• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.63-63
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• 2001

본 실험은 소에서 체외수정란과 복제수정란을 modified CRlaa (mCRlaa) medium에서 각각 배양하였을 때 배반포단계까지 발육율을 비교하였다. 체외성숙은 TCM-199 medium에 10 %의 Fetal Bovine Serum (FBS)를 첨가한 배양액에서 실시하였고 이를 이용해 체외수정과 복제를 시행하였다. 체외성숙란과 복제수정란의 배반포까지 발육율은 각각 22.5 %, 19.4 %이었다. 복제수정란을 생산하기 위해서는 제핵을 실시한 난자내로 공여핵를 도입하는 과정이 필요한 데 할구보다 체세포를 이용하는 경우 융합율이 저하되는 것으로 보고되고 있다. 따라서 본 연구에서는 공여핵을 제공하는 체세포와 수핵란간에 융합을 기존에 많이 사용한 chamber와 비교해 Needle을 사용했을 때 복제수정란의 발육율간의 차이를 서로 비교하였다. Ear skin cell을 이용해 핵이식을 실시한 다음 Zimmerman cell fusion medium에서 융합하였다 융합된 핵이식란은 5 % $CO_2$, 5 % $O_2$, 90 % Na, 38.5$^{\circ}C$ 조건에서 배반포까지 배양하였다. Chamber와 Needle을 사용한 경우 융합율은 각각 46.1 %, 70.4 %, 분할율은 62.6 %, 76.4 %로 Needle의 경우가 유의적으로 높았다.

• 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.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.235-243
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• 2002

This study was designed to examine the ability of the bovine (MII) oocytes cytoplasm to support several mitotic cell cycles under the direction of differentiated somatic cell nuclei of bovine, porcine, mouse and human. Bovine GV oocytes were matured in TCM-199 supplemented with 10% FBS. At 20h after IVM, recipient oocytes were stained with 5 $\mu\textrm{g}$/$m\ell$ Hoechst and their 1st polar body (PB) and MII plate were removed by enucleation micropipette under UV filter. Ear skin samples were obtained by biopsy from an adult bovine, porcine, mouse and human and cultured in 10% FBS added DMEM. Individual fibroblast was anlaysed chromosome number to confirm the specificity of species. Nuclear transferred (NT) units were produced by electrofusion of enucleated bovine oocytes with individual fibroblast. The reconstructed embryos were activated in 5 $\mu$M ionomycin for 5 min followed by 1.9 mM 6-dimethylaminopurine (DMAP) in CR1aa for 3 h. And cleaved NT embryos were cultured in CR1aa medium containing 10% FBS on monolayer of bovine cumulus cell for 8 days. Also NT embryo of 4~8 cell stage was analysed chromosome number to confirm the origin of nuclear transferred somatic cell. The rates of fusion between bovine recipient oocytes and bovine, porcine, mouse and human somatic cells were 70.2%, 70.2%, 72.4% and 63.0%, respectively. Also, their cleavage rates were 60.6%, 63.7%, 54.1% and 62.7%, respectively, there were no differences among them. in vitro development rates into morula and blastocyst were 17.5% and 4.3% in NT embryos from bovine and human fibroblasts, respectively. But NT embryos from porcine and mouse fibroblasts were blocked at 16~32-cell stage. The chromosome number in NT embryos from individual fibroblast was the same as chromosome number of individual species. These results show that bovine MII oocytes cytoplasm has the ability to support several mitotic cell cycles directed by newly introduced nuclear DNA.

• 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

• Korean Journal of Animal Reproduction
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• v.25 no.1
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• pp.51-61
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• 2001

This study was conducted to determine developmental ability of reconstructed embryos by nuclear transfer using somatic cell of Korean bull with high genetic value. Fibroblast cells obtained from ear biopsy of the bull were cultured in Dulbecco's Modified Eagle's medium (DMEM) at 37$^{\circ}C$ in air containing 5% $CO_2$. The cummulus-oocyte complexes were collected from slaughterhouse and were matured in vitro for 20 h in TCM 199 culture medium and the oocytes were then enucleated in modified phosphate buffered saline with cytochalasin B. Matured bovine oocytes were enucleated by aspirating the first polar body and metaphase chromatin using a beveled pipette in modified phosphate buffered saline. The ear fibroblast cells were fused into enucleated oocyte by electrical stimulation. The reconstructed oocytes were activated with ionomycine and 6-dimethylaminopurine, and then cultured in CR1aa medium for 7.5 days. Out of 524 bovine eggs reconstructed by nuclear transfer 65.6%(277/422) embryos were cleaved, and 30.7% (85/277) cleaved embryos were developed to the morula to blastocysts. There was no difference of developmental ability in vitro of reconstructed embryos regardless of donor cell passages. In order to determine fate of foreign mitochondria of donor nucleus, the Mito Tracker stained cells were fused into enucleated oocytes. The donor mitochondria were detected early stage of embryos, but disappeared rapidly. The developmental ability of reconstructed embryos was not impaired by Mito Tracker treatments. The results indicate that viable reconstructed embryos can be producted by nuclear transfer using somatic cell of Korean bulls.bulls.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.238-238
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• 2004

