In general, cloned pigs have been produced using the somatic cell nuclear transfer (SCNT) technique with various types of somatic cells; however, the SCNT technique has disadvantages not only in its low efficiency but also in the development of abnormal clones. This study aimed to compare early embryonic development and quality of SCNT embryos with those of induced pluripotent stem cells (iPSCs) NT embryos (iPSC-NTs). Ear fibroblast cells were used as donor cells and iPSCs were generated from these cells by lentiviral transduction with human six factors (Oct4, Sox2, c-Myc, Nanog, Klf4 and Lin28). Blastocyst formation rate in iPSC-NT (23/258, 8.9%) was significantly lower than that in SCNT (46/175, 26.3%; p < 0.05). Total cell number in blastocysts was similar between two groups, but blastocysts in iPSC-NT had a lower number of apoptotic cells than in SCNT (2.0 ± 0.6 vs. 9.8 ± 2.9, p < 0.05). Quantitative PCR data showed that apoptosis-related genes (bax, caspase-3, and caspase-9) were highly expressed in SCNT than iPSC-NT (p < 0.05). Although an early development rate was low in iPSC-NT, the quality of cloned embryos from porcine iPSC was higher than that of embryos from somatic cells. Therefore, porcine iPSCs could be used as a preferable cell source to create a clone or transgenic animals by using the NT technique.

This experiment was conducted to analyse the effects of flavone supplementation on the preimplantation development of in-vitro produced porcine embryos. During in-vitro development, immature oocytes and early embryos were exposed to different concentrations of flavone (0, 1μM, 25μM, 50 μM, and 100 μM respectively). Results showed that 100 μM of flavone significantly reduced the intracellular ROS levels of oocytes accompanied with a significant rise in GSH level. In parthenogenesis, no significant change was observed in the cleavage rates whether flavone was supplemented in IVM or IVC media. In IVM supplemented group, the blastocyst development rate was significantly enhanced by 1 μM concentration than other groups (51.5% vs. 41.3%, 44.0%, 36.3%, 31.7%; P<0.05) respectively. However, in IVC group 1 μM concentration significantly improved the blastocysts production than 50 μM and control groups (50.0% vs. 40.5%, 38.0%; P<0.05) respectively. Following nuclear transfer, the cleavage rate of IVM group was significantly more in 1 μM than 50 μM and 100 μM groups (92.9% vs. 89.7%, 87.8%; P<0.05), followed by similar pattern of cloned blastocysts production being significantly higher in 1 μM group than 50 μM, 100 μM and control groups (16.8% vs. 9.0%, 7.1%, 12.8%; P<0.05) respectively. In IVC group, 1 μM concentration resulted in significantly higher cleavage rate than 25 μM and 50 μM groups (91.7% vs. 87.8%, 88.8%; P<0.05) respectively. However, the blastocysts production was significantly higher in 100 μM group than others (26.2% vs. 13.6%, 14.0%, 18.2%; P<0.05) respectively. The optimal concentrations of flavone significantly enhanced the percentages of ICM:TE than control group (43.8% vs. 37.6%; P<0.05) accompanied with significantly higher expression levels of reprogramming related genes. In conclusion, the optimal concentrations of 1 μM during IVM and 100 μM during IVC can significantly improve the production of porcine in-vitro embryos.

Mitochondrial dysfunction is found in oocytes and transmitted to offspring due to maternal obesity. Treatment of obese mothers with endoplasmic reticulum (ER) stress inhibitors such as salubrinal (SAL) can reverse the mitochondrial dysfunction and result in normal embryonic development. Pig oocytes have also shown ER stress mostly in metaphase II stage. ER stress in oocytes may hinder the in vitro production of pig embryos. This study investigated the effect of ER stress inhibition by SAL treatment during in vitro maturation (IVM) of porcine oocytes at 1, 10, 50 and 100 nM concentrations. Firstly, we tested various concentrations of SAL. SAL at 10 nM showed higher (P < 0.05) developmental competence to the blastocyst stage (55.6%) after parthenogenesis (PA) than control (44.2%) while not different from other concentrations (49.2, 51.6, and 50.8% for 1, 50, and 100 nM, respectively). Secondly, we performed time-dependent treatment at 10 nM of SAL for IVM of oocytes. It revealed that treatment with SAL during 22 to 44 h of IVM significantly improved PA embryonic development to the blastocyst stage compared to control (40.5, 46.3, 51.7 and 60.2% for control, 0 to 22 h, 22 to 44 h and 0 to 44 h of IVM, respectively, P < 0.05). Glutathione (GSH) content is an indicator of cytoplasmic maturation of oocytes. Reactive oxygen species (ROS) have a harmful effect on developmental competence of oocytes. For this, we determined the intraoocyte levels of GSH and ROS after 44 h of IVM. It was found that SAL increased intraoocyte GSH level and also decreased ROS level (P < 0.05). Finally, we performed somatic cell nuclear transfer (SCNT) after treating oocytes with 10 nM SAL during IVM. SAL treatment significantly improved blastocyst formation of SCNT embryos compared to control (39.6% vs. 24.7%, P < 0.05). Our results indicate that treatment of pig oocytes with ER stress inhibitor SAL during IVM improves preimplantation development PA and cloned pig embryos by influencing cytoplasmic maturation in terms of increased GSH content and decreased ROS level in IVM pig oocytes.

