활성화를 통한 수핵란의 대량확보를 위해 44시간동안 체외 성숙된 돼지 난자를 ethanol, Ca/sup 2+/-ionophore, 6-DMAP 및 cycloheximide의 화학물질들을 사용하여 단위발생을 유기한 후 그들의 가장적합한 처리농도 및 노출 시간을 규명하였다. 1. Ethanol은 10%, 10분 처리가 전핵형성율, 난할율 및 배발달율에 있어 각각 약 53.4%, 51.6%, 그리고 39.9%로 가장 적합한 조건으로 판명되었다. 2. Ca/sup 2+/-ionophore 가장 적합한 난활성화 조건은 25μM에서 2분간 처리한 것이며, 전핵형성율, 난할율 및 배발달율은 각각 약 59.7%, 62.2%, 그리고 43.9%를 보였다. 3. 6-DMAP를 처리하여 돼지 난자의 활성화를 유기하였을 경우 2mM의 농도에서 각각 약 57.3%, 58.4% 및 29.0%의 전핵형성율, 난할율, 그리고 배발달율을 보여 가장 적합한 조건을 보였으며 2시간∼4.5시간 사이의 노출에는 영향을 받지 않았다. 4. Cycloheximide는 5㎍/ml의 농도가 전핵형성율 2.1%, 난할율 47.7%, 배발달율 31.8%로 가장 은 효율을 보였고, 노출시간에서는 4시간∼6시간 동안 처리하였을 때 60.5∼65.8%, 63.6∼66.7% 및 39.0∼39.5%로 가장 적합한 조건으로 판명되었다. 이상의 결과들은 돼지 체외 성숙 난자의 활성화에 있어 각 화학물질들의 적합한 조건을 바탕으로한 중복처리 및 병용처리 조건 확립 및 효율적인 수핵란의 확보에 기여할 수 있을 것이다.

한우 태아 섬유아세포 유래 체세포복제수정란 이식으로 임신된 수란우가 분만예정일이 도래하였으나 분만징후가 없어 수정란이식 후 272일째에 dexamethasone 20 mg을 근육주사하고 24시간후 PG 25 mg과 estradiol 20 mg을 근육주사하여 분만유도를 실시하였다. dexamethasone 투여후 48시간만 분만징후를 나타내었으며, dexamethasone 투여후 50시간째에 40 kg의 수송아지를 견인에 의해 정상적으로 분만시켰다. 그러

인체 트롬보포이에틴(hTPO)은 megakaryopoiesis 과정에 주요한 역할을 하는 사이토카인이다. 따라서 이러한 트롬보포이에틴을 유선조직에서 직접적으로 발현시키기 위하여 소 베타 카제인 프로모터, 인체 트롬보포이에틴 cDNA 및 네오유전자로 구성된 발현벡터를 구축하였다. 소 귀조직 세포로부터 유도된 섬유아세포에 lipoffctamine을 이용하여 발현벡터(pBT-L n대)의 삽입을 유도하였다. G4l8 저항성을 지닌 세포의 콜로니 형성을 유도하기 위하여 2주 이상 배양을 실시하였다. 형질전환 콜로니는 PCR에 의해 동정하였으며, 이들 콜로니를 핵치환 전까지 계속적으로 증식을 유도하였다. 형질전환 세포에 의해 재구성된 난자는 전기적인 융합과 calcium ionophore와 6-DMAP를 이용한 활성화를 실시하였으며, 체외에서 7일간 배양을 실시하였다. 총 35개의 콜로니를 PCR에 의해 분석한 결과, 이 중 29(82.9%)개가 형질전환된 콜로니였다. 형질전환된 세포로 재구성된 난자의 난할율 및 배반포로의 발달율은 65.1%와 23.8%로 나타났다. 형질전환된 세포로 재구성된 난자로부터 발달한 29개의 배반포 중 27개가 형질전환으로 확인되었다. 따라서 이러한 결과들은 형질전환 소 수정란을 형질전환된 세포를 이용한 체세포 복제 기법을 통해 효과적으로 생산할 수 있다는 것을 제시하고있다.

