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 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 552.4 in in vitro and in vivo cultured mouse embryos, respectively, but significantly(P<0.05) decreased to 350.7 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%).

This study was to investigate an effective biological control of forage diseases and provide a basic data and a model in improving variety of antagonistic bacteria, with growth promoting effect on forage, through cell fusion. The results obtained were sum

This study evaluated the influence of cell stage of donor nucleus on nuclear injection, electrofusion and in vitro development in the rabbit to improve the efficiency of nuclear transplantation in the rabbit. The embryos of 8-, 16- and 32-cell stage were collected from the mated does by flushing viducts with Dulbecco's phosphate buffered saline(D-PBS) containing 10% fetal calf serum(FGS) at 44, 54 and 60 hours after hCG injection. The blastorneres separated from these embryos were used as donor nucleus. The ovulated oocytes collected at 14 hours after hCG injection were used as recipient cytoplasm following removing the nucleus and the first polar body. The separated blastomeres were injected into the enucleated oocytes by micromanipulation and were electrofused in 0.28 M mannitol solution at 1.5 kV /cm, 60 sec for three times. The fused oocytes were cocultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FGS for 72~120 hours at 39 in a 5% incubator. The cultured nuclear transplant embryos were stained with Hoechst 33342 solution and the number of cells were counted by fluorescence microscopy. The successful injection rate of 8-, 16- and 32-cell-stageblastomeres into enucleated oocytes was 86.7, 91.0 and 93.9%, respectively. The electrofusion rate of 8-, 16- and 32-cell-stage blastomeres with enucleated oocytes was 93.3,89.3 and 79.0%, respectively. Development of blastomeres to blastocyst was similar with 8-,16- and 32-cell-stage donor nuclei(26.2, 25.8 and 26.6%, respectively, P<0.05). The mean number of cell cycle per day during in vitro culture in nuclear transplant embryos which received 8-, 16- and 32-cell- stage nuclei was 1.87, 1.81 and 1.43, respectively.

This study was carried out to investigate the aggregation rate of isolated mouse 2-, 4- and 8-cell stage blastomeres in phytohemagglutinin(PHA) solution. The morphologically normal embryos were collected from the oviduct of superovulated female mouse by flushing with M2 and the zona pellucida of embryos were removed with 0.5% pronase. The blastomeres were isolated by pipetting after plunging into Ca++-Mg++free PBS for 20 min. The result showed that aggregation rate in 0.5% (84.9~93.1%) was higher than that in 1.0% PHA(76.0~82.1%). Optimal aggregation time was 60min (83.9~100.0%) when compared with 30min (78.8~87.5%). Developmental to blastocyst in recombinated blastomeres was higher under conditions of 0.5% PHA solution and 60-min aggregation than that under other conditions.

The long term goal of this research is to develop an efficient procedure for large scale production of genetically identical or cloned animals. To improve nuclear transpalntation efficiency in the rabbit, this study evaluated the age of nuclear recipient oocytes on the different steps of nuclear transplantation. The ovulated oocytes in different ages were collected from the superovulated does by flushing oviducts with Dulbecco's phosphate buffered saline(D-PBS) supplemented with 10% fetal calf serum(FCS) from 13 to 15, 17 to 20 and 23 to 26 hours after hCG 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 enucleated oocytes receiving a blastomere in the perivitteline space were fused in the 0.28 M mannitol solution at 1.5 kV/cm, 60 sec for three times. The fused oocytes were co-cultured with the monolayered rabbit oviductal epithelial cells in TGM-199 solution with 10% FCS for 72 hours at 37 in a 5% incubator. The cultured nuclear transplant embryos and in vivo developed embryos collected at 72 hours after hCG injection were stained with Hoechst 33342 dye. Their cell numbers were counted under a fluorescent microscope. The results obtained were summarized as follows ; 1. The aged oocytes(20 hrs. post hCG) showed significantly(P<0.05) higher fusionrates(70 ~ 90%) than the recently ovulated oocytes(30.8%) 2. The aged oocytes which were electrically activated and fused at 20 hours developed to blastocyst at significantly(P<0.05) high rate, while none of the recently ovulated oocytes developed to blastocyst. 3. Even though the aged oocytes at 23~26 hours showed higher fusion rate(85.7%), not only they were inadequate to manipulate but also their developmental potential to blastocyst was highly impaired. 4. The developmental potential in vitro of nuclear transplant embryos was significantly retarded than in vivo deveolped embryos.

