The development of embryos reconstructed by nuclear transfer is dependent upon numerous factors including the type of recipient cell, method of enucleation, the type of donor cell, method of reconstruction, activation, the cell cycle stage of both the donor nucleus and the recipient cytoplasm and the method of culture of the reconstructed embryos. Many of these points which have been reviewed extensively elsewhere (Sun and Moor, 1995; Colman, 1999; Oback and Wells, 2002; Renard et al., 2002; Galli et al., 2003b), here we will concentrate on main area, the production of suitable cytoplast and nuclear donor, nuclear-cytoplasmic coordination, oocyte activation, culture of reconstructed embryos, and the effects that this may have on development.

This study was carried out to investigate effective condition for producing somatic cell nuclear transfer (SCNT) embryos of Jeju native cattle. As donor cells for SCNT, ear skin cells from Jeju native cattle were used. In experiment 1, the effect of recipient oocyte sources on the development of Jeju native cattle SCNT embryos were examined. Fusion rate of recipient oocyte and donor cell was not different between the Hanwoo and Holstein recipient oocytes (86.0% vs 89.9%). The rate of embryos developing to the blastocyst stage was significantly (p<0.05) higher in Hanwoo recipient oocytes than in Holstein recipient ones (28.2% vs 14.7%). Blastocysts derived from Hanwoo recipient oocytes contained higher numbers of total cells than those derived from Holstein ones ( vs ), although there were no significant difference. The mean proportion of apoptotic cells in blastocyst was not different between the sources of recipient oocytes. In experiment 2, the development of Jeju native cattle and Hanwoo SCNT embryos were compared. Hanwoo oocytes were used as the recipient oocytes. Fusion rate was not different between the Jeju native cattle and Hanwoo SCNT embryos (92.1% vs 92.9%). The blastocyst rate of SCNT embryos was significantly (p<0.05) lower in Jeju native cattle than in Hanwoo (16.9% vs 31.0%). Blastocysts derived from Jeju native cattle SCNT embryos contained smaller numbers of total cells than those derived from Hanwoo ones ( vs ), but there were no significant difference. The mean proportion of apoptotic cells in blastocyst was not different between the Jeju native cattle and Hanwoo SCNT embryos. The present study demonstrated that Hanwoo recipient oocytes were more effective in supporting production of Jeju native cattle SCNT embryos, although Jeju native cattle SCNT embryos showed reduced developmental capacity when compared to Hanwoo SCNT embryos.

This study was conducted to examine the optimal concentration and treatment time of antioxidants for inhibition of the ROS generation in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine oocytes were activated parthenogenetically, during which oocytes were treated with various antioxidants to determine the optimal concentrations and kind of antioxidants. Determined antioxidants were applied to oocytes during in vitro maturation (IVM) and/or SCNT procedures. Finally, antioxidant-treated SCNT embryos were compared with in vitro fertilized (IVF) embryos. H2O2 levels were analyzed in embryos at 20 h of activation, fusion or insemination by staining of embryos in 10 μM 2'7'-dichlorodihydrofluorescein diacetate (H2DCFDA) dye, followed by fluorescence microscopy. H2O2 levels of parthenogenetic embryos were significantly lower in 25 μM β- mercaptoethanol (β-ME), 50 μM L-ascorbic acid (Vit. C), and 50 μM L-glutathione (GSH) treatment groups than each control group (24.0±1.5 vs 39.0±1.1, 29.7±1.0 vs 37.0±1.2, and 32.9±0.8 vs 36.3±0.8 pixels/embryo, p<0.05). There were no differences among above concentration of antioxidants in direct comparison (33.6±0.9～35.2±1.1 pixels/embryo). Thus, an antioxidant of 50 μM Vit. C was selected for SCNT. H2O2 levels of bovine SCNT embryos were significantly lower in embryos treated with Vit. C during only SCNT procedure (26.4±1.1 pixels/embryo, p<0.05) than the treatment group during IVM (29.9±1.1 pixels/embryo) and non-treated control (34.3±1.0 pixels/embryo). Moreover, H2O2 level of SCNT embryos treated with Vit. C during SCNT procedure was similar to that of IVF embryos. These results suggest that the antioxidant treatment during SCNT procedures can reduce the ROS generation level of SCNT bovine embryos.

