The establishment of porcine embryonic stem cells (ESCs) from porcine somatic cell nuclear transfer (SCNT) blastocysts is influenced by in vitro culture day of porcine reconstructed embryo and feeder cell type. Therefore, the objective of the present study was to determine the optimal in vitro culture period for reconstructed porcine SCNT embryos and mouse embryonic fibroblast (MEF) feeder cell type for enhancing colony formation efficiency from the inner cell mass (ICM) of porcine SCNT blastocysts and their outgrowth. As the results, porcine SCNT blastocysts produced through in vitro culture of the reconstructed embryos for 8 days showed significantly increased efficiency in the formation of colonies, compared to those for 7 days. Moreover, MEF feeder cells derived from outbred ICR mice showed numerically the highest efficiency of colony formation in blastocysts produced through in vitro culture of porcine SCNT embryos for 8 days and porcine ESCs with typical ESC morphology were maintained more successfully over Passage 2 on outbred ICR mice-derived MEF feeder cells than on MEF feeder cells derived from inbred C57BL/6 and hybrid B6CBAF1 mice. Overall, the harmonization of porcine SCNT blastocysts produced through in vitro culture of the reconstructed embryos for 8 days and MEF feeder cells derived from outbred ICR mice will greatly contribute to the successful establishment of ESCs derived from porcine SCNT blastocysts.

The osmolarity of a medium that is commonly used for in vitro culture (IVC) of oocytes and embryos is lower than that of oviductal fluid in pigs. In vivo oocytes and embryos can resist high osmolarities to some extent due to the presence of organic osmolytes such as glycine and alanine. These amino acids act as a protective shield to maintain the shape and viability in high osmotic environments. The aim of this study was to determine the effects of glycine or/and alanine in medium with two different osmolarities (280 and 320 mOsm) during IVC on embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) in pigs. To this end, IVC was divided into two stages; the 0-2 and 3-7 days of IVC. In each stage, embryos were cultured in medium with 280, 320, or 360 mOsm and their combinations with or without glycine or/and alanine according to the experimental design. Treatment groups were termed as, for example, "T(osmolarity of a medium used in 0-2 days of IVC)-(osmolarity of a medium used in 3-7 days of IVC)" T280-280 was served as control. When PA embryos were cultured in medium with various osmolarities, T320-280 showed a significantly higher blastocyst formation (29.0%) than control (22.2%) and T360-360 groups (6.9%). Glycine treatment in T320-280 significantly increased blastocyst formation (50.4%) compared to T320-280 only (36.5%) while no synergistic was observed after treatment with glycine and alanine together in T320-280 (45.7%). In contrast to PA embryonic development, the stimulating effect by the culture in T320-280 was not observed in SCNT blastocyst development (27.6% and 23.7% in T280-280 and T320-280, respectively) whereas the number of inner cell mass cells was significantly increased in T320-280 (6.1 cells vs. 9.6 cells). Glycine treatment significantly improved blastocyst formation of SCNT embryos in both T280-280 (27.6% vs. 38.0%) and T320-280 (23.7% vs. 35.3%). Our results demonstrate that IVC in T320-280 and treatment with glycine improves blastocyst formation of PA and SCNT embryos in pigs.

Polo-like kinase 1 (Plk1) has multiple roles in somatic cell and mammalian oocyte division. In mice, Plk1 distributes to the centromeres from prophase to anaphase and compose spindle apparatus in mitosis stages. Somatic cell nuclear transfer (SCNT) has diverse advantages. However, low cloning efficiency of SCNT procedure causes difficulty to application. The causes of this low efficiency are still unclear. However, they are attributed to the cumulative results of several biological and technical factors. In this study, Plk1, a biological factor, was investigated. B6D2F1 mice (7 weeks old) were superovulated with 10 IU of pregnant mare’s serum gonadotropin and 9 U of human chorionic gonadotropin (HCG) 48 hr later. The oocytes were collected 14 hr after HCG injection and cultured on potassium simplex optimized medium. The BI2536, Plk1-specific inhibitor, was used to understand the influence of Plk1. Also, the embryos were assessed by immunofluorescence. All BI2536-treated embryos failed to the first mitotic division. It showed Plk1 has a critical role in the first mitotic division of the mouse embryo. Moreover, there were significant differences between the control and SCNT embryos in the patterns of Plk1. All SCNT embryos which failed 2-cell development presented incorrect positioning and low expression of Plk1. On the other hand, the control embryos which failed to 2-cell division showed only low expression of Plk1. Taken together, this results demonstrate that Plk1 is critical for successful mitotic division of mouse embryos. Also, correct localization of Plk1 has crucial effect in the development of murine SCNT embryos.

