• Asian-Australasian Journal of Animal Sciences
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• v.18 no.9
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• pp.1242-1248
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• 2005

This study was designed to investigate the in vitro developmental ability and apoptosis of bovine embryos nucleartransferred (NT) with frozen-thawed or cooled donor cells. Cultured adult bovine ear cells were used as donor cells after sub-culturing to confluence (CC), cooling to 4$^{\circ}C$ for 48 h, or freezing-thawing (FT). Apoptotic cells in blastocysts were evaluated for apoptosis by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method. Fusion, cleavage and blastocyst rates were 69.0 (167/242), 68.8 (115/167), and 29.9 (50/167) with CC cells, 70.4 (88/125), 69.3 (61/88), and 29.6 (26/88) with cooled cells and 66.1 (117/177), 70.1 (82/117), and 13.7 (16/117) with FT cells, respectively. Blastocyst rates of NT embryos derived from FT cells were significantly lower than those from CC or cooled cells (p<0.05). In addition, NT blastocysts produced by using FT cells showed significantly higher apoptosis rates (6.4${\pm}$4.0%) than those produced by CC (2.8${\pm}$1.7%) or cooled (2.3${\pm}$1.3%) cells. However, cooling of donor cells had no significant adverse effect on blastocyst rate as well as apoptosis rate. Therefore, our results suggest that cooled cells may be used as an alternative to freshly cultured confluent culture cells, as donor cells, for the production of Somatic nuclear cloned cattle.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.7-8
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• 2003

Since it was first reported in 1997, somatic cell cloning has been demonstrated in several other mammalian species. On the mouse, it can be cloned from embryonic stem (ES) cells, fetus-derived cells, and adult-derived cells, both male and female. While cloning efficiencies range from 0 to 20%, rates of just 1-2% are typical (i.e. one or two live offspring per one hundred initial embryos). Recently, abnormalities in mice cloned from somatic cells have been reported, such as abnormal gene expression in embryo (Boiani et al., 2001, Bortvin et al., 2003), abnormal placenta (Wakayama and Yanagimachi 1999), obesity (Tamashiro et ai, 2000, 2002) or early death (Ogonuki et al., 2002). Such abnormalities notwithstanding, success in generating cloned offspring has opened new avenues of investigation and provides a valuable tool that basic research scientists have employed to study complex processes such as genomic reprogramming, imprinting and embryonic development. On the other hand, mouse ES cell lines can also be generated from adult somatic cells via nuclear transfer. These 'ntES cells' are capable of differentiation into an extensive variety of cell types in vitro, as well assperm and oocytes in vivo. Interestingly, the establish rate of ntES cell line from cloned blastocyst is much higher than the success rate of cloned mouse. It is also possible to make cloned mice from ntES cell nuclei as donor, but this serial nuclear transfer method could not improved the cloning efficiency. Might be ntES cell has both character between ES cell and somatic cell. A number of potential agricultural and clinical applications are also are being explored, including the reproductive cloning of farm animals and therapeutic cloning for human cell, tissue, and organ replacement. This talk seeks to describe both the relationship between nucleus donor cell type and cloning success rate, and methods for establishing ntES cell lines. (중략)

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.75-75
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• 2002

The present study was conducted for the production of transgenic cloned cows by somatic cell nuclear transfer (SCNT) that secrete human prourokinase into milk. To establish an efficient production system for bovine transgenic SCNT embryos, the offset was examined of various conditions of donor cells including cell type, size, and passage number on the developmental competence of transgenic SCNT embryos. An expression plasmid far human prourokinase (pbeta-ProU) was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and a human prourokinase target gene into a pcDNA3 plasmid. Three types of bovine somatic cells including two adult cells (cumulus cells and ear fibroblasts) and fetal fibroblasts were prepared and transfected using a lipid-meidated method. In Experiment 1, developmental competence and rates of GFP expression in bovine transgenic SCNT embryos reconstructed with cumulus cells were significantly higher than those from fetal and ear fibroblasts. In Experiment 2, the effect of cellular senescence in early (2 to 4) and late (8 to 12) passages was investigated. No significant differences in the development of transgenic SCNT embryos were observed. In Experient 3, different sizes of GFP-expressing transfected cumulus cells [large (>30 ${\mu}{\textrm}{m}$) or small cell (<30 ${\mu}{\textrm}{m}$)] were used for SCNT. A significant improvement in embryo development and GFP expression was observed when small cumulus cells were used for SCNT. Taken together, these results demonstrate that (1) adult somatic cells could serve as donor cells in transgenic SCNT embryo production and cumulus cells with small size at early passage were the optimal cell type, and (2) transgenic SCNT embryos derived from adult somatic cells have embryonic development potential.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.140-140
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• 2003