Epigenetic modification including genome-wide DNA demethylation is essential for normal embryonic development. Insufficient demethylation of somatic cell genome may cause various anomalies and prenatal loss in the development of nuclear transfer embryos. Hence, the source of nuclear donor often affects later development of nuclear transfer (NT) embryos. In this study, appropriateness of porcine embryonic germ (EG) cells as karyoplasts for NT with respect to epigenetic modification was investigated. These cells follow methylation status of primordial germ cells from which they originated, so that they may contain less methylated genome than somatic cells. This may be advantageous to the development of NT embryos commonly known to be highly methylated. The rates of blastocyst development were similar among embryos from EG cell nuclear transfer (EGCNT), somatic cell nuclear transfer (SCNT), and intracytoplasmic sperm injection (ICSI) (16/62, 25.8% vs. 56/274, 20.4% vs. 16/74, 21.6%). Genomic DNA samples from EG cells (n=3), fetal fibroblasts (n=4) and blastocysts from EGCNT (n=8), SCNT (n=14) and ICSI (n=6) were isolated and treated with sodium bisulfite. The satellite region (GenBank Z75640) that involves nine selected CpG sites was amplified by PCR, and the rates of DNA methylation in each site were measured by pyrosequencing technique. The average methylation degrees of CpG sites in EG cells, fetal fibroblasts and blastocysts from EGCNT, SCNT and ICSI were 17.9, 37.7, 4.1, 9.8 and 8.9%, respectively. The genome of porcine EG cells were less methylated than that of somatic cells (p<0.05), and DNA demethylation occurred in embryos from both EGCNT (p<0.05) and SCNT (p<0.01). Interestingly, the degree of DNA methylation in EGCNT embryos was approximately one half of SCNT (p<0.01) and ICSI (p<0.05) embryos, while SCNT and ICSI embryos contained demethylated genome with similar degrees. The present study demonstrates that porcine EG cell nuclear transfer resulted in hypomethylation of DNA in cloned embryos yet leading normal preimplantation development. Further studies are needed to investigate whether such modification affects long-term survival of cloned embryos.

The reactive oxygen species (ROS) generated during the somatic cell transfer nuclear (SCNT) procedures may cause the mitochondrial dysfunction and DNA damage, which may result in restricts the reprogramming of SCNT embryos and play a key direct role in apoptosis. The present study was conducted to investigate the effect of antioxidant treatment during the SCNT procedures on the inhibition of mitochondria and DNA damages in bovine SCNT embryos. The reconstituted oocytes were treated with antioxidants of 25 μM β-mercaptoethanol (β-ME) or 50 μM vitamin C (Vit. C) during the SCNT procedures. In vitro fertilization (IVF) was performed for controls. Mitochondrial morphology and membrane potential (ΔΨ) were evaluated by staining the embryos with MitoTracker Red or JC-1. Apoptosis was analyzed by Caspase-3 activity assay and TUNEL assay, and DNA fragmentation was measured by comet assay at the zygote stage. Mitochondrial morphology of non-treated SCNT embryos was diffused within cytoplasm without forming clumps, while the IVF embryos and antioxidant treated SCNT embryos were formed clumps. The ΔΨ of β-ME (1.3±0.1, red/green) and Vit. C-treated (1.4±0.2, red/green) SCNT embryos were significantly higher (p<0.05) than that of non-treated SCNT embryos (0.9±0.1, red/ green), which similar to that of IVF embryos (1.3±0.1, red/green). Caspase-3 activity was not difference among the groups. TUNEL assay also revealed that little apoptosis was occurred in SCNT embryos as well as IVF embryos regardless of antioxidant treatment. Comet tail lengths of β-ME and Vit. C-treated SCNT embryos (337.8±23.5 μm and 318.7 ±27.0 μm, respectively) were shorter than that of non-treated SCNT embryos (397.4± 21.4 μm) and similar to IVF embryos (323.3±10.6 μm). These results suggest that antioxidant treatment during SCNT procedures can inhibit the mitochondrial and DNA damages of bovine SCNT embryos.

