Cloning or somatic cell nuclear transfer (SCNT) using adult somatic cell to derive cloned embryos is a promising new technology with potential applications in both agriculture and regenerative medicine. Mammalian embryos derived by nuclear transfer are capable of development to the blastocyst stage with a relatively high efficiency of 30～ 50%. However, in full-time development, usually only 2% of NT embryos can result in live births due to abnormalities in placenta formation. In SCNT embryos, the donor cell nucleus is epigenetically reprogrammed by oocyte cytoplasm during development. Incomplete reprogramming of the donor cell genome is considered a major reason for low cloning efficiency. Aberrant epigenetic modifications include DNA methylation, histone modification and X-chromosome-inactivation. Due to a lack of basic knowledge regarding the embryos following nuclear transfer, the success rate of cloning is low. Therefore, elucidation of the molecular mechanism of SCNT embryo development will be of great value for further research. MicroRNAs (microRNA) are single-strand RNA molecules of about 19 23 nucleotides in length, which regulate gene expression by imperfect base pairing with target mRNA, subsequently guiding mRNA cleavage or translational repression. Since the first discovery and functional annotation in 1993 of the small RNA, lin-4 and let-7, which are involved in developmental timing and gene regulation during C. elegans larval development, microRNAs have received scientific attention. Now hundreds of microRNAs have been identified in various multicellular organisms, and many microRNAs have been shown to be evolutionarily conserved. The roles proposed for this novel class of tiny RNA molecules are diverse. They are likely to be involved in developmental timing, differentiation, cell proliferation, signaling pathways, apoptosis, metabolism, heterochromatin formation, genome rearrangement, brain development and carcinogenesis. Currently (2006- present) we are working to determine the role of microRNAs on the epigenetic regulation of fertilized and cloned embryo development. The general hypothesis of our research is that genetic and epigenetic factors regulate the development of preimplantation mammalian embryos, and aberrant modulations in cloned embryos are causes of abnormal development and low success rate of cloned embryos.

One-step dilution and direct transfer would be a practical technique for the field application of frozen embryo. This study was to examine whether Jeju Black Cattle (JBC, Korean Cattle) can be successfully cloned from vitrified and one-tep diluted somatic cell nuclear transfer (SCNT) blastocyst after direct transfer. For vitrification, JBC-SCNT blastocysts were serially exposed in glycerol (G) and ethylene glycol (EG) mixtures〔10% (v/v) G for 5 min., 10% G plus 20% EG (v/v) for 5 min., and 25% G plus 25% EG (v/v) for 30 sec.〕which is diluted in 10% FBS added D-PBS. And then SCNT blastocysts were loaded in 0.25 ml mini straw, placed in cold nitrogen vapor for 3 min. and then plunged into LN2. One-step dilution in straw was done in 25℃ water for 1 min, by placing vertically in the state of plugged- end up and down for 0.5 min, respectively. When in vitro developmental capacity of vitrified SCNT blastocyst was examined at 48 h after one-step dilution, hatched rate (56.4%) was slightly lower than that of control group (62.5%). In field trial, when the vitrified-thawed SCNT blastocysts were transferred into uterus of synchronized 5 recipients, a cloned female JBC was delivered by natural birth on day 299 and healthy at present. In addition, when the short tandem repeat marker analysis of the cloned JBC was evaluated, microsatellite loci of 11 numbers was perfectly matched genotype with donor cell (BK94-14). This study suggested that our developed vitrification and one-step dilution technique can be applied effectively on field trial for cloned animal production, which is even no longer in existence.

This study was to investigate the effect of flavonoid treatment on in vitro development of bovine somatic cell nuclear transfer (SCNT) embryos, and their pregnancy and delivery rate after embryo transfer into recipient. In experiment 1, to optimize the flavonoid concentration, parthenogenetic day 2 (≥ 2-cell) embryos were cultured in 0 (control), 1, 10 and 20 μM flavonoid for 6 days. In the results, in vitro development rate was the highest in 10 μM flavonoid group (57.1%) among treatment groups (control, 49.5%; 1 μM, 54.2%; 20 μM, 37.5%), and numbers of total and ICM cells were significantly (p<0.05) higher in 10 μM flavonoid group than other groups. We found that 10 μM flavonoid treatment can significantly (p<0.05) decrease the apoptotic index and derive high expression of anti-oxidant, anti-apoptotic, cell growth and development marker genes such as Mn-SOD, Survivin, Bax inhibitor, Glut-5, In-tau, compared to control group. In experiment 2, to produce the cloned Jeju Black Cattle, beef quality index grade 1 bull somatic cells were transferred into enucleated bovine MII oocytes and reconstructed embryos were cultured in 10 μM flavonoid added medium. When the in vitro produced day 7 or 8 SCNT blastocysts were transferred into a number of recipients, 10 μM flavonoid treatment group presented higher pregnancy rate (10.2%, 6/59) than control group (5.9%, 2/34). Total three cloned Jeju Black calves were born. Also, two cloned calves in 10 μM flavonoid group were born and both were all healthy at present, while the one cloned calf born in control group was dead one month after birth. In addition, when the result of short tandem repeat marker analysis of each cloned calf was investigated, microsatellite loci of 11 numbers matched genotype between donor cell and cloned calf tissue. These results demonstrated that the flavonoid addition in culture medium may have beneficial effects on in vitro and in vivo developmental capacity of SCNT embryos and pregnancy rate.

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

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 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%).