• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.92-92
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• 2003

Accurate analysis of nuclear status is needed when biopsied-blastomeres are used for embryo sexing. In this study, the nuclear status of blastomeres derived from 8- to 16-cell stage IVF bovine embryos was analyzed to evaluate the representative of single blastomere for embryo sexing. When 55 embryos were analyzed by PCR following biopsy, the coincident rate of sex determination between biopsied-single blastomere and matched blastocyst by PCR was 80 %. Karyotyping of biastomeres in 8- 16-cell stage bovine embryos was conducted to assess chromosome status of IVF embryos. To establish karyotyping of blastomeres, concentrations of vinblastine sulfate and duration of exposure time for metaphase plate induction with 8- to 16-cell stage bovine embryos were tested. The most effective condition for induction of metaphase plate (>45%) was 1.0 ug/ml vinblastine sulfate treatment for 15 h. In 22 embryos under the condition, only 8 embryos out of ten that had a normal diploid chromosome complement showed a sex-chromosomal composition of XX or XY (36.4%) and 2 diploid embryos showed mosaicism of the opposite sex of XX and XY in blastomeres of embryo (9.1%). One haploid embryo contained only one X-chromosome (4.5%). Four out of the other 11 embryos having a mixoploid chromosomal complement contained haploid blastomere with wrong sex chromosome (18.2%). These results suggested that morphologically normal bovine embryos derived from IVF had considerable proportion of mixoploid and sex-chromosomal mosaicism which could be the cause of discrepancies of the sex between biopsied-single blastomere and matched blastocyst by PCR analysis.

• Journal of Embryo Transfer
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• v.8 no.1
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• pp.37-44
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• 1993

Nuclear transplantation techinque has been found to be the most potential and efficient method for producing a large number of genetically identical animals from a single embryo. The technical development of nuclear transplantation in mammals and its application to the production of cloned animals were reviewed. For the efficient and successful production of cloned embryos by nuclear transplantation, selection and micromanipulation of recipient eggs or embryos as capacious recipient cytoplasm, and benefitial preparation of multiple totipotent embryonic cells as donor nuclei, and also fusion technique are very critical. Recent works approaching to these critical points were introduced and discussed.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.2
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• pp.194-200
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• 2009

The aim of this study was to investigate the effects of donor cell passage, size and type on the development of nuclear transfer embryos. Porcine cumulus cells, fetal fibroblasts and oviductal epithelial cells from 1-2, 3-6 and 7-10 passages were used for the nuclear transfer. In the oocytes with the cumulus donor cells, fusion and cleavage rates of oocytes and cell numbers per blastocyst among the three different passage groups did not show any differences, but the rates of blastocyst formation from 1-2 and 3-6 passage groups were higher than those from 7-10 passage group. The rates of fusion, cleavage and blastocyst formation, and the cell numbers per blastocyst were higher in the embryos with the sizes of <20 and 20 ${\mu}m$ cumulus donor cells compared to the >20 ${\mu}m$ cumulus donor cell. In the oocytes with the fetal fibroblast donor cells, the rate of blastocyst formation from the 3-6 passage group was higher than from 1-2 and 7-10 passage groups. The embryos with the size of 20 $\mu{m}$ fetal fibroblast donor cell showed higher rate of blastocyst formation compared to those with <20 and >20 ${\mu}m$ donor cells. In the oocytes with the oviductal epithelial cells, the rates of blastocyst formation from 1-2 and 3-6 passage groups were higher compared to those from 7-10 passage group. The embryos with the sizes of <20 and 20 ${\mu}m$ oviductal epithelial donor cells had a higher rate of blastocyst formation compared to those with >20 ${\mu}m$ donor cell. Fusion and cleavage rates of oocytes, and cell numbers per blastocyst among the three different donor cell types from the 3-6 passage did not show any differences. However, the rate of blastocyst formation of somatic cell nuclear transfer (SCNT) embryos with the fetal fibroblast donor cell was higher than that of blastocyst formation of SCNT embryos with the cumulus and oviductal epithelial donor cells.

