동물자원연구 제29권 2호 (p.56-66)

미세주입된 소 수정란에서 유전자적중 효율 및 배반포 품질 확인

Determination of Gene Target Efficiency and Blastocyst Quality in Microinjected Bovine Embryos
키워드 :
CRISPR/Cas9,Microinjection,Cytochalasin B,Melatonin,Bovine embryo

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
Ⅵ. 참고문헌

초록

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.