This study was undertaken to optimize enucleation and reconstitution methods for the production of cloned mice by somatic cell nuclear transfer Outbred ICR mouse oocytes at the metapahse- II stage were retrieved from female mice superovulated by PMSG and hCG. In Experiment 1, oocytes were enucleated in medium supplemented with cytochalasin B (CCB) of 3 levels (0, 7.5 or 15 /mL), and higher rate of encleation was obtained at 7.5 and 15 /mL than at /mL. In Experiment 2, oocytes enucleated in 7.5 /mL CCB-containing medium were reconstituted with different types of somatic cell by following methods; 1) cumulus cells by direct cell injection, 2) cumulus cells by electric fusion (1.25 kV/cm, 2 pulses for each 70 ) or 3) STO cells by the electrofusion. Electrofusion of STO cells with enucleated oocytes yielded the greatest (P<0.05) rate of reconstitution without lysis (76%) than any other combinations. Although significant decrease in the rate of somatic cell introduction was found, the electrofusion of cumulus cells yielded better rate of reconstitution than direct injection (0 vs. 18%). In Experiment 3, the duration of electric stimulation for the fusion was changed to either 50 or 90 , but no significant improvement of reconstitution efficacy was obtained. In conclusion, this study showed that ICR mouse oocytes could be used for the production of reconstituted oocytes and a fusion method of 1.25 KV/cm with 2 pulses using 570 cell was the optimal.

This study was conducted to investigate the effects of quiescent treatment of donor cells and activation treatment time of recipient cytoplasm on nuclear remodeling and in vitro development of somatic cell-cloned bovine embryos. Serum starved, confluent and nonquiescent cycling adult skin cells were teansferred into enucleated oocytes. Nuclear transfer oocytes were activated at 30 min, 1 and 2 hrs after electrofusion. Some nuclear transfer embryos(23% to 35%) extruded a polar body, which was not affected by quiescent treatment of donor cells and activiation time of recipient cytoplasm. About 68% of nuclear transfer embryos fused with a serum starved cells has a chromatin clump, but which was not different from embryos fused with confluent(51%) and nonquiescent(47%) cells. The proportion of embryos with a single chromatin clump was sightly increased when nuclear transfer embryos were activated within 30 min after fusion(69%) compared to those were activated at 1 and 2 hrs after fusion, but there was not significantly different. Development rates to the blastocyst stage were 8.6% and 15.9% when serum starved and confluent cells were transferred, which were higher than that of control group. Developmental rate to the blastocyst stage was higher in embryos were activated within 30 min after fusion (17.3%) compared to those of embryos were activated at 1 and 2 hrs after fusion (P<0.05). From the present result, it is suggested that quiescent treatment of donor cells and activation time of recipient cytoplasm can affect the in vitro development. Quiescent plasm activation within 30 min after fusion could increase the number of embryos with a normal chromation structure, which results in increased in vitro development.

This study investigates the endoplasmic reticulum (ER) stress and subsequent apoptosis in duced during somatic cell nuclear transfer (SCNT) process of porcine SCNT embryos. Porcine SCNT and in vitro fertilization (IVF) embryos were sampled at 3 h and 20 h after SCNT or IVF and at the blastocyst stage for mRNA extraction. The x-box binding protein 1 (Xbp1) mRNA and the expressions of ER stress-associated genes were confirmed by RT-PCR or RT-qPCR. Apoptotic gene expression was analyzed by RT-PCR. Before commencing SCNT, somatic cells treated with tunicamycin (TM), an ER stress inducer, confirmed the splicing of Xbp1 mRNA and increased expressions of ER stress-associated genes. In all the embryonic stages, the SCNT embryos, when compared with the IVF embryos, showed slightly increased expression of spliced Xbp1 (Xbp1s) mRNA and significantly increased expression of ER stress-associated genes (p<0.05). In all stages, apoptotic gene expression was slightly higher in the SCNT embryos, but not significantly different from that of the IVF embryos except for the Bax/Bcl2L1 ratio in the 1-cell stage (p<0.05). The result of this study indicates that excessive ER stress can be induced by the SCNT process, which induce apoptosis of SCNT embryos.

