• The Korean Journal of Zoology
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• v.32 no.3
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• pp.258-263
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• 1989

Nuclear Transplantation between Rana pipiens and Rana dybowskii When diploid blastula nuclei of Rana pipiens are traraplanted into enucleated eggs of Rana dybowskii the resulting nucleocytoplasmic hybrids are lethal-those development were arrested around the stage of the dorsal lip formation For the improvement of developmental capacity, serial nuclear transplantation was carried out. Even though serial transplantation of 15 generations showed normal development in each generation until gastrula stage, there was no sign of fundamental improvement in development afterward. This results implied that up to gastrulation normal DNA replication and cell division can take place in foreign cytoplasm. Since chromosomal aberrations both in shape and number were usually observed, the nuclei must have been modifted while resided in the foreign cytoplasm. Those nuclei didn't participate in normal development and led the embryos to early death. Tissue graft experiment indicated that the abnormal behavior of this lethal nucleocytoplasmic hybrid is an inherent property which is not corrected by the contact with its own tissue.

• Korean Journal of Animal Reproduction
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• v.18 no.3
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• pp.217-228
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• 1994

These experiments were carried out to establish the optimal condition of electrostimulatin inducing cell fusion and oocyte activation for nuclear transplantation in mouse embryos. Eggs selected for cell fusion or activation by electrostimulation were equilibrated for 5~10 min. in 0.3M sucrose solution and electrostimulated for 60$\mu$sec using 1 pulse of 60, 70, 80, 90 or 100 volts DC with electrodes 0.2 mm apart. Then they were cultured in 20${mu}ell$ dropsof Tyrode's solution. The results of these experiments are as follows : 1. When one pulse of 60, 70, 80, 90 or 100 volts DC for 60$\mu$sec were applied to 2-cell embryos for fusion of blastomeres, fusion rates were 50.0, 81.7, 91.7, 100 and 100%, respectively ; and developmental rates of fused embryos to blastocyst were 76.7 to 81.5%. Higher fusion rates were observed in 90V and 100V. 2. The average cell number in fused embryos developed to blastocyst was about half of the cell number in diploid controls; and the cell number decreased with increasing of voltages. 3. When pulse numbers were increased, fusion rates improved, but developmental rates were not signficiantly different from the group for which the number of pulse was not increased. And the cell number of blastocyst decreased even more. 4. Oocytes aged for 6hrs after ovulation were electrostimulated for oocyte activation by the same method used for cell fusion. Rates of oocyte activated by electrostimulation were 45.3 to 60.4%, and fragmentation rates were 7.5~15.1%. The lysis rates were 17.0~34.0%. The results of these experiments indicate that the optimal condition for achieving cell fusion and activation is 1 pulse, duration 60$\mu$sec in 90 Volt. The results also show that this condition is suitable for nuclear transplantation using mouse eggs.

• Korean Journal of Veterinary Research
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• v.30 no.2
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• pp.123-127
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• 1990

By nuclear transplantation technology twenty eight mice have been produced after transfer of heterozygous biparental eggs. Also heterozygous gynogenetic eggs with two female pronuclei and heterozygous androgenetic eggs with two male pronuclei have been obtained by injecting a male or female pronucleus with Sendai virus into the perivitelline space of enucleated haploid zygotes at pronuclear stage. The success rate of enucleation, karyoplast injection and fusion of both the pronuclei was 80.3, 83.4 and 81.8%, respectively. The overall pronuclei fusion rates by this technique were 56, 50 and 56% in biparental, gynogenetic and androgenetic eggs, respectively. The evidence was ascertained that the gynogenetic and androgenetic eggs were also able to develop in vitro up to blastocyst stage, even though their developmental potential was greatly diminished beyond 2-cell stage. The gynogenetic eggs were able to develop in vivo up to day 10 of pregnancy, while the androgenetic eggs failed to develop in vivo during the same period.

• Journal of Embryo Transfer
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• v.9 no.2
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• pp.153-160
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• 1994

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 $\mu$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$^{\circ}C$ in a 5% $CO_2$ 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.

• Journal of Embryo Transfer
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• v.10 no.1
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• pp.65-72
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• 1995

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$^{\circ}C$ 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 $\mu$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$^{\circ}C$ in a 5% $CO_2$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.

• The Korean Journal of Nuclear Medicine Technology
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• v.28 no.1
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• pp.13-17
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• 2024

Bone Mineral Densitometry (BMD) values is determined numerically with T or Z-score while the image is not considered in the interpretation. Abnormal finding was observed in the left femoral bone density image of a 64-year-old female patient who underwent a left kidney transplantation. This was due to the ethiodized oil used for embolization in the treatment of lymphatic leakage. Radiologic technologists should not only monitor changes of BMD values, but keep in mind to assess the changes in the image to provide accurate information to the patient.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.171-179
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• 1997

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 $\pi$g /ml nocodazole and 7.5 $\pi$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$_2$ condition, but no significant differences were found when compared with TCM199-BOEC and mSOF under two different oxygen ratio(5 and 20%).

• Korean Journal of Veterinary Research
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• v.37 no.3
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• pp.639-645
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• 1997

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 transplanted embryos. The oocytes collected from slaughterhouse ovaries were matured 24h in TCM199+10% FBS and exposed to $39^{\circ}C$ or room temperature to allow cytoplasmic maturation and gain activation competence. Donor embryos were treated for 12h with $10{\mu}g/ml$ nocodazole or $0.05{\mu}g/ml$ demicolcine to synchronize the cell cycle stage at 26h after the onset of culture. The blastomeres and recipient oocytes were fused by electrofusion. The cloned embryos were then cultured in various conditions to allow further development. In the treatment of oocyte activation and cell cycle regulation of donor nuclei, the room temperature exposure and nocodazole treatment group had significant effect on the developmental rates to morula/blastocyst(21.7% vs 12.1~16.7%), but had no significant effect on the fusion rates between donor blastomeres and recipient oocytes. The developmental rates of bovine nuclear transplanted embryos appeared to be higher significantly in mTALP medium under 5% $O_2$ condition and in TCM199 with bovine oviduct epithelial cell under 20% $O_2$ condition(22.2%) than other groups. In embryo transfer of nuclear transplanted embryos, there were no significant differences in calving rates between the use of excellent and good grade donor embryos.

• 대한핵의학회:학술대회논문집
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• 2004.11a
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• pp.414.2-414.2
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• 2004

• Journal of Embryo Transfer
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• v.10 no.3
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• pp.209-217
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• 1995

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 $\mu$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 55$\pm$2.4 in in vitro and in vivo cultured mouse embryos, respectively, but significantly(P<0.05) decreased to 35$\pm$0.7 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%).