The purpose of this study is to examined the electrofusion and activation conditions for the production of porcine somatic cell nuclear transfer (SCNT) embryos. In this study, immature oocytes were cultured in TCM-199 with and without hormones for 22 hours. Skin fibroblasts cells of porcine were transferred into the perivitelline space of enucleated in vitro matured oocytes. Cell fusion was performed with two different pulses that each one pulse (DC) of 1.1 kV/cm or 1.5 kV/cm for 30 μsec. After fusion subsequent activation were divided into three groups; non-treatment (control) and treatment with 2 mM 6-DMAP or 7.5 μg/ml cytochalasin B for 4 hours. Transferred embryos were cultured in PZM-3 (Porcine Zygote Medium-3) in 5% CO₂ and 95% air at 39℃ for 7 day. Apoptosis-related genes (Caspase-3, BCL-2, mTOR, and MMP-2) were analyzed by immunofluorescence staining. There was no significant difference between two different electrofusion stimuli in the cleavage rate; 64.9±4.8% in 1.1 kV/cm and 62.7±4.0% in 1.5 kV/cm. However, blastocyst formation rate (%) was significantly different among three different activation groups (no treatment, 2 mM 6-DMAP or 7.5 μg/ml cytochalasin B) combined with electrofusion of 1.1 kV/cm. The blastocyst formation rate was 12.6±2.5, 20.0±5.0, and 34.9±4.3% in control, 2 mM 6-DMAP, and 7.5 μg/ml cytochalasin B, respectively. Immunofluorescence data showed that expression levels of caspase-3 in SCNT embryos undeveloped to blastocyst stage were higher than those in the blastocyst stage embryos. Expression levels of Bcl-2 in blastocyst stage embryos were higher than those in the arrested SCNT embryos. These results showed that the combination of an electric pulse (1.1 kV/cm for 30 μsec) and 7.5 μg/ml cytochalasin B treatment was effective for production of the porcine SCNT embryos.

Somatic cell nuclear transfer (SCNT) is the technique which generates embryos by transferring diploid nucleus into an enucleated oocyte, it has produced specific animals successfully in a variety of species. However, the developmental capacity of SCNT embryos is still relatively lower than that of embryos produced in vivo. Oocyte is a kind of lipid rich cells, its quality limits the efficiency of embryo production. L-carnitine is a co-enzyme facilitating the transportation of long chain fatty acids across the inner mitochondria membrane where fatty acids are used for generating adenosine triphosphate (ATP) via beta-oxidation. It also has antioxidant actions which may protect mitochondrial membranes and DNA against damage induced by reactive oxygen species (ROS). Whether L-carnitine is functional in bovine SCNT embryos are unknown. Therefore, the objective of this study was to examine the effects of L-carnitine on oocyte maturation and developmental competence of subsequent SCNT embryos. L-carnitine was supplemented during IVM, then intracellular ROS and GSH levels, mitochondrial activity, gene expression of COCs were analyzed at the end of IVM. SCNT embryos were produced subsequently, apoptosis detection and gene expression evaluation were performed in blastocysts. In the results, treatments with 1.5 mM and 3 mM L-carnitine significantly improved maturation rates (P<0.05). Treatments with 3 mM L-carnitine effectively induced improvement in nuclear maturation, intracellular GSH levels and mitochondrial activity, as well as a reduction in intracellular ROS levels (P<0.05). mRNA levels of CPT1A, ACAA1, ACAA2, AREG, EREG, SOD1, GPX4, GLUT1 and CDC2 transcripts were effectively up-regulated by 3 mM L-carnitine treatments (P < 0.05). Similarly, 3mM L-carnitine induced an increase in blastocyst developmental rates and an improvement in blastocyst quality (P<0.05). Our study indicates that L-carnitine treatment during IVM improves oocyte nuclear maturation and subsequent SCNT embryo development.