Polyploidy is occurred by the process of endomitosis or cell fusion and usually represent terminally differentiated stage. Their effects on the developmental process were mainly investigated in the amphibian and fishes, and only observed in some rodents as mammalian model. Recently, we have established tetraploidy somatic cell nuclear transfer-derived human embryonic stem cells (SCNT-hESCs) and examined whether it could be available as a research model for the polyploidy cells existed in the human tissues. Two tetraploid hESC lines were artificially acquired by reintroduction of remained 1st polar body during the establishment of SCNT-hESC using MII oocytes obtained from female donors and dermal fibroblasts (DFB) from a 35-year-old adult male. These tetraploid SCNT-hESC lines (CHA-NT1 and CHA-NT3) were identified by the cytogenetic genotyping (91, XXXY,-6, t[2:6] / 92,XXXY,-12,+20) and have shown of indefinite proliferation, but slow speed when compared to euploid SCNT-hESCs. Using the eight Short Tendem Repeat (STR) markers, it was confirmed that both CHA-NT1 and CHA-NT3 lines contain both nuclear and oocyte donor genotypes. These hESCs expressed pluripotency markers and their embryoid bodies (EB) also expressed markers of the three embryonic germ layers and formed teratoma after transplantation into immune deficient mice. This study showed that tetraploidy does not affect the activities of proliferation and differentiation in SCNT-hESC. Therefore, tetraploid hESC lines established after SCNT procedure could be differentiated into various types of cells and could be an useful model for the study of the polyploidy cells in the tissues.

The Korean native pig (KNP) have been considered as animal models for animal biotechnology research because of their relatively small body size and their presumably highly inbred status due to the closed breeding program. However, little is reported about the use of KNP for animal biotechnology researches. This study was performed to establish the somatic cell nuclear transfer (SCNT) protocol for the production of swine leukocyte antigens (SLA) homotype-defined SCNT KNP. The ear fibroblast cells originated from KNP were cultured and used as donor cell. After thawing, the donor cells were cultured for 1 hour with 15 μM roscovitine prior to the nuclear transfer. The numbers of reconstructed and parthenogenetic embryos transferred were 98 ± 35.2 and 145 ± 11.2, respectively. The pregnancy and delivery rate were 3/5 (60%) and 2/5 (40%). One healthy SLA homotype-defined SCNT KNP was successfully generated. The recipient-based individual cloning efficiency ranged from 0.65 to 1.08%. Taken together, it can be postulated that the methodological establishment of the production of SLA homotype-defined cloned KNP can be applied to the generation of transgenic cloned KNP as model animals for human disease and xenotransplantation researches.

The cloning of canids was succeeded in 2005, several years after the birth of Dolly the sheep and also after the cloning of numerous other laboratory and farm animal species. The delay of successful somatic cell nuclear transfer (SCNT)was due to the unique reproductive characteristics of the female dogin comparison to other domestic mammals, such as ovulation of immature canine oocyte and a requirement of 25 days for the completion of meiosis within the oviduct (Holst & Phemister, 1971). When the technology for the recovery of in vivo matured oocyte was established, the application of cloning also became possible and cloned dog offspring were obtained. This report summarizes the progress of technical procedures that are required for cloning canids and the application of this technique. The first cloned dog, Snuppy, was achieved using an in vivo-matured oocyte which was enucleated and transferred with an adult skin cell of male Afghan hound. After establishment of a criterion of well-matured oocyte for the improvement of SCNT efficiency, we obtained three cloned female Afghan hound and a toy poodle cloned from 14 year-old aged Poodle using SCNT through this factor. To date, cloned dogs appeared to be normal and those that have reached puberty have been confirmed to be fertile. Through application of canine SCNT technique, first, we demonstrated that SNCT is useful for conserving the breed of endangered animal from extinction through cloning of endangered gray wolves using inter-species SCNT and keeping the pure pedigree through the cloning of Sapsaree, a Korean natural monument. Secondly, we showed possibility of human disease model cloned dog and transgenic cloned dog production through cloning of red fluorescent protein expressing dog. Finally, SCNT can be used for the propagation of valuable genotypes for making elite seed stock and pet dog. In summary, dog cloning is a reproducible technique that offers the opportunity to preserve valuable genetics and a potential step towards the production of gene targeted transgenic cloned dogs for the study of human diseases.