• Korean Journal of Animal Reproduction
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• v.27 no.4
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• pp.349-357
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• 2003

Although nuclear transfer (NT) techniques are used to clone animals, its efficiency is very low. Moreover, nuclear transfer has resulted in offspring with severe developmental problems, probably due to incomplete nuclear reprogramming. Nuclear reprogramming is characterized by functional modification of the transferred nucleus to allow it to direct normal embryo development with the potential to grow to term. Although the nature of the reprogramming factor(s) in mammals is not clear, various nuclear as well as cytoplasmic components are involved in the processes. In this article we review recent data on factors involved in the nuclear reprogramming of cloned embryos.

• Journal of Embryo Transfer
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• v.9 no.2
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• pp.161-166
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• 1994

This experiment was carried out to produce cloned aniraals by nuclear transplantation in rabbits. The ovulated oocytes were collected from the oviducts between 14 and 15 hours after hGG injection. The denuded oocytes were used as nuclear recipient cytoplasm following enucleation by micromanipulation. The blastomeres separated from the 8-cell embryos were used as nuclear donor. The nucleated oocytes receiving a blastomere in the perivitelline space were electrically fused in the 0.28 M mannitol solution at 1.5 kV /cm, 60$\mu$sec for three times. The nuclear transplant embryos which were used and developed to 2- to 4-cell stage in vitro were transferred into the oviducts of synchronized recipient does. A total of 64 nuclear transplant embryos were transferred to 7 recipient does and produced three offspring(4.7%) from a foster mother 31 days after embryo transfer.

• Reproductive and Developmental Biology
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• v.28 no.1
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• pp.1-6
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• 2004

This study was conducted to investigate the effects of donor cell type, individual, passage number and trypsinization time on the in vitro development of bovine somatic cell nuclear transfer embryos. Three cell types (skin, muscle and cumulus cells) and cells from 3 individuals were used for nuclear transfer. Cell were passaged by 5, 15 or 30 times, and cell were trypsinized for 1 or 3 min before injection. Nuclear transfer were performed by conventional fusion method. Development rates to the blastocyst stage were not significantly different among three cell types (16.5∼23.9%) and individuals (16.4∼19.5%). Blastocyst formation rate of cloned embryos reconstituted with cells at passage 30 (5.8%) was significantly lower than those of embryos reconstituted with 5- and 15-passaged cells (25.3 and 23.5%, respectively, P＜0.05). The rate of embryos developed to the blastocyst stage was higher in embryos reconstituted with cells trypsinized for 1 min (30.7%) compared to embryos reconstituted with cells trypsinized for 3 min (P＜0.05). The result of the present study indicates that different donor cell types and individuals used in this study did not affect the development of cloned bovine embryos. However, passage number and trypsinization time of donor cells affect the in vitro development of cloned bovine embryos.

• Journal of Embryo Transfer
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• v.10 no.3
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• pp.209-217
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• 1995

The present study was carried out to develop a cloning technology of mouse embryos by nuclear transplantation with electrofusion and to produce cloned offsprings by transfer of reconstituted embryos. A single nucleus from two- and eight-cell embryos was transplanted into the enucleated two-cell embryos by rnicromanipulation. The fusion of nucleus with recipient cytoplasm and the subsequent development of reconstituted embryos in vitro as well as in vivo to term were examined to determine the optimal electrofusion parameters for nuclear transplantation in mouse embryos. The successful enucleation of donor embryos was 84.9 and 83.3% in two- and eight-cell stage, respectively, and the successful injection of nucleus from two- and eight-cell donor embryos into the perivitelline space of enucleated two-cell embryos were 85.1 and 84.7%, respectively. No significant differences were found in enucleation or injection rate between the cell stages of donor embryos. When the blastomeres of intact two-cell mouse embryos were electrofused in 0.3 M mannitol medium(100 $\mu$sec., 3 pulses), the fusion rate was similarly 93.2, 92.2 and 92.0% in 1.0, 1.5 and 2.0 kV /crn, respectively, but in vitro development to blastocyst of the fused two-cell embryos was significantly(P<0.05) lower in 2.0 kV/cm (63.4%) than in 1.0 kV/cm (91.7%) or 1.5 kV/cm (82.4%). The development in vitro to eight-cell stage of the reconstituted embryos with nucleus from two-cell stage(45.5%) was significantly(P<0.05) higher than that from eight-cell stage blastomeres (16.7%). The number of blastomeres of the intact embryos at blastocyst stage was 50i0.6 and 55$\pm$2.4 in in vitro and in vivo cultured mouse embryos, respectively, but significantly(P<0.05) decreased to 35$\pm$0.7 in nuclear transplanted blastocyst embryos. The conception rate of mice following embryo transfer was 32.1% in the reconstituted two-cell embryos using two-cell donor nuclei, which was comparable to the fresh two-cell embryos(40.6%). However, the rate of development in vivo to term following embryo transfer of the reconstituted two-cell embryos using two-cell donor nuclei (23.5%) was significantly(P<0.05) lower compared with the percentage of two-cell fresh embryos(31.5%).

