• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2005.10a
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• pp.128-128
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• 2005

• Journal of Embryo Transfer
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• v.27 no.4
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• pp.205-209
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• 2012

Transgenic rats and mice are useful experimental animal models for medical research including human disease model studies. Somatic cell nuclear transfer (SCNT) technology is successfully applied in most mammalian species including cattle, sheep, pig and mouse. SCNT is also considered to increase the efficacy of transgenic/knockout mouse and rat production. However, in the area of reproductive biotechnology, the rodent model is inadequate because of technical obstacles in manipulating the oocytes including intracytoplasmic sperm injection and SCNT. In particular, success of rat SCNT is very limited so far. In this review, the history of rodent cloning is described.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.2
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• pp.168-173
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• 2004

The ability of frozen-thawed fetal skin was examined to generate viable cell lines for nuclear transfer. Fetal skin frozen at -20$-20^{\circ}C$, $-30^{\circ}C$ or $-80^{\circ}C$ in the presence of 5% DMSO used as tissue explants to generate somatic cells. The resultant confluent cells were then used as donors for nuclear transfer (NT). Of the bovine NT embryos reconstracted from the somatic cells, 62.3%, 76.6% to 65% showed cleavage 70.5%, 81.9% to 78.5% reached the stage of morula formation and 39.7%, 43.2% or 47.6% reached the blastocyst stage. There was no significant difference in development when the NT embryos were compared with those reconstracted from fresh somatic cell derieved skin tissues (72%, 75.3%, and 45.2%, for cleavage, and development to morula and blastocyst stage, respectively). NT embryos were then placed in a portable $CO_2$ incubator and carried to China from Japan by air. After reaching to farm, two NT embryos were transferred to each of 5 recipients. We obtained 2 NT calves which birth weights is 30kg and 36kg female, and gestation periods is 281 and 284 days, respectively. There were no observation any abnormality from those calves. The results indicated that cell lines derieved from bovine fetal skin cryopreserved by a simple method could be used as donors in nuclear transfer using the portable $CO_2$ incubator.

• Journal of Embryo Transfer
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• v.25 no.4
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• pp.215-219
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• 2010

The objective of this study was to examine the reproductive characteristics of cloned miniature piglets produced from surrogate domestic pigs. Somatic cell nuclear transfer (SCNT) miniature pig embryos were transferred into domestic pigs. As controls, domestic pigs of the same breed with surrogates for SCNT embryos and miniature pigs of the same breed with the somatic cell donor were bred by artificial insemination and natural mating, respectively. Surrogate domestic pigs that farrowed cloned miniature piglets had a significantly longer gestation length (118.1 days) than conventionally bred domestic (115.4 days) and miniature (115.5 days) pigs. Furthermore, the birth weight of cloned miniature piglets produced from domestic pigs (743 g) was significantly greater than that of miniature piglets produced by natural breeding (623 g). Also, cloned miniature piglets had a significantly lower weaning rate (49.7%) than conventionally produced domestic (91.5%) and miniature (100%) piglets. No differences were observed between female and male cloned piglets in gestation length, litter size, birth weight, or weaning rate. Our results demonstrate that gestation length is extended in domestic pigs that are transferred with SCNT miniature pig embryos and that cloned miniature piglets have increased birth weight and high pre-weaning mortality.

• Journal of Embryo Transfer
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• v.17 no.2
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• pp.109-115
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• 2002

The present study was conducted for the production of transgenic cloned cows those secrete human lactoferricin into milk by somatic cell nuclear transfer (NT). To estimate detrimental effects of gene transfection on transgenic cloned embryo production, development rates of NT embryos were compared between transfected and non-transfected cumulus and ear fibroblast cells. An expression plasmid for human lactofericin (pbeta-LFC) was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and human lactoferricin target gene into a pcDNA3 plasmid. Two bovine somatic cell lines (cumulus cell and ear fibroblast) were established and transfected with the expression plasmid using a liposomal transfection reagent, Fugene6 as a carrier. Cumulus cell and ear fibroblast were transfected at the passage of 2 to 4, trypsinized and GFP-expressing cells were randomly selected and used for somatic cell NT. Developmental competences (rates of fusion, cleavage, and blastocyst formation) in bovine transgenic somatic cell NT embryos reconstructed with non-transfectecd cells were significantly higher than those from transfected cells in cumulus cell and ear fibroblast (P＜0.05). This study indicated that transfection of done. cell has detrimental effect on embryo development in bovine transgenic NT.

