• Journal of Embryo Transfer
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• v.19 no.1
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• pp.43-51
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• 2004

The present study was conducted to investigate the effects of fusion and/or activation protocol on in vitro development of porcine somatic cell nuclear transfer (SCNT) embryos. Porcine fetal fibroblast cells were transferred into the perivitelline space of enucleated in vitro matured oocytes. Cell fusion and activation were induced simultaneous fusion/activation (SA) or delayed activation (DA) with or without cytochalasin B (CB) treatment with electic pulses in 0.28 M mannitol-based medium. The SCNT embryos were cultured in vitro for 7 days and stained with Hoechst 33342 to determine the number of nuclei. After 7 days culture, cleavage and blastocyst formation rates were 72.4% and 7.6% in SCNT and 76.3% and 20.4% in parthenotes. To examine the effect of electric field strengths on development of SCNT embryos, oocytes were fused two pulses of 110 V/mm, 130 V/mm or 150 V/mm for 30 sec post-injection. The fusion and cleavage rates in 130 V/mm group (70.2% and 72.6%) and 150 V/mm group (72.6% and 70.5%) were higher (P<0.05) than 110 V/mm group (47.1% and 48.6%), respectively. However, the rate of embryos developing to the blastocyst stage (8.1%, 9.7% and 10.7%) were not different among three groups. The cleavage rates and the blastcyst formation rates were not different among three treatment groups (SA group, 71.4% and 9.7%; SA+CB treatment group, 74.7% and 8.0%; DA+CB treatment group, 70.8% and 11.2%, respectively). And, no different in the number of cells in blastocysts was observed among the three groups (22.5$\pm$12.8, 23.3$\pm$11.2 and 21.6$\pm$10.4, respectively). These result suggest that two pulses of 130 V/mm or 150 V/mm for 30 sec with SA treatment or DA treatment are enough for fusion/activation of porcine somatic cell nuclear transfer (SCNT) embryos to develop to the blastocyst stage.

• Journal of Embryo Transfer
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• v.21 no.2
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• pp.137-146
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• 2006

The objective of this study was to examine the effect of donor cell types, the source of recipient oocytes and estrous synchronization on pregnancy and delivery rates of somatic cell nuclear transfer (SCNT) embryos in Korean native goats. Recipient oocytes were surgically collected after superovulation. Ear cells and fetal fibroblasts were collected and cultured in serum-starvation condition (TCM-199 + 0.5% FBS) for cell confluence. The zonae pellucidae of in vivo- and in vitro-matured oocytes were partially drilled using a laser system. Single somatic cell was transferred into the enucleated oocyte. The reconstructed oocytes were electrically fused with 0.3 M mannitol. After the fusion, embryos were activated by Ionomycin+6-DMAP. NT embryos were cultured in mSOF medium supplemented with 0.8% BSA at $39^{\circ}C$ in an atmosphere of 5% $CO_2$, 5% $O_2$, 90% $N_2$ for 12 to 20 hr. One hundred and two SCNT embryos were transferred into 20 recipients and pregnancy rate at days 30 was 20.0%. Of them, one developed to term and delivered 1 kid. Ear cells showed significantly higher fusion (63.8 vs. 26.5%) and pregnancy rates (20.0 vs. 0.0%) than those of fetal fibroblast (p<0.05). The recipients synchronized by CIDR showed significantly lower pregnancy rates compared to that of recipient in natural estrus ($0.0{\sim}25.0%$ vs. 100%) (p<0.05). Cloned kid was born from the recipient in natural estrus. For the synchronization of estrus between recipient and donor, there was no difference between treatments (${\pm}0$ vs. +12 hr) in pregnancy rate. The first healthy cloned kid (Jinsoonny) was produced by transfer of SCNT embryos derived from in vivo oocytes and ear cells into a recipient goat whose estrus was synchronized with the donor. These results imply that donor cells for nuclear transfer may affect the success rate, and the estrus synchronization between donor and recipient animals can also be important.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.281-281
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• 2004

최근 체외수정, 핵이식, 체세포 복제동물의 생산 등으로 수정란의 동결 보존기술의 개발이 매우 중요한 시기에 왔다. 본 실험에서는 한우의 체리수정란을 완전 무혈청 배양액으로 배양하여 수정란의 배발생율과 동결융해 한 후 생존성 및 수정란이식 한 후 수태율을 조사하였다. (중략)

• Journal of Embryo Transfer
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• v.18 no.1
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• pp.81-84
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• 2003

This study was performed to produce Hanwoo-fetal fibroblast cell clone transferred to Holstein recipient. It was coming on the parturition but didn't show parturition signs until 272 days from the embryo transfer. For the induction of normal parturition, dexamethasone (20 mg) were injected with IM on 272 day and PG $F_{2a}$ (25 mg) plus estradiol (20 mg) was injected with IM after 24hrs. Forty-eight hours after dexamethasone injection, we could find parturition signs and delivered healthy male of offspring (40 kg) about 50 hrs after it's injection. However the recipient was showed retention of afterbirth.h.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.19-19
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• 2003

