산양의 이종간 핵이식에 있어서 수핵난자에 따른 공여세포의 조건이 핵이식란의 체외발달에 미치는 영향

서지정보

ㆍ발행기관 : 한국동물번식학회 ㆍ수록지정보 : Reproductive & developmental biology / 28권 / 1호
ㆍ저자명 : 이명열, 박희성

목차

I. 서론 II. 재료 및 방법 1. 공여세포의분리 및 배양 2. 수핵난자 준비 3. 핵이식 4. 핵과 세포질의 융합 및 활성화 5. 체외수정 및 배양 6. 복제수정란의 체외배양 7. 통계학적 분석 III. 결과 및 고찰 1. 공여세포의 세포주기 동기화 방법이 이종간 행이식란의 체외발달에 미치는 영향 2. 공여세포의 계대배양이 핵이식란의 체외발달에 미치는 영향 3. 이종간 핵이식 수정란의 체외발달 IV. 요약 V. 인용문헌

영어 초록

This study was conducted to investigate the developmental ability of caprine embryos after somatic cell interspecies nuclear transfer. Donor cells were obtained from an ear-skin biopsy of a caprine, digested with 0.25% trypsin-EDTA in PBS, and primary fibroblast cultures were established in TCM-199 with 10% FBS. After maturation, expanded cumulus cells were removed by vigorous pipetting in the presence of 0.3% hyaluronidase. The matured oocytes were dipped in D-PBS plus 10% FBS＋7.5 ㎍/ml cytochalasin B and 0.05 M sucrose. The reconstructed oocytes were electrically fused with donor cells in 0.3 M mannitol fusion medium. After the electofusion, embryos were activated by electric stimulation. Interspecies nuclear transfer embryos with bovine cytoplasts were cultured in TCM-199 medium supplemented with 10% FBS including bovine oviduct epithelial cells for 7∼9 day. On the other hand, the NT embryos with porcine cytoplasts were cultured in NCSU-23 medium supplemented with 10% FBS for 6∼8 day at 39℃, 5% CO₂ in air. In caprine-bovine NT embryos, the cleavage(2-cell) rate was 36.8% in confluence and 43.8% in serum starvation. The developmental rate of morula- and blastocyst-stage embryos was 0.0% in confluence and 18.8% in serum starvation. In caprine-porcine NT embryos, the cleavage(2-cell) rate was 76.7% in confluence and 66.7% in serum starvation. The developmental rate of morula and blastocyst stage embryos was 3.3% in confluence and 3.0% in serum starvation, and no significant difference was observed in synchronization treatment between donor cells. In caprine-bovine NT embryos, the cleavage(2-cell) rate of cultured donor cells was 30.8% and 17.6% in 5∼9 and 10∼14 passage(P＜0.05). The developmental rate of morula and blastocyst stage embryos were significantly higher(P＜0.05) in 5∼9 passage(23.1%) than in 10∼14 passage(0.0%) of cultured donor cells. In caprine-porcine NT embryos, the cleavage rate was significantly higher(P＜0.05) in 5∼9 passage(86.7%) than in 10∼14 passage(50.0%) of cultured donor cells. The developmental rate of morula and blastocyst stage embryos were 3.3 and 0.0% in 5∼9 and 10∼14와 passage of cultured donor cells. In caprine-bovine NT embryos, the developmental rate of morula and blastocyst stage embryos were 22.6% in interspecies nuclear transfer, 33.9% in in vitro fertilization and 28.1% in parthenotes, which was no significant differed. The developmental rate of morula and blastocyst stage embryos with caprine-porcine NT embryos were lower(P＜0.05) in interspecies nuclear transfer(5.1%) than in vitro fertiltzation(26.9%) and parthenotes(37.4%).

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