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.

This study was carried out to improve a technique of cloned animal prodcution by preactivation of nuclear recipient oocytes with ionomycin and 6-dimethylaminopurine (6-DMAP) in rabbits. The oocytes were collected from the oviduct of superovulated rabbit at 19∼20 hours post hCG injection. The collected oocytes were preactivated and self-enucleated by treating 5 uM ionoycin for 5 min, and 2.0 mM 6-DMAP for two hours. Microsurgical removel of the chromation complex in the second polar bodies was effectively performd and single blastomere separated from 32-cell stage rabbit embryos was injected into the perivitelline space of the enculeated recipient oocyts. Follwoing electrofusion and in vitro culture for 18 hours, the nuclear transplant(NT) embryos were transferred into the uterine horns of naturally mated or synchronized recipient does. When 32 NT embryous reconstituted with preactivated oocytes were transferred to 2 recipient does, one foster doe delivered two offspring (6.3%), while not a offspring was delivered from three foster does which received 17 NT embryos reconstituted with non-preactivated oocytes. A total of 68 NT embryos reconstituted with preactivated oocytes were transferred into the uterine horns of 7 synchronized ecipient does. Among them, two recipients were pregnant and delivered three offspring(5.9%).

본 연구는 토끼난자에 있어서 배란 후 경과시간에 따른 탈핵률을 조사하고 ionomycin과 DMAP을 사용하여 활성화와 아울러 자체 탈핵을 유도하는 방법을 고안하였으며 아울러 이들의 탈핵효율과 핵이식후 체외발달을 확인한 결과는 다음과 같다. 1. hCG 주사후 15∼16 시간에 채란된 토끼난자는 73.4%의 탈핵률을 보였고, 16∼17시간에는 75.8%, 19∼20 시간에는 58.5%의 탈핵률을 보여 토끼난자의 탈핵은 hCG 주사후 17시간 이내에 실시

The objectives of the present study were improvements in the efficiency of developmental rates to morula and blastocyst stages to produce a large number of genetically identical nuclear transplant embryos. The oocytes collected from slaughterhouse ovaries were matured for 24 h and then enucleated and cultured to allow cytoplasmic maturation and gain activation competence. And then the donor embryos were treated for 12 h with 10 g /ml nocodazole and 7.5 g /ml cytochalasin B to synchronize the cell cycle stage at 26 h after the onset of culture. The blastomeres were transferred into the perivitelline space of the enucleated nocytes and blastomeres and oocytes were fused by electrofusion. The cloned embryos were then cultured in various conditions to allow further development. The age of the recipient(30 vs 40 h) had no significant effect on the fusion rates(82.4 vs 82.1%) and the developmental rates to morula /blastocyst(9.8 vs 11.0%). Effect of Nocodazole treatment on the donor cell cyle synchronization to improve the developmental rates of bovine nuclear transplant embryos was significantly higher than control group(21.4 vs 10.1%, p<0.05). Significant differences were in the percentage of fusion rates(72.9,77.1vs 61.9%) in three types of fusion medium(PBS(+), mannitol and sucrose, p<0.01). The developmental rates of bovine nuclear transplant embryos appeared to be highest in mSOF medium under 5% 0 condition, but no significant differences were found when compared with TCM199-BOEC and mSOF under two different oxygen ratio(5 and 20%).

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%).

large scale production of cloned embryos requires the technology of multiple generation nuclear transplantation(NT) using NT embryos as the subsequent donor nuclei. The purposes of this study were producing the second generation cloned rabbit embryos, and also to determine the electrofusion rate and in vitro developmental potential comparatively in the cloned embryos of the first and second NT generation. 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 In the first generation NT, the nuclear donor embryos were synchronized in the phase of Gi /S transition of 32-cell stage. The first generation NT embryos which were developed to 8-cell were synchronized in Gi /S transition phase of the following 16-cell stage and used as donor nuclei for second generation Synchronization of the cell cycle of blastomeres was induced, first, using an inhibitor of microtuble polymerization, colcemid for 10 hours to arrest blastomeres in M phase, and secondly, using a DNA synthesis inhibitor, aphidicolin for 1.5 to 2 hours to arrest them in Gi /S transition boundary. The recipient cytoplasms were obtained by removing the nucleus and the first polar body from the oocytes collected at 14 hours after hCG injection. The separated donor blastomeres were injected into the enucleated recipient oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 sec at 1.25 kV /cm in 0.28 M rnannitol 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 first and second generation cloned embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The results obtained were summarized as follows: 1. The electrofusion rate was found to be similar as 79.4 and 91.5% in the first and second generation NT rabbit embryos, respectively. 2. The in vitro developmental potential to blastocyst stage of the second generation NT embryos (23.3%) was found significantly(p<0.05) lower, compared with that of the first generation NT embryos (56.8%). 3. The mean blastomeres counts of embryos developed to blastosyst stage following in vitro culture for 120 hours and also their daily cell cycles during the culture period were decreased significantly (p<0.05) to 104.3 cells and 1.33 cylces in the second NT generation, compoared with 210.4 cells and 1.54 cycles in the first NT generation, respectively.

