• 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.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.67-67
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• 2001

전기자극은 핵이식 시 수핵난자와 공여핵의 융합을 위해 보편적으로 사용되는 방법이다(Robl, 1999). 그러나 부적절한 전기자극은 수핵난자 세포질에 해를 입히고, 이후의 배발달에 좋지 않은 영향을 준다. 본 실험은 체세포 핵이식을 \circled1 1.9㎸/cm, 20$\mu\textrm{s}$ 2회, \circled22.0㎸/cm, 20$\mu\textrm{s}$ 2회, \circled32.2㎸/cm, 10$\mu\textrm{s}$ 2회, 및 \circled41.9㎸/cm, 30$\mu\textrm{s}$ 2회의 전기자극으로 융합을 실시하여 각 자극 별 융합율과 난자의 lysis율을 비교하고, 배양 후 배반포 발생율을 조사하였다. 공여핵은 칡소의 귀 세포를 10% FBS가 첨가된 DMEM에서 39$^{\circ}C$, 5%$CO_2$의 incubator에서 배양하여 monolayer confluent형성 후 0.5% FBS가 첨가된 DMEM에서 3-4일간 배양 후 trypsin처리하여 제핵된 체외성숙 난자에 핵이식하였다. \circled1,\circled2,\circled3,\circled4의 핵이식 조건을 이용하여 공여핵의 체세포와 수핵란 세포질간의 융합을 유도한 결과, 융합율은 각각 50.7%, 48.1%, 65.5%, 및 33.3%였으며, 수핵난자의 세포질 lysis율은 39.%1, 41.7%, 22.6%,및 52.7%으로 \circled32.2㎸/cm, 10$\mu\textrm{s}$ 2회의 조건에서 융합율이 유의적으로 높았고, 수핵난자의 세포질 lysis율에 있어서도 다른 군에 비하여 낮았다 각각의 핵이식 조건별로 융합한 후 난할율 및 배반포 발생율은 각각 65.7%, 73.5%, 77.2%, 및 53.3%과 47.8%, 52.0%, 49.7%, 및 21.8%로 나타나 난할율 및 배반포 발생율에 있어서 융합조건에 따라 큰 차이는 없었으나 1.9㎸/cm, 30$\mu\textrm{s}$ 2회의 조건이 다른 조건들에 비하여 유의적으로 낮았다. 따라서, 체세포와 수핵란 세포질간의 융합율과 배반포 발생에 미치는 영향은 전압보다는 시간에 더 크게 받음을 알 수 있었으며, 이와 같은 결과에서 융합시 시간을 오래 주는 것보다 전압을 높이는 것이 수핵난자의 세포질에 상해를 줄이고 이후 배반포 발생에 유리할 것으로 사료되었다.

• Journal of Embryo Transfer
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• v.18 no.3
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• pp.171-178
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• 2003

This study was conducted to investigate the effect of electric fusion methods on cell fusion rate and embryo development by somatic cell nuclear transfer in Korean Native Cattle. The KNC ear cell was cultured in vitro for confluence in serum starvation condition(DMEM＋0.05％ FBS) for cell confluence. The zona pellucida of IVM oocytes were partially dissection using micro pipette. Ear cells were transferred into an enucleated oocyte. The reconstructed embryos were electrically fused with Zimmermann Cell Fusion Medium(ZCFM). Nuclear transfer embryos were activated with a combination of 10${\mu}{\textrm}{m}$ calcium ionophore(5 min) and 2.0mM 6-DMAP(3 hr). The activated embryos were cultured in CR1 -aa medium contains 0.3％ BSA or 10％ FBS at 37$^{\circ}C$, 90％ $N_2$, and 5％ $CO_2$in incubator for 6 days. The fusion rates were 51.6％(chamber) and 68.9％(needle), respectively and there were significantly difference between the fusion method(P<0.05). But, lysis rates were not significantly different(10.7％, 11.5％), respectively. The cleavage rates were significantly different between the chamber method(73.2％) and needle method(80.3％), respectively(P<0.05). The rates of early embryos(2∼4cells) and blastocysts of chamber and needle methods were 54.1％, 61.1％ and 18.4％, 26.3％ respectively, and needle method was significantly higher than chamber method(P<0.05). But, morulae formation rate were not significantly differences between the chamber(6.7％) and needle(6.2) method(P <0.05). These result suggest that electric fusion of needle method was to be profitable for nuclear transfer embryo fusion rate, blastocyst formation rate and reduce of oocyte lysis.

