핵이식(NT) 기술을 이용하여 여러 동물 종에서 성공적으로 복제산자가 보고되고 있지만, 아직까지 비효율적인 기술로 남아있다. 본 연구에서는 돼지 체세포 복제 생산 효율성을 증진시키기 위한 방안으로 수핵난자의 품질에 초점을 맞추어 Brilliant cresyl blue (BCB) 염색을 통하여 발육능이 우수한 미성숙 난자를 선발하고, 난자의 감수분열 재개에 관여하는 단백질 합성을 비특이적으로 억제하는 cycloheximide (CHXM)을 이용하여 돼지 난자의 감수분열 재개를 억제시켜 난자의 성숙 동기화를 유도하였다. 또한 핵초기화에 밀접한 영향을 주는 핵막붕괴(NEBD)와 조기염색체응축 (PCC)을 유도하는 MPF의 활성화를 높이기 위하여 단백질 phosphatase 억제제인 caffeine을 첨가하여 수핵난자의 품질을 향상시키고자 하였다. 실험 방법으로는 13 mM BCB 첨가된 배양액에 90분 동안 미성숙난자를 배양하여 BCB 용액의 착색 여부를 구분하여 선발하고, 5 ㎍/ml CHXM를 체외 성숙액에 첨가하여 난자성숙 동기화를 유도하였다. 또한 탈핵 후 탈핵난자를 caffeine을 처리하여 세포주기 관련 단백질의 활성화를 인위적으로 조절하여 체세포복제 수핵난자로 사용하였다. 실험 결과로서 BCB 염색 돼지 미성숙 난자를 대조구와 비교할 때 제2 감수분열 중기(MII)에 도달하는 체외성숙율과 단위발생란의 배반포기까지의 체외 발육율이 유의적으로 증가하는 것이 관찰되었다. 또한 미성숙 돼지 난자의 초기 성숙 (12∼16시간)에 CHXM를 처리하였더니 난자 감수분열 재개가 억제되어 GV기에 핵 성숙이 정지되어 동기화가 유도되었다. GV기에 세포주기 동기화된 난자들은 CHXM를 제거하였을 때 난자 성숙의 진행속도도 일치하는 것이 관찰되었다. 이런 결과는 가장 적합한 탈핵시기인 제1 감수분열 후/말기(AI/TI)에 난자들이 다수 분포하여 대조구에 비하여 높은 탈핵율 (87.9%)을 얻을 수 있었다 (P < 0.05). 덧붙여 5 mM의 caffeine을 돼지 난자에 12시간 처리하였을 때 난자 MPF의 활성화가 증가하는 것이 관찰되었지만 (P < 0.05), 10 mM caffeine 농도를 처리하였을 때 MPF의 활성화가 오히려 감소되어 단위발생란의 배반포기까지의 체외발육에도 악영향을 주는 것이 관찰되었다.

The technique of SCNT is now well established but still remains inefficient. The in vitro development of SCNT embryos is dependent upon numerous factors including the recipient cytoplast and karyoplast. Above all, the metaphase of the second meiotic division (MII)　oocytes have typically become the recipient of choice. Generally high level of MPF present in MII oocytes induces the transferred nucleus to enter mitotic division precociously and causes NEBD and PCC, which may be the critical role for nuclear reprogramming. In the present study we investigated the in vitro development and pregnancy of White-Hanwoo SCNT embryos treated with caffeine (a protein kinase phosphatase inhibitor). As results, the treatment of 10 mM caffeine for 6 h significantly increased MPF activity in bovine oocytes but does not affect the developmental competence to the blastocyst stage in bovine SCNT embryos. However, a significant increase in the mean cell number of blastocysts and the frequency of pregnant on 150 days of White-Hanwoo SCNT embryos produced using caffeine treated cytoplasts was observed. These results indicated that the recipient cytoplast treated with caffeine for a short period prior to reconstruction of SCNT embryos is able to increase the frequency of pregnancy in cow.

Since embryonic stem cells (ESCs) were first established from explant cultures of in vivo day 3.5 mouse embryos, the establishment of ESCs from species such as primates and rat has been developed. However, this success relies on the development of culture medium suitable for human and rat cells, which has different requirements from the murine ESC. In general, the establishment of ESC from pig and cow is of great interest both the agricultural perspective and for biomedical application. Large animal models, particularly pig, are likely to provide models of human genetic diseases and transplantation research where rodent models are inappropriate. However, establishment of ESCs establishment from pigs has remained an elusive goal. In the present study, we focused on signaling transduction regulation in pig epiblast stem cells (pEpiSCs). Pig epiblasts were isolated from early tubular stage embryos collected in vivo day 10.5～12 after insemination. Epiblasts were separated from trophoblast and underlying primitive endoderm using 21G needles and fine forceps. Epiblasts were cultured on mitomycin C (10 μl/ml) treated mouse embryonic feeder cells in Dulbecco’s modified Eagle’s medium (DMEM) containing 1% minimal essential medium (MEM) nonessential amino acids, 1% penicillin/ streptomycin, 1% glutamine, 0.007% β-mercaptoethanol, 5 ng/ml bFGF and 1 ng/ml LIF. After plating rapid differentiation of isolated epiblasts to extraembryonic cell types was visualized in most cultures but stem cells were enclosed by these differentiated cells. We have established seven pig epiblast stem cells lines (pEpiSC1-7) from Days 10.5–12 pig embryos. pEpiSC expressed the pluripotent markers including OCT4, NANOG, SOX2 and NODAL at 3-5 passage. In addition, the modification of culture condition by the inclusion of particular protein kinase inhibitor such as Akt inhibitor, PD0325091(PD), delyed rapid differentiation of pEpiSCs. These results showed that stemness of pEpiSCs can be maintained by regulation of signaling pathway. * This work was partly supported by a grant from the NPR (2011-0013703) and the Next-Generation BioGreen 21 Program (No. PJ008209), Rural Development Administration, Republic of Korea.