The study evaluated the effect of donor cell treatments for G0/Gl synchronization and the donor ceil type on development and incidence of apoptosis in cloned cattle embryos. Primary cultures were established from a female fetus on day 50 of gestation and adult ear skin biopsies. Cells were randomly allocated into 3 experimental treatment groups after 6~8 passages. Group 1 (Confluent), cells were cultured in DMEM supplemented with 10% FBS until 90% confluent. Group 2 (Serum-starvation), cells were cultured in DMEM Supplemented With 0.5% FBS for 5 days. Group 3 (Roscovitine), Cells were cultured in DMEM supplemented with 10% FBS and 30 $\mu$M Roscovitine for 12 h. Cell cycle and apoptosis were analyzed using flow cytometry after labelling with DAPI and YO-PRO-1. At 19 h post-maturation (hpm), enucleated oocytes were reconstructed with donor cells and fused by a single DC pulse (1.6 kV/cm, 60 $\mu$sec). (중략)

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.245-254
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• 2018

Pigs are considered as optimal donor animal for the successful xenotransplantation. To increase the possibility of clinical application, genetic modification to increase compatibility with human is an important and essential process. Genetic modification technique has been developed and improved to produce genetically modified pigs rapidly. CRISPR/Cas9 system is widely used in various fields including the production of transgenic animals and also can be enable multiple gene modifications. In this study, we developed new gene targeting vector and enrichment system for the rapid and efficient selection of genetically modified cells. We conducted co-transfection with two targeting vectors for simultaneous inactivation of two genes and enrichment of the genetically modified cells using MACS. After this efficient enrichment, genotypic analysis of each colony showed that colonies which have genetic modifications on both genes were confirmed with high efficiency. Somatic cell nuclear transfer was conducted with established donor cells and genetically modified pigs were successfully produced. Genotypic and phenotypic analysis of generated pigs showed identical genotypes with donor cells and no surface expression of ${\alpha}$-Gal and HD antigens. Furthermore, functional analysis using pooled human serum revealed dramatically reduction of human natural antibody (IgG and IgM) binding level and natural antibody-mediated cytotoxicity. In conclusion, the constructed vector and enrichment system using MACS used in this study is efficient and useful to generate genetically modified donor cells with multiple genetic alterations and lead to an efficient production of genetically modified pigs.

• Journal of Embryo Transfer
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• v.26 no.1
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• pp.85-89
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• 2011

Somatic cell nuclear transfer (SCNT) is a useful tool for reproducing genetically identical animals or producing transgenic animals. Many reports have demonstrated that the efficiency of animal cloning by SCNT requires reprogramming of the somatic nucleus to a totipotent like-state. The SCNT-related reprogramming might mimic the natural reprogramming process that occurs during normal mammalian development. However, recent evidence indicates that the reprogramming event by SCNT is incomplete. In this study, the traditional SCNT procedure (TNT) was modified by injecting donor nuclei into recipient cytoplasm prior to the enucleation process to expose the donor nucleus before removing the karyoplast containing the chromosomes of the oocytes which might possess additional reprogramming factors, and this modified technique was named as reversing the usual order of SCNT (RONT). Other procedures including activation and in vitro culture were the same as TNT. Contrary to expectations, the rate of blastocyst development was not different significantly between RONT and TNT (8.6% and 7.9%, respectively). However, duration of micromanipulation performed by the same technician and equipments was remarkably reduced because the ruptured oocytes after nuclear injection were excluded from the enucleation process. This study suggests that RONT, a simplified SCNT protocol, shortens the duration of SCNT procedure and this less time-costing protocol may enable the researchers to perform murine SCNT easier.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.67-75
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• 2011