소 난자 체외성숙에 있어 소 태아혈청 (fetal bovine serum: FBS)첨가시 높은 성숙율을 보이며 핵 이식 후 배 발달율에 있어서도 높다. 하지만 소 태아혈청은 hormone, growth factor, vitamine 그리고 다수의 어떤 잘 알려지지 않은 인자를 포함한 복잡한 배지로 알려져 있으며, 수정란 이식 후 태아의 발육중 및 태어난 직후에 발생되는 송아지에서 나타나는 몇몇 비정상적인 현상들의 원인인 것으로 보고되고 있다. 본 실험은 체외 배양시 소 태아혈청과 이의 대체물질로서 BSA 또는 PVA가 첨가된 배양조건에서의 복제 수정란의 배 발달율을 비교함과 동시에 배반포의 세포수 그리고 세포자연사 (Apoptosis)를 각각의 조건에서 비교함으로써 배발달에 미치는 효과를 알아보기 위하여 실시하였다. 핵이식은 소 체세포를 이용하였으며, 핵 이식 후 CR1aa 기본 배양액으로 FBS, BSA, 그리고 PVA를 첨가하여 5% $CO_2$, 5% $O_2$, 39$^{\circ}C$ 조건하에서 7~8일간 배양하였다.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2003.10a
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• pp.90-90
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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.87-87
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• 2004

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.135-135
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• 2003

본 연구는 체세포 핵이식 복제 수정란의 이식에 의한 고능력 한우를 다량 증식하기 위한 방안을 확립하기 위하여 수행되었다. 본 실험에 공여된 체세포는 육질과 육량 등급이 국내에서 100위 이내의 암소 귀세포를 채취하여 동결 및 계대배양하여 사용하였다. 한편, 핵이식 수정란의 준비를 위하여 도축장에서 채취한 난소에서 난자를 회수하여 22시간 성숙배양 후 난구세포를 제거하고 극체가 존재하는 난자만을 선별하여 recipient cytoplasm으로 이용하였다. 난자의 제핵, 체세포 핵이식, 전기융합 및 활성화 처리는 본 실험실의 방법에 준하여 실시하였으며, 핵이식란은 CR1aa 배양액 내에서 5% $CO_2$, 95% Air 및 39$^{\circ}C$의 기상조건하에서 7일간 배양 후 이식에 이용되었다. 한편, 수란축은 2회 이상 정상 발정주기가 확인된 경산우와 미경산우에 25mg의 PG $F_{2}$$\alpha$/를 투여하여 발정을 유기하거나 자연발정우를 선발하여 수란축으로 이용하였다. 그 결과, 배반포기배를 이식한 경우 14두중 5두에서 임신이 확인되었으며 그중 4두에서 유산되었고, 1두는 임신 6개월령으로 정상 발육되고 있는 것이 확인되었지만 상실배기단계에서 이식된 경우는 임신이 되지 않았다. (중략)

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.55-55
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• 2002

본 연구는 체세포 복제 가축의 정상 분만율이 낮은 원인을 규명하고, 복제란 이식우의 분만전후 생리적인 특성을 검토하기 위하여 혈중 또는 태반내 TGF-β1 과 progesterone 농도를 분석하여 검토하였다. 축산기술연구소에서 자체 생산된 체세포 복제란을 자연 발정이 유기된 수란우에 이식하였으며, 분만 예정일 하루 전에 제왕절개수술을 실시하여 태반 및 혈액을 채취하여 실험에 공시하였으며, 대조구의 시료는 분만직전에 제왕절개수술을 실시하여 공시하였다. (중략)

• Korean Journal of Veterinary Research
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• v.30 no.4
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• pp.355-360
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• 1990

Single nuclei from two-, four- and eight-cell mouse embryos were transplanted into enucleated two-cell embryos by micromanipulation and Sendai virus mediated fusion. The developmental potential of these reconstituted embryos in vitro and in vivo was examined. It was found that the single nuclei which were transplanted to enucleated two-cell embryos were not only able to develop to the blastocyst stage in vitro(two-cell nuclei, 76.5%; four-cell nuclei, 68.4%; eight-cell nuclei, 48.3%), but also able to develop to full term in vivo after transfer to recipient mice(two-cell nuclei, 37.1%; four-cell nuclei, 29.6%; eight-cell nuclei, 16.3%). Although the proportion of live young produced after transfer of nucler of nuclear transplant embryos which received eight-cell nuclei was significantly (p<0.05) reduced, it would be suggested that the overall efficiency in producing identical offspring is greater when eight-cell embryos were selected for nuclear donor than two- or four-cell embryos were selected.