• Korean Journal of Animal Reproduction
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• v.26 no.2
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• pp.125-132
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• 2002

This study was conducted to examine the effects of electric stimulation conditions on in vitro developmental ability of procine embryos after somatic cell nuclear transfer, The porcine ear cell was cultured in vifro for confluency in serum-starvation condition (TCM-199＋0.5% FBS) for cell confluency. The zona pellucida of IVM oocytes were partially drilled using laser system. Single somatic cell was individually transferred into the enucleated oocyte. The reconstructed embryos were electrically fused with 0.3M mannitol. After electric fusion, the embryos were activated and cultured in NCSU-23 medium containing 10% FBS at 39$^{\circ}C$, 5% $CO_2$ in air for 6 to 8 days. Nuclear transferred(NT) oocytes which fused at a field strength of 1.90kv/cm showed a higher (P<0.05) fusion rate(49.5%, 50/101) compared to 2.10 kv/cm(25.8%, 24/93) or 2.50kv/cm(30.3%, 27/89). After electric activation, the cleavage rate of NT embryos was 48.0(24/50), 66.6(16/24) and 70.3% (19/27), respectively and these were not different. There was no significant difference in fusion rate by duration and pulse of electric stimulation. In cleavage rate, however, more NT embryos(76.3%, 45/59) cleaved at 60 $\mu$sec twice than other embryos(49.1 to 56.5%) with different conditions of electric stimulation(P<0.05). NT embryos activated at a field strength of 1.50kv/cm showed a higher developmental rate(9.8%, 5/51) than those embryos activated at 1.25kv/cm(0%) or parthenotes(6.4%, 7/109). These results suggest that some factors such as field strength, duration and pulse of electric stimulation could be affected to in vitro developmental ability of nuclear transplanted porcine embryos.

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.33-39
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• 2002

This study was conducted to investigate the effect of $Ca^{2+}$ concentration in fusion medium on the fusion, nuclear morphology and the development of bovine somatic cell nuclear transfer embryos. Bovine skin cells were transferred into an enucleated oocyte and fused with cytoplasm in the fusion medium containing with 0.05 to 1.0 mM Cacl$_2$. Nuclear transfer embryos were activated with a combination of A23187 and cycloheximide. Nuclear transfer embryos were fixed at 3 h after fusion or cultured for 7 ~8 days. Fusion rate was significantly (P＜0.01) increased by increasing the $Ca^{2+}$ concentrations in the fusion medium from 0.05 mM (56.6%) to 0.5 mM (50.1%) and 1.0 mM (84.3%). More than 80% of reconstituted embryos underwent premature chromosome condensation (PCC) with 0.05, 0.1 mM CaCl$_2$, whereas 54.5% and 59.3% of embryos formed pronucleus (PN) directly without PCC in the 0.5 and 1.0 mM CaCl$_2$, groups. Blastocyst formation rates were significantly (P＜0.05) different between 0.1 mM and 1.0 mM CaCl$_2$groups. From the present result, it is suggested that the elevated $Ca^{2+}$ concentrations in fusion medium can enhance the fusion and blastocyst formation rates of bovine nuclear transfer embryos.bryos.

