We attempted to control the maturation promoting factors (MPF) activity and nuclear remodeling of somatic cell nuclear transfer (NT) bovine embryos. Bovine ear skin fibroblasts were fused to enucleated oocytes treated with either 5 mM caffeine for 2.5 h or 0.5 mM vanadate for 0.5 h and activated. The nuclear remodeling type of the reconstituted embryos was evaluated 1.5 h after activation. MPF activity was assessed in enucleated and chemical treated oocytes before the injection of a donor cell. Effect of chemicals on the embryonic development was evaluated with parthenogenetic embryos. MPF activity increased significantly by caffeine treatment, but decreased by vanadate treatment (p<0.05). Caffeine or vanadate had no deleterious effect on the parthenogenetic embryo development. In caffeine treated group, premature chromosome condensation (PCC) was occurred in 72.2% of NT embryos (p<0.05). In contrast, vanadate induced the formation of a pronucleus-like structure (PN) in a high frequency (68.9%, p<0.05) without PCC (NPCC). Blastocyst development of NT embryos increased by treating with caffeine (30.3%), whereas decreased by treating with vanadate (11.4%) compared to control (22.1%, p<0.05). The results indicate that caffeine or vanadate can control of MPF activity and remodeling type of NT embryos, resulting in the increased or decreased in vitro development.

Our goal was to examine the effects of early denudation on the enucleation efficiency and developmental competence of embryos following somatic cell nuclear transfer (SCNT) and parthenogenetic activation (PA). Oocytes were denuded following 30 h of in vitro maturation (IVM) and then cultured with (D+) or without (D-) their detached cumulus cells for additional h. Control oocytes were denuded after h of IVM. The size of the perivitelline space was larger at 40 h of IVM () than at 30 h ( p<0.01). The distances between the metaphase II (M II) plates and the polar bodies (PBs) were shorter in D+ () and D- oocytes () than in control oocytes ( p<0.01). Enucleation rates following blind aspiration at 40 h of IVM were higher (p<0.01) in D+ (92%) and D- oocytes (93%) compared to controls (82%). Early denudation did not affect oocyte maturation or the in vitro development of SCNT and PA embryos. When SCNT embryos from D+ oocytes were transferred to four gilts, pregnancy was established in two pigs, and one of them farrowed three live piglets. In conclusion, early denudation of oocytes at 30 h of IVM could improve the enucleation efficiency by maintaining the M II plate and the PB within close proximity and support the in vivo development of SCNT embryos to term.

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