• Journal of Embryo Transfer
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• v.31 no.3
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• pp.179-183
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• 2016

Even though klotho deficiency in mice exhibits multiple aging-like phenotypes, studies using large animal models such as pigs, which have many similarities to humans, have been limited due to the absence of cell lines or animal models. The objective of this study was to generate homozygous klotho knockout porcine cell lines and cloned embryos. A CRISPR sgRNA specific for the klotho gene was designed and sgRNA (targeting exon 3 of klotho) and Cas9 RNPs were transfected into porcine fibroblasts. The transfected fibroblasts were then used for single cell colony formation and 9 single cell-derived colonies were established. In a T7 endonuclease I mutation assay, 5 colonies (#3, #4, #5, #7 and #9) were confirmed as mutated. These 5 colonies were subsequently analyzed by deep sequencing for determination of homozygous mutated colonies and 4 (#3, #4, #5 and #9) from 5 colonies contained homozygous modifications. Somatic cell nuclear transfer was performed to generate homozygous klotho knockout cloned embryos by using one homozygous mutation colony (#9); the cleavage and blastocyst formation rates were 72.0% and 8.3%, respectively. Two cloned embryos derived from a homozygous klotho knockout cell line (#9) were subjected to deep sequencing and they showed the same mutation pattern as the donor cell line. In conclusion, we produced homozygous klotho knockout porcine embryos cloned from genome-edited porcine fibroblasts.

• Reproductive and Developmental Biology
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• v.35 no.2
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• pp.175-183
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• 2011

The present study compared the developmental potential, telomerase activity and transcript levels of X-linked genes (ANT3, HPRT, MeCP2, RPS4X, XIAP, XIST and ZFX) in the bovine somatic cell nuclear transfer (SCNT) embryos derived from different age and cell cycle of female donor nucleus. In experiment 1, the fusion rate, cleavage rate to 2-cell stage, developmental rate to blastocyst stage, and the mean number of total and ICM cells was slightly increased in embryos cloned with fetal fibroblasts compared to those with adult fibroblasts, but there was no significantly (p<0.05) differences. Telomerase activity was also similar in blastocysts cloned with fetal and adult fibroblasts. Up-regulated RPS4X and down-regulated MeCP2, XIAP, and XIST transcript level were observed in blastocysts cloned with adult fibroblasts, compared to those with fetal fibroblasts. In experiment 2, the fusion rate, cleavage rate to 2-cell stage, developmental rate to blastocyst stage, and the mean number of total and ICM cells was significantly (p<0.05) increased in embryos cloned with fetal fibroblasts at early G1 phase of the cell cycle, compared to those of fetal fibroblasts at late G1 phase. DNMT1 transcript was observed to significantly (p<0.05) increased in the fetal fibroblasts at 3 hrs after trypsin treatment of confluent culture. Further, level of telomerase activity and transcribed X-linked genes was also significantly (p<0.05) higher in the early G1 SCNT blastocysts than those of late G1. The results imply that fetal fibroblasts at early G1 phase induces the enhanced developmental potential and up-regulated telomerase activity and X-linked gene, but aberrant transcript pattern of X-linked genes may be displayed in the SCNT embryos.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.123-129
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• 2011

The pig has been considered to serve as an appropriate model of human disease. Therefore, establishment of porcine embryonic stem cell lines is important. The purpose of the present study was to further work in this direction. We produced porcine parthenogenetic embryos, and separately aggregated two of each of two-cell ($2{\times}2$), four-cell ($2{\times}4$), and eight-cell ($2{\times}8$) embryos derived by parthenogenesis. After culture for 4 days, the developmental ability of the aggregates and total blastocyst cell numbers were evaluated. The percentage of blastocysts was significantly higher in both $2{\times}4$- and $2{\times}8$-aggregated embryos ($58.3{\pm}1.9%$ and $37.2{\pm}2.8%$, respectively) than in the control or $2{\times}2$-aggregated embryos ($23.6{\pm}1.1%$ and $12.5{\pm}2.4%$, respectively). Total blastocyst cell numbers were increased in the $2{\times}4$- and $2{\times}8$-aggregated embryos (by $44{\pm}3.0%$ and $45{\pm}3.3%$, respectively) compared with those of control or $2{\times}2$-aggregated embryos ($30.5{\pm}2.1%$ and $30.7{\pm}2.6%$, respectively; p<0.05). The levels of mRNA encoding Oct-4 were higher in both the $2{\times}4$- and $2{\times}8$-aggregated embryos than in the control. When blastocysts derived from $2{\times}4$- aggregated embryos or intact normal embryos were cultured on mouse embryonic fibroblast feeder cells to obtain porcine stem cells, blastocysts from aggregated embryos formed colonies that were better in shape compared with those derived from intact blastocysts. Together, the data show that aggregation of porcine embryos not only improves blastocyst quality but also serves as an efficient procedure by which porcine embryonic stem cells can become established.