• Korean Journal of Animal Reproduction
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• v.24 no.4
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• pp.365-373
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• 2000

The purpose of this study was to investigate the effects of the fusion pulses and fusion media on fusion rate and the development of embryos produced by somatic cell nuclear transfer in Hanwoo (Korean cattle). Nuclear donor cumulus and fetal fibroblast cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum at 38.5$^{\circ}C$ in a humidified atmosphere of 5% $CO_2$in air. The in vitro matured oocytes were enucleated and then the isolated donor cells were introduced. The cumulus cell and cytoplast were fused using one pulse of 70 volts for 40$mutextrm{s}$, two pulses of 70 volts for 40$mutextrm{s}$ and one pulse of 180 volts for 15$mutextrm{s}$. The fetal fibroblast cell and cytoplast were fused using one pulse of 180 volts for 15$mutextrm{s}$ or 30$mutextrm{s}$. The cumulus cell and cytoplast were fused using mannitol and Zimmerman cell fusion medium (ZCFM) as a fusion medium. The fused embryos were activated after the fusion with 10 $\mu$M calcium ionophore for 5 min and 2 mM 6-dimethyl- aminopurine for 3 h. The nuclear transfer embryos were cultured in 500 ${mu}ell$ well of modified CR1aa supplemented with 3 mg/$m\ell$ BSA in th $\varepsilon$ four well dish cove red with mineral oil. After 3 days culture, culture medium was changed into modified CRlaa medium containing 1.5 mg/$m\ell$ BSA and 5% FBS for 4 days. The incubation environment was 5% $CO_2$, 5% $O_2$, 90% $N_2$ at 38.5$^{\circ}C$. When the cumulus cells were fused with enucleated oocytes by three different fusion pulses, one pulse of 180 volts for 15 $mutextrm{s}$ yielded the highest fusion rate and developmental rate to blastocyst among the pulses (P＜0.05). When the fetal fibroblast cells were fused with enucleated oocytes, one pulse of 180 volts for 30$mutextrm{s}$ yielded significantly higher fusion rate compared with that for 15 $mutextrm{s}$(P＜0.05). The present result indicates that the fusion rate between karyoplast and cytoplast was affected by the cell type and the optimal fusion condition was different according to cell type or size. When the fusion was conducted by the use of mannitol and ZCFM, the fusion rate was 71.2% and 65.8%, respectively. The developmental rates to blastocyst were 37.8% and 39.8%, respectively. There was no significant difference between two fusion media in the developmental rate of cumulus cell nuclear transfer embryos. These results indicate that optimal electric current should be selected according to cell type.

• 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.

• Proceedings of the Korea Technology Innovation Society Conference
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• 2017.05a
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• pp.175-192
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• 2017

4차 산업혁명의 도래와 더불어 ICT 기계, ICT 금융, ICT 의료, ICT 나노 등과 같이 기술분야별 영역의 장벽이 허물어지고, 학제간 연구(Interdisplinary Research)가 일상화됨에 따라 무형자산의 가치평가시에도 융합기술의 가치평가 모형에 대한 니즈가 증대되고 있다. 특히, 기술의 매매, 현물출자, 기술금융(투자유치, 담보 보증), 인수 합병, 청산 소송 등 다양한 용도로 사용되고 있는 기술가치평가 모형은 융합기술의 입력변수 결정에 대한 체계적인 로직을 제공하지 못하고 있는 실정이다. 일반적으로 실제 거래사례가 존재할 경우, 국제가치평가기준(IVS)에 의하면 시장접근법이 우선적으로 적용될 수 있다고 권고된다. 그러나 융합기술의 이전거래를 비롯한 평가 활용사례를 수집하기도 어렵고 그렇다할 평가모델이 존재하지 않는 것이 사실이다. 융합기술에 대한 기술 및 시장의 사업화 환경을 고려하는 경우 소득접근법 기반의 평가기법이 유용하게 활용될 수 있는데, 기술수명, 매출액추정, 할인율, 산업기술요소 등의 핵심변수 결정에 관한 정형화된 로직이 존재하지 않으므로 본 고에서 융합기술 사례에 대해 실용적으로 활용가능한 변수추정 로직을 제시하고자 한다. 기술수명의 경우, 복수 개의 국제특허분류(IPC)별 피인용특허수에 따라 가중 적용하여 수명 추정을 위한 기준값을 정하며, 사업화소요기간 및 비용의 경우 평가대상 융합기술이 속하는 업종별 메타데이터값을 가중평균하여 현금흐름 추정기간을 최종 도출할 수 있다. 소득접근법에서의 매출추정, 할인율, 산업기술요소 변수 추정 이외에도 로열티공제법 적용을 위한 로열티율 결정에 있어서, 융합기술이 응용가능한 산업(업종)별 매출액 기반으로 가중 적용하여 각 변수값을 산출할 수 있다. 본 연구에서 개발된 융합기술 가치평가 모형은 향후 기술의 융복합화 특성을 반영하여 적정 가치를 산출하는 평가 가이드라인을 제공할 수 있을 것으로 기대된다.