Petunia hybrida와 Nicotiana sanderae의 원형질체(原形質體) 융합(融合)에 의한 체세포잡종식물(體細胞雜種植物)을 얻기 위하여 원형질체융합(原形質體融合)에 미치는 융합제(融合濟)의 종류, PEG의 처리시간(處理時間) 및 융합시(融合時) 온도(溫度), 융합용액(融合溶液)과 희석용액내(稀釋溶液內)의 의 양(量) 및 희석용액(稀釋溶液)의 pH에 대해 실험(實驗)하였으며 그 결과(結果)는 다음과 같다. 두 종(種)의 원형질체(原形質體)를 5.5mM이 함유(含有)된 PEG 6,000 30%용액(溶液)으로 에서 10분간 처리(處理)한 후 50mM이 함유(含有)된 희석용액(稀釋溶液)의 pH를 9.0으로 조절(調節)한 용액(溶液)으로 희석(稀釋)시켰을 때 원형질체(原形質體) 융합율(融合率)이 가장 높았다.

완두엽육세포(葉肉細胞)의 원형질체분리(原形質體分離) 및 융합(融合)에 있어서 효소(酵素)의 처리시간(處理時間), 의 효과(效果), 효소(酵素)의 농도(濃度)와 엽(葉)의 위치(位置)에 따른 원형질체(原形質體)의 분리(分離)에 미치는 영향(影響)과 Mole 농도(濃度)에 따른 원심분리효과(遠心分離效果), PEG용액(溶液)의 처리시간(處理時間), 산도(酸度)에 따른 융합효과(融合效果) 그리고 dimethyl sulfoxide 첨가효과(添加效果)를 구명(究明)하기 위하여 실시(實施)한 실험(實驗)의 결과(結果)는 다음과 같다. 1. 원형질체(原形質體)의 분리(分離)에는 4시간(時間)의 산소처리(酸素處理)가 가장 양호(良好)했고 의 첨가(添加)에 따라 원형질체(原形質體)의 분리속도(分離速度)가 늦어졌으며 활성도(活性度)는 4시간(時間)까지 변동(變動)이 없었고 의해서 활성도(活性度)가 장기간(長期間) 유지(維持)되었다. 2. 산소(酸素)의 농도(濃度)는 1% 이상의 처리(處理)에서 거의 동일(同一)한 원형질체분리율(原形質體分離率)을 나타냈으며 상위(上位) 1~2엽(葉)이 가장 좋은 원형질체분리율(原形質體分離率)을 보였으며 원심분리효과(遠心分離效果)는 0.4M, 0.5M에서 양호(良好)하였다. 3. 원형질체융합(原形質體融合)은 PEG 6000용액(溶液) 75분(分) 처리(處理)에서 좋은 융합율(融合率)을 보였다. 그러나 30분(分) 이내(以內)의 처리(處理)가 활성도(活性度)에 영향(影響)을 미치지 않았으며 pH 5.8인 PEG 6000의 0.2M용액(溶液)에서 가장 높은 62.84%의 융합율(融合率)을 나타내었다. dimethyl sulfoxide에 의한 효과(效果)는 융합율(融合率)이 3~7% 감소(減少)하지만 binucleate의 유도(誘道)에 유효(有效)한 것으로 추정(推定)된다.

