This study was conducted to examine the viability of Korean native striped cattle (Bos namadicus Falconer, Chikso) clone embryos after embryo transfer. Chikso somatic cell nuclear transfer (SCNT) embryos were produced by fusion of ear skin cells derived from a female Chikso with enucleated oocytes matured in vitro for 18-24 hr. After in vitro culture of SCNT embryos for 7 to 8 days, fresh or vitrified blastocysts derived from SCNT were transferred into a uterine horn of recipient cows. Fifteen of total 43 recipients were pregnant at Day 50 and 4 recipients were maintained to term. Three IVF-derived calves and 1 clone Chikso calf were born. Pregnancy rate was higher when fresh embryos were transferred to recipients compared to vitrified embryos, but development to term was not different between both groups. The clone Chikso calf died at 5 days after birth due to the fullness of amniotic fluid in rumen and the infection of umbilical cord. The result of the present study shows that clone Chikso calf can produced from the embryo transfer of SCNT embryos, however, solution of abortion problem is necessary to improve the cloning efficiency.

This study was conducted to evaluate the effect of cytochalasin B (CB) treatment in the activation medium on the development of somatic cell nuclear transfer (SCNT) rat embryos. Fetal fibroblast cells were isolated from a Day 14.5 fetus, and the oocytes for recipient cytoplasm were recovered from 4-week old Sprague Dawley rats. After enucleation and nuclear injection, the reconstructed oocytes were immediately exposed to activation medium consisting of 10 mM SrCl2 with or without CB for 4 hr, and formation of pseudo-pronucleus (PPN) was checked at 18 hr after activation. Then, they were transferred into day 1 pseudopregnant recipients (Hooded Wistar) or cultured for 5 days to check their developmental competence in vivo or in vitro. The number of PPN was not affected by CB treatment during the activation. However, CB treatment supported pre-implantation development of rat SCNT embryos. Embryos generated by the procedures of SCNT were also capable of implanting, with 1 implantation scar found from a recipient following the transfer of 87 SCNT embryos to four foster mothers. The result of the present study shows that rat SCNT embryo can develop to post-implantation stage following treatment with CB.

본 연구는 재래 산양의 체세포 핵이식에 의하여 생산한 복제 산양(진순이)의 조직으로부터 공여 핵을 배양하여 다시 핵이식을 실시하여 재복제에 따른 융합율과 분할율, 이식 후의 수태율 등을 조사하여 재복제 가능성 여부를 검토하기 위하여 실시하였다. 공여 세포는 귀 유래 섬유아세포를 분리 배양하여 사용하였으며, 체내 성숙 난자는 성숙한 미경산 재래 산양에 과배란을 유기하여 외과적인 방법으로 난관 관류를 통해 회수하여 핵이식을 실시하였다. 핵이식란의 융합은 전

본 연구는 소 체외수정란과 체세포 복제란의 초자화 동결 및 융해 후 생존능을 검토하였다. 배반포로 발육된 체외수정란 및 체세포 복제란을 초자화 동결법에 의해 동결하였다가 융해하여 생존율 및 배양 후 부화율을 검사하였다. 체외수정란 배반포를 초자화 동결 융해한 결과, 확장배반포가 배반포기 난자에 비하여 생존율(82.1%, 96/117)과 부화율(64.1%, 75/117)에서 모두 유의적으로 높았다(p<0.05). 핵이식 배반포 복제란을 초자화 동결 융해한 경우도 체외수정란과 비슷한 경향을 보여 확장 및 부화배의 생존율과 부화율이 각각 81.1%(30/37)와 78.3%(29/37)로, 배반포(각각 71.8 및 53.8%)에 비하여 다소 높게 나타났다. 본 연구의 결과는 초자화 동결 방법에 의해서 소 체외수정란과 체세포 복제란을 성공적으로 동결할 수 있으며, 특히 후기 배반포기 단계에서 초자화 동결 시 높은 생존율과 부화배 형성율을 얻을 수 있음을 보여준다.