• Reproductive and Developmental Biology
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• v.30 no.2
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• pp.119-124
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• 2006

The present study compared the developmental potential of cloned porcine embryos with mesenchymal stem cells (MSCs), fetal fibroblasts (FFs) and cumulus cells (CCs) by assessing the cleavage and blastocyst rate, total cell number, inner cell mass (ICM) ratio and apoptosis. MSCs were isolated by ficoll gradients from femur of -6 month old female pig, and maintained for primary cultures. FFs from a female fetus at ${\sim}30$ day of gestation were established, and CCs were obtained from cumulus oocyte complexes (COCs) aspirated from $3{\sim}6$ mm follicles in diameter. Donor cells at $3{\sim}4$ passage were employed for nuclear transfer (NT). COCs were matured and fertilized in vitro(IVF) as control. Cleavage rate was significantly (P<0.05) higher in IVF than in NT embryos with MSCs, FFs and CCs ($82.7{\pm}8.9%\;vs\;70.6{\pm}5.4,\;68.7{\pm}5.1\;and\;63.4{\pm}5.6%$, respectively). However, blastocyst rates in IVF and NT embryos derived from MSCs ($24.5{\pm}2.8\;and\;20.4{\pm}8.3%$) did not differ, but were significantly (P<0.05) higher than NT derived from FFs and CCs ($10.6{\pm}2.7\;and\;9.8{\pm}2.1%$). Total cell number and the ratio of ICM to total cells among blastocysts cloned from MSCs ($35.4{\pm}5.2\;and\;0.40{\pm}0.09%$, respectively) were significantly (P<0.05) higher than those from FFs and CCs ($24.9{\pm}6.2%\;vs\;0.19{\pm}0.16,\;23.6{\pm}5.5\;and\;0.17{\pm}0.16%$, respectively). Proportions of TUNEL positive cells in NT embryos from FFs and CCs ($6.9{\pm}1.5\;and\;7.4{\pm}1.7%$, respectively) were significantly (P<0.05) higher than in MSCs ($4.8{\pm}1.4%$) and IVF ($2.3{\pm}0.9%$). The results demonstrate that MSCs have a greater potential as donor cells than FFs and CCs in achieving enhanced production of cloned porcine embryos.

• Journal of Embryo Transfer
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• v.25 no.2
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• pp.117-125
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• 2010

Several cloned animals have been produced using somatic cell nuclear transfer (SCNT) and have interested in producing the transgenic cloned animals to date. But still its efficiency was low due to a number of reasons, such as sub-optimal culture condition, aberrant gene expression and nuclear reprogramming. The purpose of this study was to analyze gene expression pattern in in vitro fertilized (IVF) or SCNT pre-implantation embryos. IVF- or SCNT-embryos were cultured in media supplemented with different proteins (FBS and BSA) or energy sources (glucose or fructose). Blastocysts from IVF or SCNT were analyzed using semi-quantitative RT-PCR in terms of developmentor metabolic-related genes. Culture medium supplemented different proteins or energy sources had affected on the expression of developmental or metabolic genes in the SCNT blastocysts.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2003.10a
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• pp.92-92
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• 2003

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2004.10a
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• pp.102-102
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• 2004

• 대한생식의학회:학술대회논문집
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• 2006.06a
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• pp.92-94
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• 2006

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2005.10a
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• pp.138-138
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• 2005