• Korean Journal of Animal Reproduction
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• v.13 no.2
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• pp.93-97
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• 1989

These experiments were carried out to develop the technique of nuclear transplantation necessary for elevating utilization efficiency of high quality embryos and the production of clone animals. Embryos of pronucleus stages were obtained from ICR mice. Removal of pronuclei and their transfer to recipient embryos were carried out by micromanipulation and virus mediation. The results obtained in these experiments were summarized as follows ; 1. Total 337 pairs of pronuclear stage embryos were subjected to nuclear transplantatin and 247 pairs(73.3%) of them were successfully transplanted and the number of fused embryos between transplanted nucleus and cytoplasm was 188 pairs(55.8%). 2. Of the 188 fused embryos cultured in vitro, 174(92.4%), 131(69.7%) and 117(62.2%) embryos were developed to 2-cell, morula and blastocyst stages, respectively. 3. When total 104 nuclear transplanted embryos were transferred to uteri of recipient mice on day 2-3 of pseudopregnancy, 4 of 12 recipient mice were pregnant and the number of embryos developed to young was 28(26.9%).

• 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.

• Proceedings of the KSAR Conference
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• 2001.10a
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• pp.62-62
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• 2001

Development of effective activation protocols is of great importance for improving the success of cloning and subsequent transgenic. Three methods for oocyte activation, including 5μM ionomycin (5 min) alone, ionomycin＋1.9 mM 6-dimetylaminopurine (DMAP, 3 hrs) and ionomycin＋10㎍/㎖ cycloheximide(CHX, 3 hrs) were compared for their effects of pronuclei(PN) formation, development, developmental velocity and ploidy of parthenotes to IVF control in bovine. In group of ionomycin＋DMAP, the oocytes having more 3 PN were significantly(P〈0.05) higher than in groups of ionomycin alone and of ionomycin＋CHX (45.5% vs. 0 and 0%, respectively). Activation with the ionomycin alone, ionomycin＋DMAP and ionomycin＋CHX resulted in cleavage rates of 30, 85.5 and 57.9%, respectively. The blastocysts rate of parthenotes activated by ionomycin＋DMAP treatment was significantly higher (12.3%, P〈0.05) than those of other treated groups. Chromosome analysis shows that ionomycin＋DMAP treatment greatly increases the incidence of chromosomal abnormality of the parthenotes. When compared the developmental velocity at 24 hrs after insemination and activation, 27% eggs in IVF control and 55% in DMAP treatment out of total cleaved eggs developed to 2-cell stage, respectively. Developmental velocity of parthenotes activated by ionomycin ＋DMAP treatment was significantly (P〈0.05) faster than others. From the results, we may conclude that DMAP treatment to the oocytes accelerates developmental velocity resulting in both the higher incidence of chromosome abnormality and of PN formation suggesting that CHX combined with ionomycin is suitable DMAP for the purpose of successful nuclear transplantation.

• Korean Journal of Animal Reproduction
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• v.22 no.4
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• pp.419-424
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• 1998

The present study was carried out to examine the efficiency of cloning of transgenic embryos by nuclear transfer(NT) using gene-injected rabbit embryos. The rabbit embryos at pronuclear stage were microinjected with methallothionein-human growth hormone(MT-hGH) gene and cultured to 8- and 16-cell in TCM-199 containing 10% FCS with a monolayer of rabbit oviductal epithelial cells in a 5% $CO_2$incubator. The recipient oocytes were collected from the oviducts 14~16 h after hCG injection. The oocytes were enucleated and activated with 5$\mu$M ionomycin and 2mM 6-dimethylaminopurine. Blastomeres form gene-injected embryos were transferred into the enucleated oocytes by micromanipulation. The nuclear transplant oocytes were electrofused and co-cultured with rabbit oviductal cells. Following 120 h of culture, blastocysts were prepared for gene analysis by polymerase chain reaction(PCR). In previous experiment, the rate of gene-positive embryos detected by the nested PCR analysis was significantly decreased while developing to blastocyst(25%)(Kang et al., 1998). The fusion rate of gene-injected blastomeres was significantly(P＜0.05) lower than non-injected blastomeres(66% vs 80%). However, the NT embryos that were derived from gene-injected donor embryos did not differ from control embryos in development to the blastocyst stage(39% vs 31%). Of the 43 NT blastocysts developed from the gene-injected donor embryos, twelve(28%) were positive for the injected DNA. The results indicate that NT with gene-injected embryos can be successfully used for cloning and multiplication of transgenic embryos, furthermore applicable to improvement of transgenic animal production.

• Journal of Embryo Transfer
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• v.16 no.2
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• pp.107-115
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• 2001

Development of effective activation protocols is of great importance for improving the success of cloning and subsequent transgenic. Three methods for oocyte activation, including 5$\mu$M ionomycin (5 min) alone, ionomycin + 1.9 mM 6-dimetylaminopurine (DMAP, 3 hrs) and ionomycin + 10 $\mu\textrm{g}$/ml cycloheximide (CHX, 3 hrs) were compared for their effects of pronuclei (PN) formation, development, developmental velocity and ploidy of parthenotes to IVF control in bovine. In group of ionomycin + DMAP, the oocytes having more 3PN were significantly (P<0.05) higher than in groups of ionomycin alone and of ionomycin + CHX (45.5% vs. 0 and 0%, respectively). Activation with the ionomycin alone, ionomycin + DMAP and ionomycin + CHX resulted in cleavage rates of 30, 85.5 and 57.9%, respectively. The blastocysts rate of parthenotes activated by ionomycin + DMAP treatment was significantly higher (12.3%. p<0.05) than those of other treated groups. Chromosome analysis shows that ionomycin + DMAP treatment greatly enhances the incidence of chromosomal abnormality of the parthenotes. From the results, we may conclude that DMAP treatment to the oocytes accelerates developmental velocity resulting in both the higher incidence of chromosome abnormality and of PN formation, and strongly suggest that CHX combined with ionomycin is better than DMAP for the purpose of successful nuclear transplantation. Developmental velocity of parthenotes activated by ionomycin + DMAP treatment was significantly (P<0.05) faster than others.