세포 내에서 발현되고 있는 protein들의 양상을 분석하기 위한 기법으로 최근 proteomics에 기초하여 2차 전기영동과 MALDI-TOF MS에 의한 protein 분석방법이 개발되었는데, 특정 조직에 또는 특정 발생시기에 특이적으로 발현되는 protein의 발현양과 발현양상을 비교ㆍ분석하는데 매우 효과적으로 이용될 수 있다. 최근 체세포 핵이 식기술을 이용하여 동물의 복제가 성공하고 있지만, 임신 중이나 분만시 유사산이 많이 나타나 전반적인 효율이 크게 낮아 실용화에 지장을 초래하고 있다. (중략)

• Journal of Animal Science and Technology
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• v.50 no.5
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• pp.641-648
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• 2008

There has been great success for making transgenic animals using somatic cell nuclear transfer(SCNT) up to this time. However, the success rates of the production of live transgenic animals are still very low. The current research has been carried out for delineation of differentially expressed genes between SCNT and normal placenta in cattle. In the present observations, high expression has been observed for CTSZ, LOC509426 and ELF1 genes in normal placenta. On the other hand, TIMP2, PAG1B, PAG-21, LOC782894, SERPINB6 and mKIAA2025 protein were highly expressed in SCNT placenta. Five genes, which were highly expressed in SCNT placenta, have been further investigated using semi-quantitative real-time PCR. The results were similar to that we observed using ACP. In the future, all genes affecting the SCNT and normal placenta have to be discovered and their networks will be fully investigated. The genes were identified in this study would be great help for identifying differential gene expressions in SCNT placenta.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.131-138
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• 2017

In most mammals, metaphase II (MII) oocytes having high maturation promoting factor (MPF) activity have been considered as good oocytes and then used for assisted reproductive technologies including somatic cell nuclear transfer (SCNT). Caffeine increases MPF activity in mammalian oocytes by inhibiting p34cdc2 phosphorylation. The objective of this study was to investigate the effects of caffeine treatment during in vitro maturation (IVM) on oocyte maturation and embryonic development after SCNT in pigs. To this end, morphologically good (MGCOCs) and poor oocytes (MPCOCs) based on the thickness of cumulus cell layer were untreated or treated with 2.5 mM caffeine during 22-42, 34-42, or 38-42 h of IVM according to the experimental design. Caffeine treatment for 20 h during 22-42 h of IVM significantly inhibited nuclear maturation compared to no treatment. Blastocyst formation of SCNT embryos was not influenced by the caffeine treatment during 38-42 h of IVM in MGCOCs (41.1-42.1%) but was significantly improved in MPCOCs compared to no treatment (43.4 vs. 30.1%, P<0.05). No significant effects of caffeine treatment was observed in embryo cleavage (78.7-88.0%) and mean cell number in blastocyst (38.7-43.5 cells). The MPF activity of MII oocytes in terms of p34cdc2 kinase activity was not influenced by the caffeine treatment in MGCOCs (160.4 vs. 194.3 pg/ml) but significantly increased in MPCOCs (133.9 vs. 204.8 pg/ml). Our results demonstrate that caffeine treatment during 38-42 h of IVM improves developmental competence of SCNT embryos derived from MPCOCs by influencing cytoplasmic maturation including increased MPF activity in IVM oocytes in pigs.

• Reproductive and Developmental Biology
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• v.30 no.3
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• pp.175-180
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• 2006

This study was conducted to examine the effect of pre activation treatment and activation time of recipient cytoplasm on the development of bovine somatic cell nuclear transfer(NT) embryos. Donor cells were transferred and electrofused to enucleated oocytes before(pre-AC) or after activation(post-AC). Activation was induced with a combination of $Ca^{2+}$-ionophore(A23187) and DMAP. NT embryos were cultured in CR1aa containing 3 mg/ml BSA for 9 days. Some NT embryos were fixed at 0.5 to 2.5 hr after fusion(for post-AC) or activation(for pre-AC) for confocal microscopy. Developmental rate to the blastocyst stage was slightly high in the post-AC group(20.6%) compared to that of pre-AC group(15.3%). However, developmental speed of embryos in the pre-AC group was faster than that of embryos in the post-AC group. Development rates to the blastocyst stage were similar among different activation time before fusion(0.5,2 and 4 hr). The result of the present study suggests that development and nuclear morphology are affected f the activation status of the recipient cytoplasm before fusion.

• The Science & Technology
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• v.32 no.4 s.359
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• pp.16-17
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• 1999

세계에서 5번째로 체세포로 동물 복제에 성공, 젖소 영롱이를 탄생시킨 서울대 수의과대 황우석교수는 "어려운 여건 속에서도 연구가 결실을 거둠으로써 축산농민들에게 희망을 안겨주게되어 보람을 느낀다"고 말했다. 황교수는 "기존 복제술에다 핵이식 전 세포의 전염병과 기형검사를 추가해서 유산을 미리 막고 성공률도 높였다"고 자신의 복제방법을 설명했다.