Somatic cell nuclear transfer (SCNT) and induced pluripotent stem cell (iPS) experiments have generally demonstrated that a differentiated cell directly converts into a undifferentiated or pluripotent state. In SCNT experiment, nuclear reprogramming is induced by exposure of introduced donor cell nuclei to the recipient cytoplasm of matured oocytes. Although nuclear reprogramming of cells by the ex-ovo methods is not always consistent or efficient, it has been suggested that a combination of nuclear reprogramming technique may improve the efficiency or frequency of normal development of SCNT embryos. Here we hypothesized that treatment of somatic cells with extracts prepared from GV stage sturgeon's oocytes prior to their use as nuclear donors for SCNT will improve subsequent development. We reported a reversible permeabilization protocol with digitonin to deliver sturgeon oocyte exteact (SOE) to porcine fetal fibroblast cell nuclei ex ovo. Porcine fibroblasts were permeabilized by 4 μg/ml of digitonin for 2 min at 4℃ and then incubated in SOE for 7h at 15 18℃ followed by resealing of cell membrane. As results, no difference was observed in the number of fused couplets or the number of fused couplets that cleaved between the extract treated or control group. However, there was a significantly decrease in the percentage of fused couplets that developed to the blastocyst stage in the SOE treated group (p<0.05). Histone acetylation status was determined using an antibody to acetylation at lysine 9 on histone 3 (H3K9Ac). The intensity of H3K9Ac staining in 1-cell stage NT embryos was significantly increased when treated with the SOE (p<0.05), similar to that in 1-cell stage IVF embryos. In addition, porcine NT embryos reconstructed by using donor cell exposed to SOE prior to cell fusion significantly decreased developmental competence to the blastocyst stage but increased pluripotent gene expressions (Sox2, Nanog and Oct3/4) when compared with those in normal NT embryos (p<0.05).

Although somatic cell nuclear transfer (SCNT) has successfully been produced cloned animals in several species, the cloning efficiency is extremely low. It is generally believed that the low cloning efficiency is mainly attributed to faulty epigenetic modifications underlying the aberrant reprogramming of donor cell nuclei in recipient cytoplasm after SCNT. The nuclear reprogramming process involves epigenetic modifications, such as DNA demethylation and histone acetylation, which may be a key factor in improving the cloning efficiency. Recently, the histone deacetylase inhibitors (HDACi), such as trichosatin A (TSA) and m-carboxycinnamic acid bishydroxamide (CBHA), to increase histone acetylation have been used to improve the developmental competence of SCNT embryos. Therefore, we compared the effects of TSA with CBHA on the in vitro developmental competence and pluripotency-related gene expressions (Nanog, Oct3/4 and Sox2) in porcine cloned blastocysts. The porcine cloned embryos were treated with a 50 nM concentration of TSA or a 100 μm concentration of CBHA during the in vitro early culture (10h) after cell fusion and then were assessed to cleavage rate, development to the blastocyst stage and pluripotency-related gene expressions in NT blastocysts. All data was analyzed by chi-square. Following 4-5 replicates (245, 200 and 222 for NT, TSA and CBHA treated NT embryos respectively) there was no difference between normal NT and CBHA treated NT embryos, whereas TSA treated NT embryos was significantly decreased for cleavage rate (p<0.05). The developmental competence to the blastocyst stage in CBHA treated NT embryos (18.9%) significantly increased than that of normal NT and TSA treated NT embryos (9.4% and 11.5%) (p<0.05). In addition, all of pluripotent transcription factors (Nanog, Oct3/4 and Sox2) were highly expressed in the CBHA treated NT embryos, however, Sox2 and Oct3/4 were expressed in TSA treated NT embryos and Sox2 was only expressed in normal NT embryos (p<0.05). In conclusion, the treatment of CBHA as a histone deacetylase inhibitor significantly increased the developmental competence of porcine NT embryos and pluripotency- related gene expressions (Nanog, Oct3/4 and Sox2) in NT blastocysts.