Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
권호 표지

Improvement of Somatic Cell Nuclear Transfer Technology for the Production of Disease Model Mouse: I. Optimization of Oocyte Enucleation and Reconstruction

질환모델마우스 생산을 위한 체세포핵이식방법 개선; I. 탈핵 및 재조합난자 생산기법 최적화

  • Jun, S. H. (College of Medicine, Pochon CHA University and Infertility Medical Center of CHA General Hospital) ;
  • Shim, H. S. (College of Medicine, Pochon CHA University and Infertility Medical Center of CHA General Hospital) ;
  • Chung, H. M. (College of Medicine, Pochon CHA University and Infertility Medical Center of CHA General Hospital) ;
  • Lee, B. C. (Col. Vet. Med., Seoul Nat'l Univ.) ;
  • Lee, E. S. (Dept. Vet. Med., Kangwon Nat'l Univ.) ;
  • Ko, J. J. (College of Medicine, Pochon CHA University and Infertility Medical Center of CHA General Hospital) ;
  • Shin, T. (Col. Vet. Med., Texas A &M Univ.) ;
  • Park, C. (College of Medicine, Pochon CHA University and Infertility Medical Center of CHA General Hospital) ;
  • Hwang, W. S. (Col. Vet. Med., Seoul Nat'l Univ.) ;
  • Cha, K. Y. (College of Medicine, Pochon CHA University and Infertility Medical Center of CHA General Hospital) ;
  • Lim, J. M. (School Agr. Biotech., Seoul Nat'l Univ.)
  • 전수현 (포천중문의과대학교 의학과-차병원 여성의학연구소) ;
  • 심호섭 (포천중문의과대학교 의학과-차병원 여성의학연구소) ;
  • 정형민 (포천중문의과대학교 의학과-차병원 여성의학연구소) ;
  • 이병천 (서울대학교 수의과대학) ;
  • 이은송 (강원대학교 수의학과) ;
  • 고정재 (포천중문의과대학교 의학과-차병원 여성의학연구소) ;
  • 신태형 (텍사스 A ＆ M 대학교 수의과대학) ;
  • 박찬 (포천중문의과대학교 의학과-차병원 여성의학연구소) ;
  • 황우석 (서울대학교 수의과대학) ;
  • 차광렬 (포천중문의과대학교 의학과-차병원 여성의학연구소) ;
  • 임정묵 (서울대학교 농생명공학부)
  • Published : 2000.12.01
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Abstract

This study was undertaken to optimize enucleation and reconstitution methods for the production of cloned mice by somatic cell nuclear transfer Outbred ICR mouse oocytes at the metapahse- II stage were retrieved from female mice superovulated by PMSG and hCG. In Experiment 1, oocytes were enucleated in medium supplemented with cytochalasin B (CCB) of 3 levels (0, 7.5 or 15 $\mu\textrm{g}$/mL), and higher rate of encleation was obtained at 7.5 and 15 $\mu\textrm{g}$/mL than at $\mu\textrm{g}$/mL. In Experiment 2, oocytes enucleated in 7.5 $\mu\textrm{g}$/mL CCB-containing medium were reconstituted with different types of somatic cell by following methods; 1) cumulus cells by direct cell injection, 2) cumulus cells by electric fusion (1.25 kV/cm, 2 pulses for each 70 $mutextrm{s}$) or 3) STO cells by the electrofusion. Electrofusion of STO cells with enucleated oocytes yielded the greatest (P<0.05) rate of reconstitution without lysis (76%) than any other combinations. Although significant decrease in the rate of somatic cell introduction was found, the electrofusion of cumulus cells yielded better rate of reconstitution than direct injection (0 vs. 18%). In Experiment 3, the duration of electric stimulation for the fusion was changed to either 50 $mutextrm{s}$ or 90 $mutextrm{s}$, but no significant improvement of reconstitution efficacy was obtained. In conclusion, this study showed that ICR mouse oocytes could be used for the production of reconstituted oocytes and a fusion method of 1.25 KV/cm with 2 pulses using 570 cell was the optimal.

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