초자화 동결과 1-단계 융해된 체세포 핵이식란의 직접 이식 기술로 제주흑우 복제소 생산

Production of Cloned Jeju Black Cattle (Korean Cattle) from SCNT Embryo using Vitrification, One-Step Dilution and Direct Transfer Technique

  • 김은영 (미래생명공학연구소) ;
  • 박민지 (미래생명공학연구소) ;
  • 김재연 (미래생명공학연구소) ;
  • 박효영 (미래생명공학연구소) ;
  • 노은지 (미래생명공학연구소) ;
  • 노은형 (미래생명공학연구소) ;
  • 송동환 ;
  • 오창언 (제주대학교 줄기세포연구센터) ;
  • 김영훈 (제주특별자치도 축산진흥원) ;
  • 문성호 (제주대학교 줄기세포연구센터) ;
  • 이동선 (제주대학교 생명자원과학대학 생명공학부) ;
  • 고문석 (국립축산과학원 난지축산시험장) ;
  • 류기중 (제주대학교 생명자원과학대학 생명공학부) ;
  • 박세필 (미래생명공학연구소)
  • 투고 : 2011.02.25
  • 심사 : 2011.03.03
  • 발행 : 2011.03.31

초록

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 $LN_2$. One-step dilution in straw was done in $25^{\circ}C$ 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.

키워드

참고문헌

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