• Reproductive and Developmental Biology
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• v.28 no.4
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• pp.241-245
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• 2004

This study was conducted to investigate nuclear remodeling and developmental rate following nuclear transfer of fetal fibroblast cells, ear skin cells and oviduct epithelial cells into porcine recipient oocytes. To test par-thenogenetic activation, oocytes were treated with a 6-dimethylaminopurine (6-DMAP), a single DC-pulse (DC), calcium ionomycin (ionomycin), DC+6-DMAP and ionomycin + 6-DMAP after in vitro maturation. For nuclear transfer, in vitro matured oocytes were enucleated, and donor cells were transferred into oocytes. Cloned embryos were fused and stimulated with 6-DMAP for 4 h and cultured in vitro for 6 days. Among treatments for parthenogenesis, the activation rate of DC +6-DMAP treatment was significantly higher than that of single treatment roups (p<0.01), except for DC treatment group. However, the difference was not significant in activation rate compared to other complex treatment groups. Nuclear swelling of the cloned embryos was initiated at 60 min after stimulation and increased afterwards. Fusion rates were not different among different donor cells. Cleavage rates of DC treatment groups were significantly higher than those of DC+6-DMAP treatment groups (p<0.05) in case that fetal fibroblast and ear cells were used for nuclear donor. The cloned embryos from developed to blastocysts in oviduct epithelial cell nuclear transfer with DC+6-DMAP treatment was significantly higher compared to those with DC only treatment (p<0.05). However, no blastocyst was developed from nuclear transfer of fetal fibroblast and ear cells regardless of activation treatments. Based on these results, a proper activation stimulation may be necessary to increase the activation rate and the development to blastocyst in cloned porcine embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.5
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• pp.648-656
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• 2008

The objective of this study was to establish conditions for transfection of a foreign gene into somatic cells using cationic lipid reagents and to evaluate the effects of transfection on in vitro development of somatic cell nuclear transfer (SCNT) embryos. Green fluorescent protein (GFP) gene was used as a foreign gene and a non-transfected somatic cell was utilized as a control karyoplast. Monolayers of porcine cells were established and subsequently transfected with a GFP-expressing gene (pEGFP-N1) using three types of transfection reagents (LipofectAMINE PLUS, FuGENE 6 or ExGen500). Donor cells used for SCNT included transfected fetal or adult fibroblasts and oviduct epithelial cells, either serum-fed or serum-starved. Oocytes matured in vitro for 42 h were reconstructed with either transfected or non-transfected porcine somatic cells by electric fusion and activation using a single DC pulse of 1.8 kV/cm for $30{\mu}s$ in $Ca^{2+}$ and $Mg^{2+}-containing$ 0.26 M mannitol solution. Reconstructed oocytes were subsequently cultured in NCSU-23 medium for 168 h and the developmental competence and cell number in blastocyst were compared. There were no significant differences (P>0.05) in fusion, cleavage rates or development to the blastocyst stage between non-transfected, transfected, serum-fed and serum-starved cells. However, the rates of GFP-expressing blastocysts were higher in the FuGENE 6 group (71.4%) among transfection reagents and in the fetal fibroblasts group (70.4%) for donor cells. These results indicate that fetal fibroblasts transfected with FuGENE 6 can be used as donor cells for porcine SCNT and that GFP gene can be safely used as a marker of foreign genes in porcine transgenesis.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2005.10a
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• pp.84-85
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• 2005

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2005.10a
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• pp.113-113
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• 2005

• Development and Reproduction
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• v.19 no.2
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• pp.79-84
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• 2015

The Korean native pig (KNP) have been considered as animal models for animal biotechnology research because of their relatively small body size and their presumably highly inbred status due to the closed breeding program. However, little is reported about the use of KNP for animal biotechnology researches. This study was performed to establish the somatic cell nuclear transfer (SCNT) protocol for the production of swine leukocyte antigens (SLA) homotype-defined SCNT KNP. The ear fibroblast cells originated from KNP were cultured and used as donor cell. After thawing, the donor cells were cultured for 1 hour with 15 ${\mu}M$ roscovitine prior to the nuclear transfer. The numbers of reconstructed and parthenogenetic embryos transferred were $98{\pm}35.2$ and $145{\pm}11.2$, respectively. The pregnancy and delivery rate were 3/5 (60%) and 2/5 (40%). One healthy SLA homotype-defined SCNT KNP was successfully generated. The recipient-based individual cloning efficiency ranged from 0.65 to 1.08%. Taken together, it can be postulated that the methodological establishment of the production of SLA homotype-defined cloned KNP can be applied to the generation of transgenic cloned KNP as model animals for human disease and xenotransplantation researches.