Epigenetic status of the genome of a donor nucleus has an important effect on the developmental potential of cloned embryos produced by somatic cell nuclear transfer (SCNT). In our previous study has results showed that the donor cells treated with 5-aza-2’- deoxyctidine (5-aza-dC, DNA methylation inhibitors) and Trichostatin A (TSA, histone deacetylase inhibitors) could improve the development of porcine nuclear transfer embryos in vitro. In this study we want to investigate why these two drugs treatment with the donor cell can improve the cloning efficiency, whether they can alter the epigenetic status of the genome of the donor nucleus. This study included 6 groups: control group, the donor cell (porcine fetal fibroblast cell) with no treatment; 2.5 nM 5-aza-dC group, the donor cells treated with 2.5 nM 5-aza-dC for 1h; 5-aza-dC group, the donor cells treated with 5 nM 5-aza-dC for 1h; TSA group, the donor cells treated with 50 nM TSA for 1h; 2.5 nM 5-aza-dC+TSA group, the donor cells treated with 2.5 nM 5-aza-dC for 1h and subsequently treated with 50 nM TSA for another 1h; 5-aza-dC+TSA group, the donor cells treated with 5 nM 5-aza-dC and 50 nM TSA together for 1h. The first experiment detected the DNA methylation status in the different groups. After treatment with these two drugs, the DNA methylation level of the donor cells decreased, however there is no significant difference among the groups. This result indicated that the donor cell treatment with 5-aza-dC and TSA can partially alter the DNA methylation status of the donor cells. The second experiment checked the histone acetylation level of the donor cells treated with these two drugs by western blot. TSA, 2.5 nM 5-aza-dC+TSA, 5 nM 5-aza-aC+TSA, these three groups can significantly improve the hisone acetylation level compared with control and 5-aza-dC groups, there is no significant difference among these three groups. The results of this study suggest that the donor cells treated with 5-aza-dC and TSA can partially decrease DNA methylation and can significantly improve the histone acetylation level of the donor cells, these alterations of the epigenetic modification maybe can improve the clonging efficiency.

Cathepsins (CTSs), a family of lysosomal cysteine proteases, and their inhibitors (CSTs) play a critical role in remodeling of the uterine endometrium and placenta for the establishment and maintenance of pregnancy in many animal species including rodents, sheep, cow and pigs. It has been shown that the high rate of pregnancy failure by somatic cell nuclear transfer (SCNT) is associated with abnormal placental development. Our previous study has shown that CST6 is highly expressed in the uterine endometrium from mid to late pregnancy in pigs. In this study, to understand whether appropriate endometrial and placental tissue remodeling occurs in the uterine endometrium from gilts with conceptuses derived from SCNT during pregnancy in pigs, we investigated expression of CST6 in the uterine endometrium. Uterine endometrial tissues were obtained from gilts that carried SCNT-derived normal conceptuses (NT-No) and abnormal conceptuses (NT-Ab), and from gilts carrying conceptuses from natural mating (Non-NT) on D114 of pregnancy. Immunoblot analysis showed that CST6 protein levels in the endometrial tissues of gilts carrying NT-No were lower than those of gilts carrying Non-NT. The levels of CST6 protein in the endometrial tissues of gilts carrying NT-Ab decreased even more than those of gilts carrying NT-No. These results indicate that decreased expression of CST6 in the endometrium with NT-No and NT-Ab reflects inappropriate endometrial tissue remodeling and pregnancy failure of pigs with SCNT derived conceptuses and that CST6 plays an important role for the maintenance of pregnancy in pigs. * This work was supported by the Next Generation BioGreen 21 program (#PJ007997), RDA, Republic of Korea.

This study was to analyse the usability of morphological evaluation of vitrified-thawed oocyte before somatic cell nuclear transfer (SCNT) using Oosight imaging system to show spindle. For the vitrification, in vitro matured bovine MII oocytes were treated by two-step freezing medium without (control group) or with 5 ug/ml cytochalasin-b (CCB group). In Exp. 1, after thawing, recovered oocytes in each treatment group were assessed by live image using Oosight imaging system or/and cytoskeletal protein image using immunostaining. In Exp. 2, in each treatment group the in vitro developmental potential of frozen-thawed bovine oocytes post evaluation using Oosight imaging system and then SCNT was investigated. The SCNT embryos were cultured in CR1aa medium supplemented with 10% FBS, 1 mM EGF and 1 mM IGF at 38.5 C in 5% O2 and 5% CO2 in air for 8 days. In Exp.1, the rates of in vitro survival, morphological good grade and spindle normality of CCB treatment group (91.1%, 54.2% and 55.5%) were better than those of control group (86.1%, 48.5% and 48.5%). After SCNT using vitrified-thawed oocyte, the rates of fusion, reconstructed embryos and blastocyst development were also high in CCB treatment group (66.6%, 36.4% and 3.0%) than control group (60.0%, 27.3% and 0%). These results demonstrated that the identification of morphological spindle image of the vitrified-thawed bovine oocytes using Oosight imaging system helps to predict the SCNT embryo quality.