Bovine somatic cell nuclear transfer (bSCNT) embryos can develop to the blastocyst stage at a rate similar to that of embryos produced by in vitro fertilization (IVF). However, the efficiency of somatic cell cloning has remained low, and applications have been limited, irrespective of the nuclear donor species or cell types. One possible explanation is that the reprogramming factors of each oocyte is insufficient or not properly adapted for the receipt of a somatic cell nucleus, because it is naturally prepared only for the receipt of a gamete. Here, we would like to introduce the aggregation method (agSCNT), a new experimental system that enables and increase oocyte volume and examined its subsequent development. Judgement by the blastocyst formation rate or total cell number was significantly higher in the agSCNT group than that in the SCNT group, and was very similar to that in the control IVF group. Moreover, the cleavage formation rate in the agSCNT group (61.5 ± 1.3) was higher than that in the SCNT group (39.7 ± 2.1), while still less than that in the IVF group (75.4 ± 1.3). We also analyzed the epigenetic modifications in bovine IVF, agSCNT, and untreated SCNT embryos. In conclusion, the present study demonstrated that agSCNT improves the in vitro developmental competence and quality of cloned embryos, as evidenced by increased total cell numbers (TC).

Mitochondrial dysfunction is found in oocytes and transmitted to the offspring due to maternal obesity. This is curable by endoplasmic reticulum (ER) stress inhibitors such as salubrinal (SAL). Recently pigs are considered as a model animal for biomedical research due to its physiological similarity with human. Pig oocytes have shown ER stress mostly in metaphase II stage. ER stress is hindering the in vitro embryo production (IVP). This study investigated the effect of ER stress inhibition by using SAL during 44 h of in vitro maturation (IVM) of oocytes at 1, 10, 50 and 100 nM concentrations. Firstly, we defined the concentration of SAL during IVM of pig oocytes. SAL at 10 nM showed higher (44.2 to 55.6%, P<P0.05) development competence to the blastocyst state than control and other concentrations after parthenogenetic activation (PA). Secondly, we sorted out the time-dependent treatment at 10 nM of SAL for IVM of oocytes. It revealed that treatment with SAL during 22 to 44 h and 0 to 44 h of IVM improved PA embryonic development significantly (40.5, 51.7 and 60.2% for control, 22 to 44 h and 0 to 44 h of IVM, respectively, P<0.05). Glutathione (GSH) level is an indicator of cytoplasmic maturation of oocytes. Reactive oxygen species (ROS) have a harmful effect on development competence of oocytes. For this, we determined the intraoocyte levels of GSH and ROS after 44 h of IVM. It was found that SAL increased intraoocyte GSH level and also decrease ROS level (P<0.05). Finally, we performed somatic cell nuclear transfer (SCNT) after treating oocytes with 10 nM SAL during IVM. SAL treatment significantly improved blastocyst formation of SCNT embryos compared to control (24.7 vs. 39.6%, P<0.05). Our results indicate that treatment of pig oocytes with ER stress inhibitor SAL during IVM improves preimplantation development cloned pig embryos by influencing cytoplasmic maturation in terms of increased GSH content and decreased ROS level in IVM pig oocytes.