Polo-like kinase 1 (plk1) shows multiple events of somatic cell and mammalian oocyte division. In mice, Plk1 distributes to the centromeres from prophase to anaphase and compose spindle apparatus at different stages of mitosis in spindle organization. Somatic cell nuclear transfer (SCNT) has a number of advantages however it is difficult to apply to basic or translational researches due to its low cloning efficiency. The causes of this low cloning efficiency are unclear. However, they are attributed to the cumulative results of several biological and technical factors. In this study, a biological factor plk1 was investigated. B6D2F1 mice (7–8 weeks old) were superovulated with 10 IU of pregnant mare’s serum gonadotropin and 9 U of human chorionic gonadotropin (HCG) 48 hr later. The oocytes were then collected 14 hr after HCG injection and cultured on potassium simplex optimized medium (KSOM). The plk1-specific inhibitor BI2536 was used to understand the influence of plk1. The 2-cell stage embryos were assessed by fluorescence immunoassay. In consequence, all BI2536-treated embryos failed in the first mitotic division which showed plk1 have critical role in the first mitotic division of the mouse embryo. SCNT requires enucleation of oocyte and injecting a donor cell into the enucleated cytoplast. In this process, a respectable amount of plk1 that co-localize with nucleus may be removed together. Fluorescence immunoassay and qPCR were used to monitor the change of plk1 level during SCNT. There was significant difference between the control and enucleated embryos in the level of plk1. In all division-failure 2-cell embryos, incorrect positioning of plk1 was found. Taken together, this results demonstrate that plk1 is critical for successful mitotic division of mouse SCNT 1-cell embryos.

The nature of molecular mechanisms governing embryo development is largely unknown, but recent reports have demonstrated that proper execution of programmed cell death is crucial for this process. The main objective of this study is to examine the mode of programmed cell death during nuclear transfer embryos development in porcine. In particular, the relative employment of two major pathways in programmed cell death; e.g. apoptosis (type I) and autophagy (type II) was compared. Oocytes use in the study was matured in vitro in the presence of 10% FBS maturation medium. After nuclear transfer embryos were cultured for each programmed cell death control factor [Cysteamine(Cyst : 0.4mM), 3-methyladenine(3MA : 2.5mM) and Rapamycin(RP : 100nM)] in TCM-199 medium supplemented with 0.1% BSA. In this study results of among the blastocysts development in 3MA; PCNA, MAP1LC3A and ATG5 RNA gene expression level increased in the order of IVF<Cyst < 3MA < RP. However Casp-3 and TNF-r RNA gene expression level decreased in the order of IVF < 3MA and RP< Cyst. The expression of mTOR showed a pattern opposite to that of MAP1LC3A. That is, its expression was the lowest in Cyst group. And next experiments analysis of MMP expression patterns. Analysed this MMPs enzyme activation to evaluate the effectiveness of high quality brastocyst culture in porcine. In this results of the enzymatic activity of MMP-2 and MMP-9 was assessed in culture, the level of active MMP-9 was higher expression in the medium of each 3MA and RP treatment group, with the level of another treatment group being relatively higher. These results suggest that the autophagy activation culture medium is more effective for stable and innovative nuclear transfer embryos development.

Abnormal epigenetic reprogramming of donor nuclei is supposed to be one of the factors that causes low development efficiency of mammalian somatic cell nuclear transfer (SCNT). Trichostatin A (TSA) is an inhibitor of histone acetylase, and so development of SCNT embryos could be increased by treatment with TSA. In the present study, we examined the effect of TSA on in vitro development of porcine embryos derived from NT (nuclear transfer) by investigating the status of histone acetylation in TSA-treated and control NT embryos and the expression of developmental related genes. In this study, we found that incubating　NT embryos with 40nM TSA for 24h after activation could improved the blastocyst formation rate from 13.7% to 32.5%. Thechange in histone acetylation level as a reslut of TSA treatment were validated using immunofluorescence and confocal microscopy. Immunofluorescence results indicated that the level of aetylation at histone 3 lysine 18 (AcH3K18) was increased at early embryo development stage after TSA treatment. furthermore, we compared the expression patterns of several genes (developmental related genes; Oct4, Sox2, Nanog, Cdx2, the imprinting genes; igf2r). TSA treatment improved the expression of development related genes such as Oct4, Cdx2, Nanog as well as the imprinted genes like igf2r. In conclusion, our results demonstrated that TSA treatment improves the in vitro development of porcine NT embryos, increased the global histone acetylation (AcH3K18) and enhances the expression of some developmentally important genes (Oct4, Cdx2, Nanog) at blastocyst stages.