본 연구는 한우미경산우에 인공수정 7일째와 복제수정란 이식시 hCG를 투여에 의해 수태율 향상을 위하여 실시하였다 인공수정 후 임신 60일째에 임신감정을 실시한 결과 대조구, CIDR 그리고 hCG를 투여한 결과 53%, 46%, 71%였으며, 처리간에 유의적인 차이를 보였다(P<0.05). 복제수정 란의 이식시 hCG를 투여한 결과 대조구, hCG투여구는 각각 5.8%, 10.4%를 나타냈으며, 유의차는 인정되지 않았다. 인공수정 7일째에 hCG를 투

The principal objective of this study was to clone transgenic embryos in order to improve the efficiency of transgenic animal production by the combination of microinjection and nuclear transplantation techniques. Mature female New Zealand White rabbits were superovulated by eCG and hCG treatments, fllowed by natural mating. Zygotes were collected from the oviducts at 18∼22 h after hCG injection by flushing with D-PBS containing 5% fetal calf serum(FCS). Two to three picoliters of green fluorescent protein(GFP) gene wa microinjected into male pronucleus. The foreign gene-injected zygotes were cultured in TCM-199 or RD medium containing 10% FCS with a monolayer of rabbit oviductal epithelial cells in a 5% CO2 incubator. The morulae expressing GFP gene were selected and their blastomeres were separated for the use of nuclear donor. Following nuclear transplantation of fluorescence-positive morula stage blastomeres, 13 (21.3%) out of 61 fused oocytes developed to blastocyst stage and all of the cloned blastocysts expressed GFP. The results indicate that the screening of transgene in rabbit embryos by GFP detection could be a promisible method for the preselection of transgenic embryos. Also the cloning of preselected transgenic embryos by nuclear transplantatin could be efficiently applied to the multiple production of transgenic animals.

To improve the efficiency of production of cloned embryos and animals by nuclear transplantation in the rabbit, the effect of cell cycle of donor nuclei and type of recipient cytoplasm on the in vitro developmental potential and production efficiency of offspring was determined. The embryos of 16-cell stage were collected from the mated does at 48h post-hCG injection and they were synchronized to G phase of 32-cell stage. The oocytes collected at 14h post-hCG injection were freed from cumulus cells and then enucleated. One group of the enucleated cytoplasms was activated by electrical stimulation prior to injection of donor nucleus, and the other group was not pre-activated. The separated Gphase blastomeres of 32-cell stage embryos were injected into the perivitelline space of recipient cytoplasms. After culture for 20h post-hCG injection, the nuclear transplant oocytes were electrofused and activated by electrical stimulation and the fused nuclear transplant embryos were co-cultured for 120h and the nuclear transplant embryos developed to blastocyst stage were stained with Hoechst 33342 dye and their blastomeres were counted. Some of the nuclear transplant embryos developed in vitro to 2- to 4-cell stage were transferred into the oviducts of synchronized recipient does. The electrofusion rate was similar between the types of donor nuclei and recipient cytoplasms used. However, the nuclear transplant embryos using G phase donor nuclei were developed to blastocyst at higher rate(60.3%) than those using S phase ones(24.7%). Also, when non-preactivated oocytes were used as recipient cytplasms, the develop-mental rates of nuclear transplant embryos to blastocysts were significantly(P< 0.05) higher(57.1%) than those using preactivated ones(20.8%). The cell counts of nuclear transplant embryos developed to blastosyst stage were increased signficantly(P<0.05) more in the non-preactivated recipient cytoplasm(163.7 cells), as compared whit the preactivated recipient cytoplasm(85.4 cells), A total of 49 nuclear transplant embryos were tranferrid into 5 recipient does, of which two offsprings were produced from a foster mother 31 days after embryo transfer. these results showed that the blastomeres of G1 phase and non-preactivated oocytes might be utillzed efficiently as donor nuclei and recipient cytoplasms in the nuclear transplant procedure, thought the offspring production remained still low.