• Korean Journal of Animal Reproduction
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• v.20 no.4
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• pp.459-465
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• 1997

The present study was undertaken to determine the effect of NT time on the rate of fusion a and suhseguent development In vitro and determine the optimal strength and duration of DC pulse for electrofusion of IVF donor embryo nuclei and IVM recipient oocytes. The recipient oocytes were enucleated 25 ~ 2Sh after IVM and further cultured for 18 ~ 20h prior to fusion for oocyte aging. IVF embryos as donor nuclei were C co cultured with BOEC for 16- to 32-cell stage development. The transfer time of donor bIas tomeres was 1~3h post-enucleation in early NT group and 1 ~ 18h post-enucleation in late NT group, respectively and fusion was performed 43~4Sh post-IVM. The fusion rate did not differ between the early NT and late NT group, but the rate of cleavage and 8- to 16-cell stage embryos in the late NT group was more higher than that in the early NT group. The fusion, cleavage and M+B development was high from O.7SkV /cm DC than from 1.0kV /cm DC voltage, resulting in 17.6% M+B from 0.75kV /cm DC voltage. No difference in fusion rate was among pulse durations, but 50 and 70 usec pulse duration showed slight high cleavage and M + B d development. The results indicate that the best NT time of IVF donor blastomeres into the enucleated oocytes was 42~44 post-IVM and the most suitable condition for electrofusion was a single 0.7SkV /cm DC voltage for SO~70$\mu$sec.

• Journal of Embryo Transfer
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• v.22 no.2
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• pp.89-95
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• 2007

The present study was conducted to examine some factors affecting in vitro development and fecundity of embryos recloned with somatic cell nuclear transfer (SCNT). Fibroblast cells retrieved from the ear of a 3-week-old, cloned Korean goat (Jinsoonny) were used as karyoplast donors and serum-starvation was conducted in tissue culture medium (TCM)-199 supplemented with 0.5% FBS. Recipient oocytes were surgically collected by flushing the oviducts 35 h after hCG injection following FSH priming. The zonae pellucidae of the oocytes were partially perforated with a laser drill and a donor cell was transferred into an enucleated oocyte. The couplets were electrically fused and activated by ionomycin (5 min) and 6-DMAP (4 h). The reconstructed embryos were cultured in mSOF medium containing 0.8% BSA at $39^{\circ}C$ in an atmosphere of 5% $CO_2$, 5% $%O_2$, 90% $N_2$ for 12 to 15 h. Re-cloned embryos (2- to 4-cell stages) were surgically transferred into the oviducts of the recipients and pregnancy was subsequently diagnosed by progesterone assay and ultrasound on Days 21 and 63 of pregnancy. The fusion rate following 1st fusion pulse was higher (p<0.05) in 2nd cloning (65.9%) compared to 1st cloning (51.0%), but it was not different in the other groups. The rate of cleavage after fusion was significantly higher (p<0.05) in 1st (77.7%) than in 2nd cloning (56.0%). A total of 175 re-cloned embryos were transferred into 28 recipients. On day 21 and 60 after transfer, 11 (39.3%) and 4 recipients (17.4%) were pregnancy, respectively. In comparison of pregnancy rate by estrous synchronization, a total of 66 and 109 re-cloned embryos were transferred into 11 recipients in natural estrus and 17 recipients in induced estrus, respectively. Five (45.4%) and 2 recipients (18.2%) in natural estrus were pregnant on days 21 and 63 while 6 (35.3%) and 2 (11.8%) recipients in induced estrus were pregnant, respectively. These results show that recloning of goat can be achieved by SCNT and estrous synchronization between donor and recipient animals may be one of the major factors affecting success rate.