Somatic cell nuclear transfer (SCNT) technique is a key point of producing transgenic animal disease models. During in vitro production of SCNT embryo, the quality of matured oocytes are one of the important factors that regulate embryo developmental capacity. In preliminary test, we confirmed the effect of fibroblast growth factor 10 (FGF10) on porcine oocyte maturation. In this study, we investigated the developmental potential of SCNT embryos treated with the 10 ng/ml FGF10 (10 F) during in vitro maturation of recipient oocytes. The polar body emission rate was significantly higher in the 10 F treated group than control group. After SCNT, although the rate of fusion was no significant difference, the rate of cleavage and blastocyst formation was significantly increased in the 10 F treated group (p<0.05). In 10 F treated group, the total cell number was increased and the percentage of apoptotic cell was decreased in the blastocyst stage at day 7 (p<0.1). The transcription level of apoptosis relative gene, Casp3 was significantly decreased, while anti-apoptosis gene BCL2l1 was increased in the 10 F treated group compared to control group. The 10 F treated group was highly expressed the reprogramming related genes, Sox2 and POU5f1. Also, the first cleaving time was more faster and the percentage of cell block was significantly lower in 10 F treated group than in control group. In this study, we confirmed that 10 ng/ml FGF10 has effect on enhance the oocyte maturation and developmental capacity. These results demonstrate that FGF10 treatment can be used for in vitro development of porcine SCNT embryos and subsequent production of transgenic animal model.

Understanding the behavior of transgenes introduced into oocyte or embryos is essential for evaluating the methodologies for transgenic animal production. To date, many studies have reported the production of transgenic pig embryos with, however, low efficiency in environment of blastocyst production. The aim of present study was to determine the expression and duration of transgene transferred by intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT). Embryos obtained from the ICSI-MGT procedure were analysed for the expression of GFP and then for the transmission of the transgene. Briefly, fresh spermatozoa were bound to exogenous DNA after treatment by Triton X-100 and Lipofectin. When ICSI-MGT was performed using sperm heads with tails removed, the yield of blastocyst (25.3%), treated with Lipofectin (18.8%) and Triton X-100 (19.2%) were observed. Treatments of Lipofectin or Triton X-100 did not further improve the rates of blastocysts. Moreover, the apoptosis rates of embryos were obtained from the control and LIpofectin groups (8.7%, 9.7%, respectively), but were significantly higher in the Triton X-100 group (13.0%). Our results demonstrated that ICSI-MGT caused minimal damage to oocytes that could develop to full term. Moreover, the embryos derived by ICSI-MGT have shown prolonged exogenous DNA expression during preimplantation stage in vivo. However, more efforts will be required to improve the procedures of both sperm treatments cause of high frequency of mosaicisms.

The cloning efficiency is extremely low despite successful somatic cell nuclear transfer (SCNT) method producing cloned animals in several mammals. In general, faulty epigenetic modifications underlying the incomplete reprogramming of donor cell nuclei after SCNT mainly results in low cloning efficiency. The nuclear reprogramming process involves epigenetic modifications, such as DNA demethylation and histone acetylation, which may be an important factor in enhancing 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 expression (Oct4, Nanog and Sox2) in porcine cloned blastocysts and histone acetylation pattern (H3K9ac). The porcine cloned embryos were treated with a 50nM concentration of TSA and 100μM 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 expression in NT blastocyst also, level of histone acetylation in zygote, 2cell, 4cell stage. As results, Although NT, TSA and CBHA treated NT embryos were not different between all groups for cleavage rates, the developmental competence to the blastocyst stage was significantly increased in CBHA treated embryos (22.7%) compared to that of normal NT and TSA treated NT embryos (8.1% and 15.4%)(p<0.05). In addition, all of pluripotent transcription factors (Oct4, Nanog and Sox2) were expressed in the CBHA treated NT embryos, however, Sox2 and Oct4 were expressed in TSA treated NT embryos and expression pattern of CBHA treated NT embryos is particularly similar to that of IVF embryos. Also, CBHA treated NT embryos were increased in level of histone acetylation (H3K9ac) at the zygote, 2-cell, 4-cell stage compared to those of NT and TSA treated NT embryos. 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(Oct4, Nanog and Sox2) in NT blastocysts and level of histone acetylation (H3K9ac).