Even though klotho deficiency in mice exhibits multiple aging-like phenotypes, studies using large animal models such as pigs, which have many similarities to humans, have been limited due to the absence of cell lines or animal models. The objective of this study was to generate homozygous klotho knockout porcine cell lines and cloned embryos. A CRISPR sgRNA specific for the klotho gene was designed and sgRNA (targeting exon 3 of klotho) and Cas9 RNPs were transfected into porcine fibroblasts. The transfected fibroblasts were then used for single cell colony formation and 9 single cell–derived colonies were established. In a T7 endonuclease I mutation assay, 5 colonies (#3, #4, #5, #7 and #9) were confirmed as mutated. These 5 colonies were subsequently analyzed by deep sequencing for determination of homozygous mutated colonies and 4 (#3, #4, #5 and #9) from 5 colonies contained homozygous modifications. Somatic cell nuclear transfer was performed to generate homozygous klotho knockout cloned embryos by using one homozygous mutation colony (#9); the cleavage and blastocyst formation rates were 72.0% and 8.3%, respectively. Two cloned embryos derived from a homozygous klotho knockout cell line (#9) were subjected to deep sequencing and they showed the same mutation pattern as the donor cell line. In conclusion, we produced homozygous klotho knockout porcine embryos cloned from genome-edited porcine fibroblasts.

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.

Low efficiency of somatic cell nuclear transfer (SCNT) is attributed to incomplete reprogramming of transfered nu-clei into oocytes. Trichostatin A (TSA), histone deacetylase inhibitor and 5-aza-2’deoxycytidine (5-aza-dC), DNA methy-lation inhibitor has been used to enhance nuclear reprogramming following SCNT. However, it was not known molec-ular mechanism by which TSA and 5-aza-dC improve preimplantation embryo and fetal development following SCNT. The present study investigates embryo viability and gene expression of cloned porcine preimplantation embryos in the presence and absence of TSA and 5-aza-dC as compared to embryos produced by parthenogenetic activation. Our results indicated that TSA treatment significantly improved development. However 5-aza-dC did not improve development. Presence of TSA and 5-aza-dC significantly improved total cell number, and also decreased the apoptot-ic and autophagic index. Three apoptotic-related genes, Bak, Bcl-xL, and Caspase 3 (Casp3), and three autophagic-re-lated genes, ATG6, ATG8, and lysosomal-associated membrane protein 2 (LAMP2), were measured by real time RT-PCR. TSA and 5-aza-dC treatment resulted in high expression of anti-apoptotic gene Bcl-xL and low pro-apoptotic gene Bak expression compared to untreated NT embryos or parthenotes. Furthermore, LC3 protein expression was lower in NT-TSA and NT-5-aza-dC embryos than those of NT and parthenotes. In addition, TSA and 5-aza-dC treated embryos displayed a global acetylated histone H3 at lysine 9 and methylated DNA H3 at lysine 9 profile similar to the parthenogenetic blastocysts. Finally, we determined that several DNA methyltransferase genes Dnmt1, Dnmt3a and Dnmt3b. NT blastocysts showed higher levels Dnmt1 than those of the TSA and 5-aza-dC blastocysts. Dnmt3a is lower in 5-aza-dC than NT, NTTSA and parthenotes. However, Dnmt3b is higher in 5-aza-dC than NT and NTTSA. These results suggest that TSA and 5-aza-dC positively regulates nuclear reprogramming which result in modulation of apoptosis and autophagy related gene expression and then reduce apoptosis and autophagy. In addition, TSA and 5-aza-dC affects the acetylated and methylated status of the H3K9.

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.

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.

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).

Pig parthenotes were able to develop in vivo for 30 days with normal morphology. In pig, during blastocyst elongation between day 10 and 12 of gestation, estrogen production and secretion by conceptus increases, serving not only as the signal for maternal recognition of pregnancy, but also as a stimulus for the production of proteins and growth factors within the uterine environment that initiate implantation. Cloning efficiency is still very low regardless of species. To increase the productive efficiency of (transgenic, TG) clones, an advanced somatic cell nuclear transfer (SCNT) method may need. Here we report the productions of transgenic cloned pigs using cloned embryos and parthenotes simultaneously. Fibroblasts were isolated from an ear skin of a 10‐day‐old NIH miniature pig. The ear fibroblast cells were transfected with the alpha1,3‐ Galactosyltransferase knock‐out/human CD46 knock‐in (GalT KO/hCD46 KI). For SCNT, the TG somatic cells were used as donor cells. Immediately after fusion confirmation, the TG cloned embryos and parthenotes were transferred into both oviducts of surrogates. The mean number of TG cloned embryos and parthenotes was 137 (±15.2) and 123(±27.1), respectively. The pregnancy and delivery rate was (55.6%, 10/ 18) (44.4%, 8/18), respectively. Totally 19 GalT KO/hCD46 KI cloned piglets were delivered. Among them, 11 piglets were survived and 8 piglets were born stillbirth. The healthy 5 piglets are still survived.