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.

This experiment was carried out to produce cloned aniraals by nuclear transplantation in rabbits. The ovulated oocytes were collected from the oviducts between 14 and 15 hours after hGG 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 nucleated oocytes receiving a blastomere in the perivitelline space were electrically fused in the 0.28 M mannitol solution at 1.5 kV /cm, 60sec for three times. The nuclear transplant embryos which were used and developed to 2- to 4-cell stage in vitro were transferred into the oviducts of synchronized recipient does. A total of 64 nuclear transplant embryos were transferred to 7 recipient does and produced three offspring(4.7%) from a foster mother 31 days after embryo transfer.

The present study was undertaken to determine the optimal condition for parthenogenetic activation of rabbit oocytes by electric stimulation in vitro in an attempt to develop nuclear transplantation techniques for cloning mammalian embryos and animals. Freshly ovulated oocytes were collected from superovulated rabbits from 13 to 26 hrs. after hCG injection. The cumulus-free oocytes were activated parthenogetically by repeated stimuli of square direct electric pulses in O.3M mannitol solution. After applying electric stimulations of different voltages, pulse durations and pulse times, all of the oocytes were cultured in TCM-199 with 10% FCS for 96 hours in a 5% incubator, and their developmental potential in vitro was examined. The higher activation rate (68.9%) was achieved at the voltage of 2.0kv/cm, the pulse duration of 60 sec and three pulse times and the activation rate of 100% was achieved at the pulse duration of 100 and 200 sec, the voltage of 1.5kv/cm and three pulse times. Although the higher rates of activation of oocytes were achieved at 100 and 200 sec, none of them developed to blastocyst in vitro. The oocytes collected 18~20 hours post hCG injection showed the highest rate of activation and development to blastocyst in vitro than the oocytes collected 13~15 or 25~26 hours post hCG injection. Therefore, it can be suggested that the application of electric stimulation of 2.0kv/cm, 60 sec and three pulse times to the oocytes collected at 18~20 hours post hCG injection would be more beneficial for the parthenogenetic activation of oocytes in rabbits.

Nuclear transplantation techinque has been found to be the most potential and efficient method for producing a large number of genetically identical animals from a single embryo. The technical development of nuclear transplantation in mammals and its application to the production of cloned animals were reviewed. For the efficient and successful production of cloned embryos by nuclear transplantation, selection and micromanipulation of recipient eggs or embryos as capacious recipient cytoplasm, and benefitial preparation of multiple totipotent embryonic cells as donor nuclei, and also fusion technique are very critical. Recent works approaching to these critical points were introduced and discussed.

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

Nuclear transplantation technique is known as the most potential and efficient method for producing a large number of genetically identical animals from a single embryo. The technical development of nuclear transplantation in mammals and its application to the production of cloned animals are described. For the efficient and successful production of cloned embryos by nuclear transplantation, the right selection and micromanipulation of recipient eggs or embryos as capacious recipient cytoplasm, the adequate and benefitlal preparation of multiple totipotent embryonic cells as donor nuclei, and also the fusion technique are very critical. Recent studies approaching to these critical points are introduced and discussed. Up to date, the overall efficiency of production of cloned embryos and offspring in livestock is estimated to be low. Further technical development of nuclear transplantation will enable large-scale production of cloned livestock and in near future the commercial cloning of animals will become a reality.