Limited success of somatic cell nuclear transfer(SCNT) is attributed to incomplete reprogramming of transferred donor cell. Several approachs, such as histone deacetylase inhibitors and DNA methyltransferase inhibitors have been used to improve the efficiency of somatic cell nuclear transfer. Recently, it is reported that pre-treatment of somatic cells with undifferentiated cell extract, such as embryonic stem cell and mammalian oocytes is an attractive alternative ways to reprogramming control. The aim of this study was to evaluate the early development of porcine cloned embryos produced with porcine ear skin fibroblasts pre-treated with extract from porcine induced pluripotent stem cell (iPSC). For transport of porcine iPSC extract into cultured porcine ear skin fibroblasts, the ChariotTM reagent system was used. Treated cells were cultured for 3 days, and used for the analysis of histone H3K9 acetylation and SCNT The acetylation status of H3K9 was increased in cells treated with iPSC extract and cultured for 3 days compared with control. But, no significant difference was observed between the extract treated and control groups. After SCNT. no difference was observed in the rate of fusion (86.6% vs 86.2%) and embryo cleavage (86.6% vs 87.1%) between the extract treated and control groups. Also, no significant difference was noted in blastocyst rates (23.4% vs 28.4%) as well as cell numbers (43.8±10.8 vs 41.2±11.6) with extract treated group compared with control group. Overall apoptosis rate in blastocyst was not differences between the extract treated and control groups (4.6±3.5% vs 6.0± 5.8%). However, blastocyst rate with high apoptotic cells(>10% appototic cells) was significantly lower in extract treated group when compared with control group (7.1% vs 21.8%).. Our results demonstrated that pre-treatment of porcine ear skin fibroblasts using porcine iPSc extract had beneficial effect on the decreasing apoptosis in the blastocyst cultured in vitro, although there was no effect on the embryonic development.

The aim of this study was to examine the effect of acteoside (the cyclin-dependent kinase inhibitor) on the SCNT efficiency with adult fibroblasts in dog. Canine adult fibroblasts were obtained from muscle and cell cycle of fibroblasts was synchronized by culturing to confluency, serum starvation and treating with 30 μM acteoside for 48 h. Cell cycle stages, cell cytotoxicity (apoptosis) and, prduction of reactive oxygen species (ROS) were analyzed using flow cytometry. The canine cells, prepared by confluent-cell culture or treating with 30 μM acteoside for 48 h, were injected into enucleated in vivo matured oocytes, the couplets were electrical fused and activated by calcium ionomycin. SCNT embryos using acteoside-treated fibroblasts were surgically transferred into oviducts of estrus cycle synchronized recipient dogs. In cell cycle synchronization (G0/G1), there was no significant difference between serum starvations (83.9%) and acteoside treated groups (81.3%) that were higher than confluent group (78.5%). In production of apoptosis, confluent and acteoside treated groups (4.3 and 4.5%, respectively) were generated less than serum starvation group (21.8%). In case of ROS, serum starvation group was induced a significantly higher than other groups. After synchronization of the donor cell cycle, either confluent or acteoside treated, cells were placed with enucleated in vivo-matured dog oocytes, fused by electric stimulation, activated, and transferred into naturally estrus-synchronized surrogates. Fusion and cleavage rate of acteoside treated group were 64.1 and 41.5%, which were higher than those of confluent group (53.9 and 20.6%, respectively). The reconstructed embryo development rates to 4-cell and 8-cell in acteoside treated group were 29.5 and 14.8%, respectively, while confluent group showed 11.1 and 3.2%, respectively. Total 54 SCNT embryos using acteoside-treated fibroblasts were transferred into oviducts of 2 recipient dogs and one recipient finally delivered one puppy, whereas din`t detected pregnancy on transfer of cloned embryos reconstructed with confluent cells in 6 surrogate dogs. In conclusion, the results of the current study demonstrated that canine fibroblasts could be successfully arrested at the G0/G1 stage with reduced the formation of ROS and apoptosis after acteoside treatment. This results may contribute to improve the effi-ciency of canine SCNT. * This research was supported by iPET (Grants 110056-3), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