The faulty regulation of imprinting gene lead to the abnormal development of reconstructed embryo after nuclear transfer. However, the correlation between the imprinting status of donor cell and preimplantation stage of embryo development is not yet clear. In this study, to determine this correlation, we used the porcine spermatogonial stem cell (pSSC) and fetal fibroblast (pFF) as donor cells. As the results, the isolated cells with laminin matrix selection strongly expressed the GFR ${\alpha}$-1 and PLZF genes of SSCs specific markers. The pSSCs were maintained to 12 passages and positive for the pluripotent marker including OCT4, SSEA1 and NANOG. The methylation analysis of H19 DMR of pSSCs revealed that the zinc finger protein binding sites CTCF3 of H19 DMRs displayed an androgenic imprinting pattern (92.7%). Also, to investigate the reprogramming potential of pSSCs as donor cell, we compared the development rate and methylation status of H19 gene between the reconstructed embryos from pFF and pSSC. This result showed no significant differences of the development rate between the pFFs ($11.2{\pm}0.8%$) and SSCs ($13.3{\pm}1.1%$). However, interestingly, while the CTCF3 methylation status of pFF-NT blastocyst was decreased (36.3%), and the CTCF3 methylation status of pSSC-NT blastocyst was maintained. Therefore, this result suggested that the genomic imprinting status of pSSCs is more effective than that of normal somatic cells for the normal development because the maintenance of imprinting pattern is very important in early embryo stage.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.218-218
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• 2004

This study was designed to investigate the in vitro developmental ability and apoptosis of embryos nuclear transferred (NT) with frozen-thawed (FT) or cooled donor cells in bovine. Cultured adult bovine ear cells were used as donor cells at confluent condition (CC), after cooling at 4℃ for 48 hour, or after FT. (omitted)

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.217-217
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• 2004

The present study was conducted to investigate the developmental competency between male- and female-somatic cell derived nuclear-transferred porcine embryos, and the productive and survival efficiency of cloned male and female piglets. The potential of eggs receiving somatic cells to develop into blastocysts was not different among donor cells of different origins. (omitted)

• Journal of Embryo Transfer
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• v.12 no.1
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• pp.1-10
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• 1997

The present study was conducted to investigate the influence of embryo cell stage at 18h post-fusion and oocyte preactivation on sebsequent in vitro developmental potential in the nuclear transplant rabbit embryos. The embryos of 16-cell stage were collected and synchronized to G$_1$ phase of 32-cell stage. The recipient cytoplasms were obtained by removing the first polar body and chromosome rnass from the oocytes collected by non-dis-ruptive microsurgery procedure. The separated G$_1$ phase blastomeres of 32-cell stage were injected into non-preactivated recipient cytoplasms. Otherwise, the enucleated recipient cytoplasms were preactivated by electrical stimulation at 18h post-hCG injection and the separated G$_1$ phase blastomeres of 32-cell stage were injected. Mter culture until 20h post-hOG injection, the nuclear transplant oocytes were electrofused by electrical stimulation. The fused nuclear transplant embryos were classified into 3~4-cell, 2-cell and 1-cell stage at 18 hrs post-fusion and cultured until the embryos reached blastocyst stage. The developmental rate to blastocyst stage was significantly (P <0.05) higher in all the reconstituted embryos of 3~4-cell stage(58.0%) than in 2 and icell stage. The developmental rate to blastocyst stage in the embryos of 3~4-cell stage at 18 hrs post-fusion was significantly (P<0.05) higher in the reconstituted without oocyte preactivation(77.8%) than in the oocyte-preactivated embryos (33.3%). These results indicated that the higher rate of in the in vitro development to blastocyst stage might be obtained form the embryos which were reconstituted with nuclear donor of G$_1$ phase and non-preactivated oocyte, and developed more rapidly for 18 hrs post-fusion.