Large scale production of cloned embryos requires the technology of multiple generational nuclear transfer(NT) by using NT embryos itself as the subsequent donor nuclei. In this work we investigated comparatively the effects of enucleated oocytes treated with ionomycin and 6-DMAP on the electrofusion rate and in vitro developmental potential in the first and second NT embryos. The embryos of 16-cell stage were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline(D-PBS) containing 10% fetal calf serum(FCS) at 47 hours after hCG injection. The recipient cytoplasms were obtained by removing the nucleus and the first polar body from the oocytes collected at 15 hours after hCG injection. The enucleated oocytes were pre-activated by 5 min incubation in 5M ionomycin and 2 hours incubation in 2 mM 6-DMAP at 19~20 hours post-hCG before microinjection. In the first and second generation NT, the unsynchronized 16-cell stage embryos were used as nuclear donor. The separated donor blastomeres were injected into the enucleated activated recipient oocytes by micromanipulation and were electrofused by electrical stimulation of single pulse for 60 sec at 1.25kV/cm in +, + - free 0.28 M mannitol solution. In the non-preactivation group, the electrofusion and electrical stimulation was given 3 pulses for 60 sec at 1.25 kV/cm in 100M +, + 0.28 M mannitol solution. The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in TCM-199 solution containing 10% FCS for 120 hours at 39 in a 5% incubator. The results obtained were summarized as follows: 1. In the first generational NT embryos, the electrofusion rate of preactivated and non-activated oocytes(80.4 and 87.8%) was not significantly different, but in the second generational NT embryos, the electrofusion rate was significantly(P<0.05) higher in the non-activated oocytes(85.7%) than in the preactivated oocytes(70.1%). 2) In the first and second generational NT embryos, the developmental potential to biastocyst stage was significantly(P<0.05) higher in the preactivated oocytes(39.3 and35.7%) than in the non-preactivated oocytes(16.0 and 13.3%). No significant difference in the developmental potential was shown between the first and second generational NT embryos derived from the preactivated oocytes. In conclusion, it may be efficient to use the oocytes preactivated with ionomycin and 6-DMAP for the multiple production of cloned embryos by recycling nuclear transfer.

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%).

The purposes of this study were to produce cloned rabbit embryos and offsprings by nuclear transplantation(NT) using in vitro matured oocytes as nuclear recipient cytoplasm and to determine the effect of frozen nuclei donor embryos on the production efficiency of cloned embryos. The 8cell embryos were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline containing 10% fetal calf serum(FCS) at 40 hours after hGG injection. A portion of collected embryos were preserved at 4 for 24 hours and a portion of them were frozen by vitrification method. The embryos used for donor nuclei were synchronized in the phase of Gi /S transition. The in vitro matured oocytes were used as recipient cytoplasm following removing the nucleus and the first polar body. The synchronized blastomeres from fresh, cooled or frozen embryos were injected into the enucleated oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 sec at 1.0 W /cm in 0.28 M mannitol solution. The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FCS for 120 hours at 39 in a 5% incubator. Following in vitro culture of the NT embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The nuclear transplant embryos developed in vitro to 2- to 4-cell stage were transferred into the oviducts of synchronized recipient does. The results obtained were summarized as follows: 1. The fusion rates of the blastomeres from fresh, cooled and frozen embryos with the in vitro matured and enucleated oocytes were 100, 95.8 and 64, 3%, respectively. 2. Development in vitro to blastocyst was significantly(p<0.05) different between the cloned embryos with the blastomeres from fresh, cooled or frozen embryos as 39.0, 20. 9 and 15.7%, respectively. 3. The mean numbers of cell cycle per day during in vitro culture of cloned embryos blastomeres from fresh, cooled or frozen embryos was 1.31, 1.29 and 1.16, respectively. 4. A total of 77 nuclear transplant embryos were transferred into 6 recipient does, of which two offsprings were produced from a foster mother 31 days after embryo transfer.

포유동물의 초기 발생단계에서 핵의 분화와 전능성을 규명하고 제2세대 핵이시 기법을 개발하고자 생쥐를 모델로 하여 공핵란은 2-세포기에 있는 수정란의 핵을 사용하였으며, 수핵란은 zygote 및 2-세포기에 있는 수정란을 탈핵하여 제2세대 핵이식을 실시하여 electrofusion system으로 핵융합을 실시하고 cloned embryo를 작출하여 이를 24-48시간동안 체외에서 배양을 시킨 다음 위임신이 유기된 수란생쥐의 난관에 체내 이식을 실시하여