages 9–13) and recipient oocytes were fused by applying a single D.C. pulse of 200 V/mm, and fused embryos were cultured with or without 15–20 electro-activated oocytes. Reconstructed embryos were distinguished from electro-activated oocytes by the slits in their zonae pellucidae.
Experiment 4 was undertaken to establish whether the genome of the donor cell could be transferred into the recipient cytoplast and expressed in the reconstructed embryo using GFP as a marker. GFP-expressing cells (passages 16–18) and enucleated oocytes were fused by applying a single D.C. pulse of 200 V/mm, and fused embryos were cultured with 15–20 electro-activated oocytes. At 7 days after culture, the expression of GFP in blastocysts that had developed from reconstructed embryos was examined under ultraviolet light.
Statistical Analysis
The proportions of total cell-oocyte complexes fused and of fused embryos developing to each stage were subjected to an arcsin transformation in each replicate. The transformed values were analyzed using one-way ANOVA. When ANOVA revealed a significant treatment effect, the treatments were compared by Duncan's multiple range test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As shown in Table 2, when a pulse of 100 V/mm was applied to cell-oocyte complexes (Fig. 3A), no fused embryos were observed. However, the percentage of fused embryos (Fig. 3B) increased significantly (P < 0.05) at 150 V/mm (28%) and 200 V/mm (64%). The fusion rate at 250 V/mm (77%) and at 300 V/mm (52%) did not significantly differ from that at 200 V/mm. At 200 V/mm, 39% of fused embryos cleaved (Fig. 3C), although this percentage did not significantly differ from that at 150 V/mm (14%) or 250 V/mm (35%). Some 3-cell embryos were obtained at 200 and 250 V/mm, but no embryos developed beyond the 3-cell stage after further cultivation.
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In the absence of electro-activated oocytes, the development of reconstructed embryos to the 4-cell stage was markedly inhibited (4%, Table 3). In contrast, 65% of embryos cleaved and 23% of them developed to the 4-cell stage in the presence of activated oocytes. These percentages were significantly (P < 0.05) higher than those (cleavage: 38%; 4-cell stage: 3%) in the absence of activated oocytes. Moreover, at 7 days after culture, one reconstructed embryo developed to the blastocyst stage in the presence of activated oocytes.
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Of 237 GFP-expressing cell-oocyte complexes, 102 (43%) fused and 3 (3%) developed to the blastocyst stage. All of the blastocysts were positive for fluorescent green under ultraviolet light (Fig. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, the culture medium was a very important factor in the colony formation of hatched blastocyst-derived cells. Modified Ham's F-10 medium was inferior to Whitten's medium in promoting the development of porcine embryos [24]. It has been reported that TCM-199 did not support the development of 1- and 2-cell porcine embryos beyond the morula stage, while 63.5% of embryos developed to the morula or blastocyst stage in Hepes-buffered Tyrode's solution [25]. Miyano et al. [26] reported a similar result indicating that Whitten's medium supported the development of 1- and 2-cell porcine embryos to the blastocyst stage more effectively than TCM-199. On the basis of these reports, it seems that some components in complex tissue culture media are detrimental to the development of porcine embryos in vitro. These components in DMEM may affect not only embryonic development to the blastocyst stage but also the proliferation of blastocyst-derived cells.
The porcine hatched blastocyst-derived cells established in the present study had a stable epithelial-like morphology. This morphology was very similar to that of ovine blastocyst-derived cells that are totipotent by nuclear transfer and can produce viable offspring [10, 11, 27, 28].
The results of the present study showed that electrofusion is effective for production of porcine nuclear transfer embryos reconstituted with hatched blastocyst-derived cells and enucleated oocytes. Electrofusion has been used for fusion of cultured cells and enucleated oocytes, and consequently clone animals have been produced in sheep [10, 11, 27, 28], cattle [12, 13, 27], and goats [17]. In general, a chamber consisting of parallel wires has been used for fusion [10, 11, 14, 17, 27, 28]. It was reported that a chamber consisting of parallel wires with a separation of 1 mm is effective for fusion with blastomeres of embryos and enucleated oocytes in the pig [23]. Therefore, we used the same chamber for fusion initially, but few fused embryos were obtained (data not shown). In contrast, when cell-oocyte complexes were sandwiched between wires arranged in a straight line, the percentage of fused embryos was high. When small cells such as hatched blastocyst-derived cells are used as donors, the orientation of the cell and enucleated oocyte is critical because cell-to-cell contact is minimal. It is suggested that our method is suitable for the orientation of the donor cell and recipient oocyte and for keeping them in contact.