We investigate the effect of L-glutathione (GSH), an antioxidant, treatment during the somatic cell nuclear transfer (SCNT) procedures on the in vitro development and DNA methylation status of bovine SCNT embryos. Bovine in vitro matured (IVM) oocytes were enucleated and electrofused with a donor cell, then activated by a combination of Ca-ionophore and 6-dimethylaminopurine. The recipient oocytes or reconstituted oocytes were treated with 50 μM GSH during these SCNT procedures from enucleation to activation treatment. The SCNT embryos were cultured for 7 days to evaluate the in vitro development, apoptosis and DNA methylation in blastocysts. The apoptosis was measured by TUNEL assay and caspase-3 activity assay. Methylated DNA of SCNT embryos at the blastocyst stages was detected using a 5-methylcytidine (5-MeC) antibody. The developmental rate to the blastocyst stage was significantly higher (P<0.05) in GSH treatment group (32.5±1.2%, 78/235) than that of non-treated control SCNT embryos (22.3±1.8%, 50/224). TUNEL assay revealed that the numbers of apoptotic cells in GSH treatment group (2.3±0.4%) were significantly lower (P<0.05) than that of control (3.8±0.6%). Relative caspase-3 activity of GSH treated group was 0.8±0.06 fold compared to that of control. DNA methylation status of blastocysts in GSH treatment group (13.1±0.5, pixels/ embryo) was significantly lower (P<0.05) than that of control (17.4±0.9, pixels/embryo). These results suggest that antioxidant GSH treatment during SCNT procedures can improve the embryonic development and reduce the apoptosis and DNA methylation level of bovine SCNT embryos, which may enhance the nuclear reprogramming of bovine SCNT embryos.

Developmental potential of cloned embryos is related closely to epigenetic modification of somatic cell genome. The present study was to investigate the effects of applying histone deacetylation inhibitor, trichostatin A (TSA) to activated porcine embryos on subsequent development of porcine parthenogenetic and nuclear transfer embryos. Electrically activated oocytes were treated with 5 nM TSA for different exposure times (0, 1, 2 and 4 hr) and then the activated embryos were cultured for 7 days. The reconstructed embryos were treated with different concentrations of 0, 5, 10 and 25 nM TSA for 1 hr. Also 5 nM TSA was tested with different exposure times of 0, 0.5, 1, 2 and 4 hr. And fetal fibroblast cells were treated with 50 nM TSA for 1, 2 or 4 hr and with 5 nM TSA for 1 hr. Cumulus-free oocytes were enucleated and reconstructed by TSA-treated donor cells and electrically fused and cultured for 6 days. In parthenogenetic activation experiments, 5 nM TSA treatment for 1 hr significantly improved the percentage of blastocyst developmental rates than the other groups. Total cell number of blastocysts in 1 hr group was significantly higher than other groups or control. Similarly, blastocyst developmental rates of porcine NT embryos following 5 nM TSA treatment for 1 hr were highest. And the reconstructed embryos from donor cells treated by 50 nM TSA for 1 hr improved the percentage of blastocyst developmental rates than the control group. In conclusion, TSA treatment could improve the subsequent blastocyst development of porcine parthenogenetic and nuclear transfer embryos.

Embryo reconstruction by somatic cell nuclear transfer (SCNT) has been used to demonstrate that mammalian somatic cells can dedifferentiate into a totipotent nucleus when introduced into an oocyte. This process of dedifferentiation of somatic cells after nuclear transplantation is defined as nuclear reprogramming, although this terminology gives little information on the molecular events that characterize this process. When planning on strategies for nuclear reprogramming by nuclear transfer (NT) one may suggest that converting the somatic nuclear configuration into an embryonic state is highly desirable, so this may promote a succession of events similar to those occurring during early embryo development. In the following part of this overview we will discuss the results of many studies that have investigated different aspects of nuclear remodelling after SCNT.

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.

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.

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.