• Journal of Veterinary Science
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• v.23 no.2
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• pp.40.1-40.13
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• 2022

Background: Somatic cell nuclear transfer (SCNT) is used widely in cloning, stem cell research, and regenerative medicine. The type of donor cells is a key factor affecting the SCNT efficiency. Objectives: This study examined whether urine-derived somatic cells could be used as donors for SCNT in pigs. Methods: The viability of cells isolated from urine was assessed using trypan blue and propidium iodide staining. The H3K9me3/H3K27me3 level of the cells was analyzed by immunofluorescence. The in vitro developmental ability of SCNT embryos was evaluated by the blastocyst rate and the expression levels of the core pluripotency factor. Blastocyst cell apoptosis was examined using a terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. The in vivo developmental ability of SCNT embryos was evaluated after embryo transfer. Results: Most sow urine-derived cells were viable and could be cultured and propagated easily. On the other hand, most of the somatic cells isolated from the boar urine exhibited poor cellular activity. The in vitro development efficiency between the embryos produced by SCNT using porcine embryonic fibroblasts (PEFs) and urine-derived cells were similar. Moreover, The H3K9me3 in SCNT embryos produced from sow urine-derived cells and PEFs at the four-cell stage showed similar intensity. The levels of Oct4, Nanog, and Sox2 expression in blastocysts were similar in the two groups. Furthermore, there is a similar apoptotic level of cloned embryos produced by the two types of cells. Finally, the full-term development ability of the cloned embryos was evaluated, and the cloned fetuses from the urine-derived cells showed absorption. Conclusions: Sow urine-derived cells could be used to produce SCNT embryos.

• Korean Journal of Animal Reproduction
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• v.24 no.4
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• pp.385-394
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• 2000

When porcine primordial germ cells (PGCs) isolated from the genital ridge and placed in culture to establish EG cells, a large proportion of PGCs are lost during the early period of culture. To characterize the in vitro death of porcine PGCs, PGCs were cultured in suspension, and apoptosis analyzed using a fluorescent activated cell sorter-based DNA fragmentation assay. The results from flow cytometric analysis showed an increase in apoptosis in cultured cells. However, the cells isolated from the genital ridges are a mixture of PGCs and somatic cells. To detect apoptotic signals specific from porcine PGCs, quantitative TUNEL assay was performed at different time of culture (0 ∼ 24 h). The proportion of apoptotic porcine PGCs determined by double staining with alkaline phosphatase activity and in situ TUNEL assay increased as the time of culture progressed and continued at least 24 h. These results demonstrate that one of the causes of loss of porcine PGCs in vitro is apoptosis.

• Reproductive and Developmental Biology
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• v.33 no.1
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• pp.13-18
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• 2009

The aim of this study was to examine the microtubule distributions of somatic cell nuclear transfer (SCNT) and parthenogenetic porcine embryos. Porcine SCNT embryos were produced by fusion of serum-starved fetal fibroblast cells with enucleated oocytes. Reconstituted and mature oocytes were activated by electric pulses combined with 6-dimethlyaminopurine treatment. SCNT and parthenogenetic embryos were cultured in vitro for 6 days. Microtubule assembly of embryos was examined by confocal microscopy 1 hr and 20 hr after fusion or activation, respectively. The proportions of embryos developed to the blastocyst stage were 25.7% and 30.4% in SCNT and parthenogenetic embryos, respectively. The frequency of embryos showing $\beta$-tubulins was 81.8% in parthenogenetic embryos, whereas 31.3% in SCNT embryos 1 hr after activation or fusion. The frequency of the embryos underwent normal mitotic phase was low in SCNT embryos (40.6%) compared to that of parthenogenetic ones (59.7%) 20 hr after fusion or activation (p<0.05). The rate of SCNT embryos with an abnormal mitosis pattern is about twice compared to that of parthenogenetic ones. The spindle assembly and its distribution of SCNT embryos in the first mitotic phase were not different from those of parthenogenetic ones. The result shows that although microtubule distribution of porcine SCNT embryos shortly after fusion is different from parthenogenetic embryos, and the frequency of abnormal mitosis 20 hr after fusion or activation is slightly increased in SCNT embryos, microtubule distributions at the first mitotic phase are similar in both SCNT and parthenogenetic embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.2
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• pp.266-277
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• 2014