The in vitro developmental ability of porcine nuclear transfer embryos reconstituted with postembryonic genome activation stage nuclei is very limited. It has been shown that porcine nuclear transfer embryos reconstituted with 8- to 16-cell stage nuclei and enucleated mature oocytes cleaved in vitro at rates of 36–43%, but developmental rates beyond the morula stage were only 4.5–7% [29, 30]. Recently, Nagashima et al. [23] reported that 28% of nuclear transfer embryos reconstituted with morula-stage nuclei developed to morulae, and 15% formed blastocysts, with use of preactivated oocytes as recipient cytoplasts and induction of fusion of blastomeres and cytoplasts at 6 h after the electric activation. However, when porcine primordial germ cells were transferred to preactivated or nontreated oocytes, reconstructed embryos could not develop beyond the 4-cell stage in vitro [31]. To our knowledge, this is the first report that describes successful development to the blastocyst stage of porcine embryos reconstituted with cultured cells and enucleated oocytes.
Cell cycle stage of donor cells and maturation/meiosis/mitosis-promoting factor (MPF) activity of recipient oocytes are very important factors for development of nuclear transfer embryos. Two distinct protocols emerge for embryo reconstruction by nuclear transfer with use of mature oocytes as recipients. The first is the transfer of nuclei in G0 or G1 phase to nontreated recipients, and the second is the transfer of nuclei in G1, S, or G2 phase to preactivated recipients after the disappearance of MPF activity [32]. In the present study, preactivated oocytes were used as recipients because the cell cycle phase of hatched blastocyst-derived cells was unknown. However, production of animals from differentiated cells has been achieved mainly by transferring nuclei in G0 or G1 phase to nontreated recipients [10–15, 17, 27, 28]. As 40–60% of embryonic cell line cells are in G1 phase at any given time [33], it is necessary to clarify the most suitable time for their development for reconstituting nuclear transfer embryos with porcine hatched blastocyst-derived cells and enucleated oocytes.
Cloned animals have been obtained from nuclear transfer embryos reconstituted with cultured cells at early passages and enucleated oocytes [10, 11, 13–17, 27, 28]. It has been reported that when bovine embryo-derived cells are cultured in vitro, the percentage of cells with abnormal karyotypes increases for late passages [34]. This is one of the chief obstacles to the development of reconstructed embryos produced by transferring cells at late passages into recipient oocytes. In the present study, however, even when the cells at early passages were transferred, development of reconstructed embryos was poor. In contrast, it was shown that coculture with electro-activated oocytes improves the developmental ability of porcine reconstructed embryos. A study using 2-cell mouse embryos indicated that culturing groups of embryos resulted in development to the blastocyst stage at almost twice the rate observed for embryos cultured individually [35]. This phenomenon is caused by dilution of autocrine growth factors that are released from embryos and that have a role in embryonic development [36]. Therefore, the improvement in development of porcine reconstructed embryos by coculture with electro-activated oocytes may be attributed to the release of growth factors from activated oocytes.
Although some porcine reconstructed embryos developed to the blastocyst stage by coculturing with electro-activated oocytes, the percentage of blastocyst formation (3%) was very low compared with that of in vitro-fertilized embryos—in which it is estimated that more than 30% of normal fertilized embryos develop to blastocysts [18]. This attrition may be attributed to lack of chromosomes by polar body formation after activation of reconstructed embryos [15]. In this context it would be of interest to examine the effect of cytochalasin B, which blocks extrusion of polar bodies [15], on the development of porcine reconstructed embryos.
The results of the present study indicated that GFP is useful as a marker of gene expression in porcine nuclear transfer embryos. The expression of GFP in blastocysts demonstrated that they developed from reconstructed embryos. Further experiments in which GFP-expressing embryos are transferred to recipient females are in progress. Although it is not clear whether GFP affects the development of porcine postimplantation embryos, GFP-expressing mice were obtained by microinjecting the transgene into fertilized eggs [22].
In conclusion, we have developed a production system of porcine nuclear transfer embryos reconstituted with cultured cells and enucleated oocytes. At the present, little is known about the development of porcine reconstructed embryos. Therefore, our system should prove very useful for further studies of the factors involved in the control of their development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants for Encouragement of Young Scientists from the Japan Society for the Promotion of Science (JSPS), JSPS-RFTF 97L00904 for "Research for the Future" Program from JSPS, and for Program for Promotion of Basic Research Activities for Innovative Bioscience. K.M. is a recipient of the JSPS Fellowship (No. 9801517). 
2 Correspondence: Kazuchika Miyoshi, Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-amamiyamachi, Aoba-ku, Sendai 981-8555, Japan. FAX: 81 22 717 8879; miyoshik{at}bios.tohoku.ac.jp
Accepted: January 20, 2000.
Received: September 23, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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