The present study was conducted to examine the generation of reactive oxygen species (ROS) during micromanipulation procedures in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine enucleated oocytes were electrofused with donor cells, activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture. Oocytes and embryos were stained in dichlorodihydrofluorescein diacetate or 3'-(p-hydroxyphenyl) fluorescein dye and the H2O2 or ˙OH radical levels were measured. In vitro fertilization (IVF) was performed for controls. The samples were examined with a fluorescent microscope, and fluorescence intensity was analyzed in each oocyte and embryo. The H2O2 and ˙OH radical levels of reconstituted oocytes were increased during manipulation (37.2~49.7 and 51.0~55.2 pixels, respectively) as compared to those of mature oocytes (p<0.05). During early in vitro culture, the ROS levels of SCNT embryos were significantly higher than those of IVF embryos (p<0.05). These results suggest that the cellular stress during micromanipulation procedures can generate the ROS in bovine SCNT embryos.

The production of transgenic animals using somatic cell nuclear transfer (SCNT) has been widely described. A critical problem in the production of transgenic animals is the uncontrolled constitutive expression of the foreign gene which occasionally results in serious physiological disorders in the transgenic animal. In this study, we designed three different expression vectors that express the hEPO gene. hEPO is a hormone produced by the kidney that promotes the formation of red blood cells by the bone marrow. For the in vitro production of transgenic embryos, the different expression vectors were transduced into holstein ear fibroblast cells, respectively, and GFP expressed donor cells were transferred into enucleated oocytes, and then the reconstructed SCNT embryos were developed into pre-implantation stage. From three replicates, GFP expressed 112 transgenic SCNT embryos were produced. When their cleavage rate and blastocyst rate were compared with non-transgenic SCNT embryos, the results were presented into 73.2% vs. 76.9% and 26.8% vs. 30.6%, respectively, there were no differences. Also, total cell number and ICM cell numbers of day 8 blastocysts were statistically not different between the transgenic SCNT groups (120.6±7.9 and 31.4±8.2) and control SCNT group (128.3±4.8 and 35.3±4.0). The GFP expression levels were presented consecutively high during the culture of transgenic SCNT embryos. By analysis of semi-quantitative RT-PCR, the relative expression levels of hEPO mRNA and pluripotent gene were determined. These results demonstrated that the hEPO expressed transgenic bovine embryos can be efficiently produced in vitro by SCNT technique, while their potential of cloned animal production have to be examined in further study.

Pigs may be considered as a suitable organ source for its characteristics in xenotransplantation if significant immunological barriers can be overcome. However, xenograft could be rejected by T cells, especially CD8+ cytotoxic T lymphocytes (CTL)-mediated response, because these elements show great cytotoxicity against xenograft by recognizing Swine Leukocyte Antigen (SLA)-I. Human cytomegalovirus (HCMV) encodes unique short (US) 11 gene, which interferes with cellular immune responses by inducing rapid degradation of newly synthesized heavy chains (HC) of MHC class I from endoplasmic reticulum (ER) to the cytosol. In this study we established two US11 clonal cell lines by transfection into minipig fetal fibroblasts and confirmed the integration of US11 gene by PCR and FISH. The reduction of Swine Leukocyte Antigen (SLA)-I which was expressed on the cell surface by US11 was also detected by flow cytometry assay. The level (14.6 % to 21.2%) of SLA-I expression in US11 clonal cell lines was decreased relative to the control. The reconstructed embryos were produced with these clonal cells and transferred to nine surrogate gilts. Ultrasound examination of recipient surrogates on days 35 after embryo transfer confirmed established pregnancies in two recipients. One recipient delivered one piglet with normal birth weight. PCR analysis revealed that transgene vector was integrated in the offspring genome. Transgene-expression analysis and CTL assay are currently underway. The present results show that transgenic pig was produced with US11 cDNA for controlling cell-mediated rejection. This result indicated that grafts of transgenic pigs expressing human cytomegalovirus protein US11 could control the cellular immune response to xenografts, and create a window of opportunity to facilitate xenograft survival. This research was supported by the BioGreen 21 Program (#20110301-061-541- 001-05-00), Rural Development Administration, Republic of Korea.