감자의 엽육조직(葉肉組織)으로부터의 원형질체(原形質體) 분리(分離)에 있어서 최적생육환경(最適生育環境), 적정효소(適正酵素) 농도(濃度), 적정효소(適正酵素) 처리시간(處理時間) 및 순수분리(純粹分離)를 위(爲)한 적정(適正) sucrose의 농도(濃度)를 밝히고 감자 및 petunia의 세포융합(細胞融合)에 있어서 PEG의 분자량(分子量), 농도(濃度) 및 처리시간(處理時間) 그리고 DMSO의 첨가(添加) 효과(效果)를 밝히기 위(爲)해 수행(遂行)되었다. 원형질체(原形質體) 분리(分離)에 있어서 생육환경(生育環境)은 고습도(高濕度), 낮은 조도(照度)에서 생육(生育)된 엽육조직(葉肉組織)이 유리(有利)하였으며 pectolyase Y-23 효소(酵素)가 기내배양(器內培養)된 엽(葉)에서 원형질체(原形質體) 분리시(分離時) 좋은 효과(效果)를 나타내었다. Cellulase의 농도(濃度)는 2%(W/V)가 좋았으며 효소처리(酵素處理) 시간(時間)은 3~4시간(時間)이 좋았고 원형질체(原形質體)의 순수분리(純粹分離)는 감자의 경우(境遇) 0.4~0.5M sucrose가 효과(效果)가 좋다. 원형질체(原形質體)의 융합(融合) 시(時) 처리시간(處理時間)은 공히 30분(分)이 적당(適當)했으며 PEG의 분자량(分子量), 농도(濃度)가 높을수록 융합율(融合率)이 높았다. 감자의 두 품종간(品種間) 융합(融合)이나 감자와 petunia의 융합(融合)에 있어서도 역시 PEG의 분자량(分子量), 농도(濃度)가 높을수록 높았다. Dimethyl sulfoxide의 첨가(添加) 효과(效果)는 DMSO를 5%에서 10%첨가(添加) 시(時), 감자와 petunia의 융합(融合)에서 2~4% 융합율(融合率)이 증가(增加)되었다.

4차 산업혁명으로 표현되는 현대 사회의 새로운 변화는 단편적인 지식의 습득을 넘어서 그러한 지식들을 창의적인 방식으로 통합할 것을 요구하고 있다. 이에 2015 개정 교육과정에서는 미래 사회가 요구하는 핵심역량을 함양하여 바른 인성을 갖춘 창의 융합형 인재를 양성하는 것을 핵심 목표로 표방하고 있다. 이것은 각 교과들을 독립적인 영역의 분과로 이해한 전통적인 방식에서 벗어나 여러 교과 영역의 융합적 학습을 지향한다는 것을 의미한다. 융합교육에 기초한 교육과정은 학교 현장에서 활용될 수 있어야하며, 그것은 단순한 학문 사이의 연결을 넘어서는 초학문적 융합에 기초해야 한다. 초학문적 융합교육에서는 교과들 간의 통합뿐만 아니라 전문적인 교과 지식과 비전문적인 학생들 사이의 의사소통을 전제한다는 점에서 높은 통합성을 전제하고 있다. 초학문적 융합교육은 다양한 도덕적 문제를 성찰하고 스스로의 삶 속에서 실천하는 것을 핵심 목표로 삼는 도덕과 교육에도 적용될 수 있다. 초학문적 융합에 기초한 도덕적 탐구 모델은 하나의 윤리적 쟁점 을 학습하는 데 있어 관련된 교과 지식뿐만 아니라 다양한 자료들을 활용한다. 이 탐구 모델은 관련된 지식과 자료를 통해 오류를 제거하고 통합적인 관점에서 도덕적 신념 체계를 확립하는 과정을 포함한다. 초학문적 융합에 기초한 도덕적 탐구 모델은 윤리적 쟁점을 다루는 도덕과 수업에 활용되어 몇 가지 이점을 가질 수 있다. 첫째, 수업을 통해 학생들이 능동적이고, 자율적으로 자신의 도덕적 신념 체계를 확립하도록 유도할 수 있다. 둘째, 윤리적 쟁점에 포함된 다양한 지식에 대한 학습을 포함하기 때문에 올바른 도덕적 신념을 기르는 객관적인 기준을 가질 수 있다. 셋째, 학생들로 하여금 개방적인 태도를 견지하도록 도울 수 있다.

The plasma cortisol of nurserypigs was examined using an outdoor efficacy testwith a digital content-based approach in animal welfare convergence types. Nine nurserypigs,without discriminating between female and male, were classified into 2 groups of 3 pigs each: control and group 1 (effect+nature), control and group 2(effect+nature+music). The control group was the same for group 1 and 2 to compare the effects using a t-test. There was no significant difference in plasma cortisol levels between the control group and group 1 until 4 h after stress induction. However, significant differences were subsequently found between the control group and group 1 from 8 h to 72 h (p<0.05). Further, plasma cortisol was not affected in group 2 at 0 h through 8 h and 72 h. At 12 h through 48 h, group 2 showed a reduction in plasma cortisol level compared to the control group(p<0.05). These results indicated that after stress induction, applying effect plus nature or effect plus nature plus music can effectively decrease plasma cortisol levels in nursery pigs within8 h through 72 h and may serve as a better model for digital content-based approach in animal welfare convergence types.