Earticle

초록

영어

This study aimed to produce high-quality blastocysts and establish appropriate microinjection conditions for the introduction of target gene. First, we identified embryo development to the blastocyst stage after microinjection using the CRISPR/Cas9 system on the Cas9 protein or mRNA. As a result, we confirmed that blastocyst development in the Cas9 mRNA injected group significantly increased when compared to the Cas9 protein injected group (p<0.05). However, the blastocyst gene targeting rate increased in the Cas9 protein injected group when compared to the Cas9 mRNA injected group (p<0.05). Next, we treated the injection medium with 10 µg/ml of cytochalasin B (CB), and the microinjected embryos were cultured in CR1-aa medium supplemented with 0.1 µM of melatonin (Mela). Consequently, the blastocyst formation rate significantly increased in the CB treated group (p<0.05). After microinjecting embryos with the CB treated injection medium, we investigated blastocyst formation and quality via Mela treatment. Consequently, the Mela treated group demonstrated significantly increased blastocyst formation rates when compared to the non-treated group (p<0.05). Furthermore, immunofluorescence assay using RAD51 (DNA repair detection protein) and H2AX139ph (DNA damage detection protein) showed an increase in RAD51 positive cells in Mela treated embryos. Therefore, we verified the improvement in knock-in efficiency in microinjected bovine embryos using Cas9 protein. These results also demonstrated that the positive effect of the CB and Mela treatments improved the embryonic developmental competence and blastocyst qualities in genetically-edited bovine embryos.

목차

ABSTRACT
 Ⅰ. 서론
 Ⅱ. 재료 및 방법
  1. 시약
  2. 난소 채취 및 체외성숙
  3. 체외수정
  4. 미세주입 및 체외배양
  5. 도입 유전자의 적중 확인
  6. 면역형광염색법
  7. 통계 분석
 Ⅲ. 결과
  1. CRISPR/Cas9 nuclease에 따른 유전자 적중율 확인
  2. 미세주입 시 cytochalasin B(CB)의 첨가 효과
  3. 미세주입 후 초기배 발달에서의 멜라토닌의 효과
  4. 미세주입된 소 배반포에서의 품질 확인
 Ⅳ. 고찰
 Ⅴ. 요약
 사사
 Ⅵ. 참고문헌

저자

• 김민지 [ Min-Ji Kim | 대구대학교 공과대학 생명공학과 ]
• 박효진 [ Hyo-Jin Park | 대구대학교 공과대학 생명공학과 ]
• 김진우 [ Jin-Woo Kim | 대구대학교 공과대학 생명공학과 ]
• 김세은 [ Se Eun Kim | 전남대학교 농업생명과학대학 동물자원학부 ]
• 박다솜 [ Da Som Park | 전남대학교 농업생명과학대학 동물자원학부 ]
• 양슬기 [ Seul-Gi Yang | 대구대학교 공과대학 생명공학과 ]
• 김인수 [ In-su Kim | 대구대학교 공과대학 생명공학과 ]
• 제갈호근 [ Ho-Geun Jegal | 대구대학교 공과대학 생명공학과 ]
• 강만종 [ Man-Jong Kang | 전남대학교 농업생명과학대학 동물자원학부 ]
• 김소섭 [ So-Seob Kim | 광개토한우농업법인 주식회사 ]
• 박정준 [ Joung Jun Park | 명품한우컨설팅 ]
• 구덕본 [ Deog-Bon Koo | 대구대학교 공과대학 생명공학과 ] Corresponding author

참고문헌