• Journal of Veterinary Clinics
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• v.12 no.1
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• pp.877-886
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• 1995

The recycling nuclear transplantation(NT) technique has the powerful potential of producing a large number of genetically identical embryos and offsprings from one embryo. Multiple generational cloning by this technique utilizes the NT embryo itself as the donor for the next generation of cloning. In this experiment, we have produced the third generational cloned embryos by recycling NT. Further we examined comparatively the electrofusion rate and in vitro developmental potential in the cloned embryos of the first second and third generations. The embryos of 16-cell stage were collected from the mated does by flushing oviducts with Dulberco's phosphate buffered saline containing 10 % fetal calf serum(FCS) at 47 hours after hCG injection. In the first generation NT, the nuclear donor embryos were synchronized in the phase of Gl/S transition of 32-cell stage. The first and second generation NT embryos developed to 16-cell were used as donor nuclei for second and third generation. The recipient cytoplasms were utilized the oocytes collected at 14 hours after hCG injection, following revoming the nucleus and the first polar body by micromanipulation. The separated blastomeres were injected into the enucleated recipient oocytes by micromanipulation and were fused by electrical stimulation. The electrofusion rate was seen to be 78.0, 88.0 and 90.3 % in the first second and third generation NT rabbit embryos, respectively. The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10 % FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$ incubator. The in vitro developmental potential to blastocyst stage was significantly(P<0.05) decreased in the third(7.2 %) generation NT embryos compared to the first(53.1 %) and second(16.1 %) generation NT embryos. Following in vitro development to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The mean blastomere numbers and cell cycle numbers of NT embryos during the culture period were significantly(p<0.05) decreased in the second(93.9 cells and 6.55 cylces) and third(81.5 cells and 1.35 cylces) generation, compared to the first(189.9 cells and 7.55 cylces) generation.

• Reproductive and Developmental Biology
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• v.36 no.4
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• pp.237-241
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• 2012

Embryonic genome activation (EGA) is the first major transition that occurs after fertilization, and entails a dramatic reprogramming of gene expression that is essential for continued development. Although it has been suggested that EGA in porcine embryos starts at the four-cell stage, recent evidence indicates that EGA may commence even earlier; however, the molecular details of EGA remain incompletely understood. The RNA polymerase II of eukaryotes transcribes mRNAs and most small nuclear RNAs. The largest subunit of RNA polymerase II can become phosphorylated in the C-terminal domain. The unphosphorylated form of the RNA polymerase II largest subunit C-terminal domain (IIa) plays a role in initiation of transcription, and the phosphorylated form (IIo) is required for transcriptional elongation and mRNA splicing. In the present study, we explored the nuclear translocation, nuclear localization, and phosphorylation dynamics of the RNA polymerase II C-terminal domain in immature pig oocytes, mature oocytes, two-, four-, and eight-cell embryos, and the morula and blastocyst. To this end, we used antibodies specific for the IIa and IIo forms of RNA polymerase II to stain the proteins. Unphosphorylated RNA polymerase II stained strongly in the nuclei of germinal vesicle oocytes, whereas the phosphorylated form of the enzyme was confined to the chromatin of prophase I oocytes. After fertilization, both unphosphorylated and phosphorylated RNA polymerase II began to accumulate in the nuclei of early stage one-cell embryos, and this pattern was maintained through to the blastocyst stage. The results suggest that both porcine oocytes and early embryos are transcriptionally competent, and that transcription of embryonic genes during the first three cell cycles parallels expression of phosphorylated RNA polymerase II.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.15-15
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• 2001

We recently demonstrated that satellite regions exhibit an aberrant DNA methylation in cloned bovine embryos. Here, we examined, using bisulfite -sequencing technology, whether the inefficient demethylation of cloned donor genomes could be rescued by the presence of oocytic nuclei. Both AciI digestion and sequencing analyses showed that satellite sequence was demethylated more efficiently in cloned tetraploid blastocysts than in diploid clones. When methyl -CpG density (the number of methyl-CpG sites per string) was scored, a significant decrease was observed In tetraploids (P<0.001). These results suggest that unknown mechanisms provided by oocytic nuclei could assist the demethylation of satellite sequences in tetraploid clones.

• 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.