Somatic cell nuclear transfer (SCNT) has generally demonstrated that a differentiated cell can convert into a undifferentiated or pluripotent state. In the SCNT experiment, nuclear reprogramming is induced by exposure of introduced donor nuclei to the recipient cytoplasm of matured oocytes. However, because the efficiency of SCNT still remains low, a combination of SCNT technique with the ex-ovo method may improve the normal development of SCNT embryos. Here we hypothesized that treatment of somatic cells with extracts prepared from the germinal vesicle (GV) stage Siberian sturgeon oocytes prior to their use as nuclear donor for SCNT would improve in vitro development. A reversible permeability protocol with $4{\mu}g/mL$ of digitonin for 2 min at $4^{\circ}C$ in order to deliver Siberian sturgeon oocyte extract (SOE) to porcine fetal fibroblasts (PFFs) was carried out. As results, the intensity of H3K9ac staining in PFFs following treatment of SOE for 7 h at $18^{\circ}C$ was significantly increased but the intensity of H3K9me3 staining in PFFs was significantly decreased as compared with the control (p<0.05). Additionally, the level of histone acetylation in SCNT embryos at the zygote stage was significantly increased when reconstructed using SOE-treated cells (p<0.05), similar to that of IVF embryos at the zygote stage. The number of apoptotic cells was significantly decreased and pluripotency markers (Nanog, Oct4 and Sox2) were highly expressed in the blastocyst stage of SCNT embryos reconstructed using SOE-treated cells as nuclear donor (p<0.05). And there was observed a better development to the blastocyst stage in the SOE-treated group (p<0.05). Our results suggested that pre-treatment of cells with SOE could improve epigenetic reprogramming and the quality of porcine SCNT embryos.

• Journal of Animal Reproduction and Biotechnology
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• v.38 no.3
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• pp.109-120
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• 2023

Background: Pluripotent stem cells (PSCs) including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer the immense therapeutic potential in stem cell-based therapy of degenerative disorders. However, clinical trials of human ESCs cause heavy ethical concerns. With the derivation of iPSCs established by reprogramming from adult somatic cells through the transgenic expression of transcription factors, this problems would be able to overcome. In the present study, we tried to differentiate porcine iPSCs (piPSCs) into endothelial cells (ECs) for stem cell-based therapy of vascular diseases. Methods: piPSCs (OSKMNL) were induced to differentiation into ECs in four differentiation media (APEL-2, APEL-2 + 50 ng/mL of VEGF, EBM-2, EBM-2 + 50 ng/mL of VEGF) on cultured plates coated with matrigel^® (1:40 dilution with DMEM/F-12 medium) for 8 days. Differentiation efficiency of these cells were exanimated using qRT-PCR, Immunocytochemistry, Western blotting and FACS. Results: As results, expressions of pluripotency-associated markers (OCT-3/4, SOX2 and NANOG) were higher observed in all porcine differentiated cells derived from piPSCs (OSKMNL) cultured in four differentiation media than piPSCs as the control, whereas endothelial-associated marker (CD-31) in the differentiated cells was not expressed. Conclusions: It can be seen that piPSCs (OSKMNL) were not suitable to differentiate into ECs in the four differentiation media unlike porcine epiblast stem cells (pEpiSCs). Therefore, it would be required to establish a suitable PSCs for differentiating into ECs for the treatment of cardiovascular diseases.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.4
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• pp.483-491
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• 2009

In vitro produced porcine embryos have potential application in reproductive biotechnology. However, their development potential has been very low. This study evaluated the in vitro developmental ability and quality of cloned and parthenogenetic porcine embryos having 2-4 cells or 5-8 cells on Day 2 of in vitro culture. Analysis of results showed that 2 to 4 cell embryos had higher ability to form blastocysts than 5 to 8 cell embryos (p<0.05). Blastocysts produced from culture of 2 to 4 cell embryos also contained higher cell numbers and had lower BAX:BCLxL transcript ratio than those produced from 5 to 8 cell embryos (p<0.05), thereby suggesting 2 to 4 cell embryos have higher development potential. Further investigation revealed that 5 to 8 cell embryos had higher incidence (100${\pm}$0.0%) of blastomeric fragmentation than 2 to 4 cell embryos (15.2${\pm}$5.5% for parthenogenetic and 27.7${\pm}$7.1% for cloned embryos). This suggests that low development potential of 5 to 8 cell embryos was associated with blastomeric fragmentation. In conclusion, we have shown that morphological selection of embryos based on cell number on Day 2 of in vitro culture could offer a practical and valuable non-invasive means to select good quality porcine embryos.