본 연구는 융합 전 수핵란의 활성화 처리 유무와 활성화 처리 시간이 소 체세포 유래 핵이식란의 체외 발육능에 미치는 영향을 검토하기 위해 수행하였다. 소 체외성숙란의 탈핵 후 체세포 핵을 이식하고 일부는 전기 융합 후 활성화를 유기하였고(pre-AC), 일부는 먼저 활성화 처리 후 융합을 실시(post-AC)하였다. 난자의 활성화는 Ca2+-ionophore(A23187)로 처리 후 DMAP로 4시간 배양하는 방법으로 유기하였다. 핵이식란은 CR1aa액에서 9일간 배양하여 발육율을 검토하였으며, 활성화 후 30분～2.5시간에 고정하여 confocal microscope 하에서 핵형 변화를 검사하였다. 배반포기까지 발육율은 post-AC구(20.6%)가 pre-AC(15.3%)보다 다소 높게 나타났다. 또한 활성화 처리를 하여 핵이식란의 배 발달을 비교한 결과 post-AC구가 더 늦은 배 발달 속도를 나타내었다. Post-AC구를 활성화 후 30분, 2시간, 4시간으로 나누어 융합하여 발육율을 검토한 결과 발육율에 차이가 없었다. 본 연구의 결과는 수핵란의 활성화 시간에 따라 핵이식란의 발육 및 핵형이 영향을 받을 수 있음을 시사한다.

본 연구는 소 형질 전환 체세포 핵이식에서 용이한 탈핵을 위해 demecolcine을 이용할 시 탈핵율과 핵이식란의 발육능을 높이기 위한 최적의 조건을 알아보고자 실시되었다. 도축장 유래 미성숙 난자를 18시간 체외성숙 후 제1극체가 확인된 성숙 난자를 0.1, 0.2, 0.4 및 0.8 ug/ml의 demecolcine이 첨가된 배지에서 1시간 더 처리한 다음 세포막이 돌출되어 있는 난자를 체세포 핵이식에 공여하여 각 군간 배반포로의 발육능을 비교하였

In the present study, performances of several in vitro maturation (IVM) systems for porcine follicular oocytes were evaluated, and an efficient chemically defined IVM system for porcine oocytes was proposed. The proposed one-step culture system supplemented with polyvinylalcohol (PVA) gave competitive efficiencies in terms of oocyte maturation and blastocyst development after parthenogenetic activation and in vitro culture, compared with the conventional two-step culture system by a supplementation of porcine follicular fluid (pFF). Additionally, it is identified that the proposed chemically defined one-step culture system yielded the comparable level of blastocyst production to the conventional maturation system in porcine somatic cell nuclear transfer (SCNT). Therefore, one can eliminate un-expected effects accompanied by supplementation of pFF. No medium replacement during whole maturation period is an additional benefit by applying this new system. Thus, these data support that the developed PVA supplemented chemically defined one-step IVM system for porcine follicular oocyte might be used in porcine SCNT program.

Methods for activation of reconstructed oocytes were examined for the production of nuclear transfer (NT) rat embryos using fetal neural stem cells as donor. Neural stem cells were isolated from Day 14.5 rat fetuses, and the oocytes for recipient cytoplasm were recovered from 4-week old Sprague Dawley rats. After enucleation and nuclear injection, the reconstructed oocytes were immediately exposed to activation medium consisting of 10 mM SrCl₂ for 4 h (immediate activation after injection; IAI), or cultured in vitro for 2~3 h before activation treatment (injection before activation; IBA). Pre-activated oocytes were also used for NT to test reprogramming potential of artificially activated oocytes. The oocytes were grouped as IIA (immediate injection after activation) and ABI (activation 2~3 h before injection). Following NT, the oocytes were cultured in vitro. Development of the NT embryos was monitored at 44 and 119 h after activation. The embryos in groups IAI, mA, and IIA were cleaved to the 2-cell stage at the rates of 36.6%(15/41), 39.5% (17/43) and 46.3% (25/54), respectively. However, in the ABI group, only one embryo (1.8%, 1/55) was cleaved after activation. After in vitro culture, two NT embryos from IAI group had developed to the morula stage (4.9%, 2/41). However, no morula or blastocyst was obtained in the other groups. These results suggest that immediate activation after injection (IAI) method may be used for the production of rat somatic cell NT embryos.