The purpose of this study is to examined the electrofusion and activation conditions for the production of porcine somatic cell nuclear transfer (SCNT) embryos. In this study, immature oocytes were cultured in TCM-199 with and without hormones for 22 hours. Skin fibroblasts cells of porcine were transferred into the perivitelline space of enucleated in vitro matured oocytes. Cell fusion was performed with two different pulses that each one pulse (DC) of 1.1 kV/cm or 1.5 kV/cm for 30 μsec. After fusion subsequent activation were divided into three groups; non-treatment (control) and treatment with 2 mM 6-DMAP or 7.5 μg/ml cytochalasin B for 4 hours. Transferred embryos were cultured in PZM-3 (Porcine Zygote Medium-3) in 5% CO₂ and 95% air at 39℃ for 7 day. Apoptosis-related genes (Caspase-3, BCL-2, mTOR, and MMP-2) were analyzed by immunofluorescence staining. There was no significant difference between two different electrofusion stimuli in the cleavage rate; 64.9±4.8% in 1.1 kV/cm and 62.7±4.0% in 1.5 kV/cm. However, blastocyst formation rate (%) was significantly different among three different activation groups (no treatment, 2 mM 6-DMAP or 7.5 μg/ml cytochalasin B) combined with electrofusion of 1.1 kV/cm. The blastocyst formation rate was 12.6±2.5, 20.0±5.0, and 34.9±4.3% in control, 2 mM 6-DMAP, and 7.5 μg/ml cytochalasin B, respectively. Immunofluorescence data showed that expression levels of caspase-3 in SCNT embryos undeveloped to blastocyst stage were higher than those in the blastocyst stage embryos. Expression levels of Bcl-2 in blastocyst stage embryos were higher than those in the arrested SCNT embryos. These results showed that the combination of an electric pulse (1.1 kV/cm for 30 μsec) and 7.5 μg/ml cytochalasin B treatment was effective for production of the porcine SCNT embryos.

Abstract
 서 론
 재료 및 방법
  1. 난포란의 채란 및 성숙배양
  2. 공여세포준비
  3. 제핵 및 핵이식
  4. 융합 및 활성화 처리
  5. 체외 배양
  6. Annexin V 염색과 Immunofluorescence
  7. 통계처리
 결 과
  1. 전기융합과 활성화 조건에 따라 핵이식란의 발달율
  2. 핵이식란의 발육 단계별 Apoptosis 분석
  3. 면역학적 분석방법을 통한 Apoptosis 분석
 고 찰
 REFERENCES

• Ji-Eun Ryu(Major in the Animal Biotechnology, Graduate School of Future Convergence Technology, Hankyong National University) | 류지은
• Jong-Taek Yoon(Department of Animal Life Science, Hankyong National University) | 윤종택 Corresponding Author