• Reproductive and Developmental Biology
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• v.32 no.2
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• pp.135-140
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• 2008

Recent studies on nuclear transfer and induced pluripotent stem cells have demonstrated that differentiated somatic cells can be returned to the undifferentiated state by reversing their developmental process. These epigenetically reprogrammed somatic cells may again be differentiated into various cell types, and used for cell replacement therapies through autologous transplantation to treat many degenerative diseases. To date, however, reprogramming of somatic cells into undifferentiated cells has been extremely inefficient. Hence, reliable markers to identify the event of reprogramming would assist effective selection of reprogrammed cells. In this study, a transgene construct encoding enhanced green fluorescent protein (EGFP) under the regulation of human Oct4 promoter was developed as a reporter for the reprogramming of somatic cells. Microinjection of the transgene construct into pronuclei of fertilized mouse eggs resulted in the emission of green fluorescence, suggesting that the undifferentiated cytoplasmic environment provided by fertilized eggs induces the expression of EGFP. Next, the transgene construct was introduced into human embryonic fibroblasts, and the nuclei from these cells were transferred into enucleated porcine oocytes. Along with their in vitro development, nuclear transfer embryos emitted green fluorescence, suggesting the reprogramming of donor nuclei in nuclear transfer embryos. The results of the present study demonstrate that expression of the transgene under the regulation of human Oct4 promoter coincides with epigenetic reprogramming, and may be used as a convenient marker that non-invasively reflects reprogramming of somatic cells.

• Reproductive and Developmental Biology
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• v.34 no.4
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• pp.283-288
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• 2010

During normal early embryonic development in mammals, the global pattern of genomic DNA methylation undergoes marked. changes. The level of methylation is high in male and female gametes. Thus, we cloned the cDNA of the porcine DNA methyltransferase 1 (Dnmt1) gene to promote the efficiency of the generation of porcine clones. In this study, porcine Dnmt1 cDNA was sequenced, and Dnmt1 mRNA expression was detected by reverse transcription-polymerase reaction (RT-PCR) in porcine tissues during embryonic development. The porcine Dnmt1 cDNA sequence showed more homology with that of bovine than human, mouse, and rat. The complete sequence of porcine Dnmt1 cDNA was 4,774-bp long and consisted of an open reading frame encoding a protein of 1611 amino acids. The amino acid sequence of porcine DNMT1 showed significant homology with those of bovine (91%), human (88%), rat (76%), and mouse (75%) Dnmt1. The expression of porcine Dnmt1 mRNA was detected during porcine embryogenesis. The mRNA was detected at stages of porcine preimplantation development (1-cell, 2-cell, 4-cell, 8-cell, morula, and blastocyst stages). It was also abundantly expressed in tissues (lung, ovary, kidney and somatic cells). Further investigations are necessary to understand the complex links between methyltransferase 1 and the transcriptional activity in cloned porcine tissues.

• Journal of Embryo Transfer
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• v.22 no.3
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• pp.179-184
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• 2007

Culture method of somatic cells is one of the important factors in the production of transgenic pigs by somatic cell nuclear transfer. In this study, we established an efficient culture method of porcine fetal fibroblasts. Porcine fetal fibroblasts were isolated from 33-day-old fetuses. The proliferation of porcine fetal fibroblasts was analyzed by different serum types and culture media. The cultures in medium supplied 15% ES screened FBS showed faster increase in cell number than 15% FBS. Also, fetal fibroblasts have been propagated continuously for $7{\sim}8$ passages in ES modified DMEM and DMEM medium. We transfected $PGK-neo^r$ vector (pKJ2) into porcine fetal fibroblasts to estimate colony formation in this culture condition. The formation of colonies was confirmed in the medium containing $300\;{\mu}g/ml$ G418 at 12 day. These data show that this culture system can be used screening of porcine somatic cells transfected transgene.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.51-51
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• 2002

Despite of importance of integrated events of nucleus and microtubule remodeling in nuclear transferred embryos with somatic cells, little information is available on this subject. In this study we compared chromatin, r-tubulin and microtubule organization in porcine oocytes following somatic cell nuclear transfer and parthenogenetically activation in order to clarify nuclear remodeling process and to demonstrate centrosome inheritance during nuclear transfer. (omitted)