5‐aza‐2’‐deoxyctidine (5‐aza‐dC) is DNA methylation inhibitor and Trichostatin A (TSA) is histone deacytlase inhibitor, both of them can alter the level of the epigenetic modification of cells. The objective of this study was to investigate the effects of treatment with 5‐aza‐dC and TSA into fetal fibroblasts on the development of porcine nuclear transfer (NT) embryos. In this study, experiments were performed in order to modify epigenetic status in donor cells and evaluate developmental potential of NT embryos. 5‐ aza‐dC or TSA or combining treatment of TSA and 5‐aza‐dC was treated into growing donor cells for 1 h exposure and development of NT embryos was evaluated. Experiment was performed with 3 groups: control group (donor cells without treatment); TSA group (donor cell treated with 50 nM TSA for 1 h); TSA + 5‐aza‐dC group (donor cells were treated with 50 nM TSA and 5 nM 5‐aza‐dC for 1 h); TSA+1/2(5‐aza‐dC) group (donor cells were treated with 50 nM TSA for 1h and subsequently treated with 2.5 nM 5‐aza‐dC for another 1h). When donor cells were individually treated with 5 nM 5‐aza‐dC or 50 nM TSA for 1h, the blastocyst rate of NT embryos increased significantly compared with control group [18.8% vs 13.4% (5 nM 5‐aza‐dC group vs control group), and 26.2% vs 11.8% (50 nM TSA group vs control group), p<0.05]. However, the blastocyst rate in combining treatment group (50 nM TSA + 5 nM 5‐aza‐dC) did not increase compare with control group (12.3% vs 11.8%, p>0.05). When the donor cell were individually treated with 50nM TSA for 1 h firstly and then treated with 2.5 nM 5‐aza‐dC for another 1h, the blastocyst rate was significantly improved compared with control and TSA group (28% vs 10.2% and 23.7%, p<0.05). The present study suggested that donor cells treated with TSA or low concentration of TSA+5‐azadC in short time exposure may enhance the development of porcine NT embryo.

The necessity of conditional gene expression in pigs for transgenic models is raised. Thus, in this study, Cre-loxP conditional expression in porcine fetal fibroblasts was investigated and the transformed fibroblasts were reprogrammed in enucleated oocytes for further early embryonic development. Fetal fibroblasts from miniature pigs were used for transfection with pCALNL-DsRed including floxed neomycin resistant gene and selected with 750 ug/mL neomycin for two weeks. The transfected cells did not express DsRed under fluorescence microscope. After transient transfection of plasmid DNA expressing Cre, the fibroblasts began to express DsRed. The cells expressing Ds- Red were employed into somatic cell nuclear transfer (SCNT). A total of 121 oocytes were used for SCNT and 76 cloned embryos (62.8%)were cleaved. Six blastocysts were grown up after SCNT and expressed DsRed. Deletion of floxed neomycin resistant gene was confirmed by RT-PCR in cloned blastocysts. Taken together, this study demonstrated that Cre-loxP recombination in miniature pig fibroblasts were successfully worked and those sequential transformed cells were developed into pre-implantation stage via SCNT.

Successful early embryogenesis of somatic cell nuclear transfer (SCNT) embryos is very important to produce cloned animals. However, poor preimplantation development of SCNT embryos has been a major obstacle to the generation of cloned animals due to a lack of understanding of developmental events and underlying mechanism(s). In the current study, we show that production of SCNT embryos with high developmental competence is dependent on the fusion method. Electrofusion causes spontaneous egg activation, accompanied by an increase in intracellular Ca2+ and improper nuclear remodeling, whereas Sendai virus (SV)-mediated fusion greatly reduces these events. In addition, SV-SCNT increased the blastocyst development rate and trophectoderm cell number compared to electrofusion-mediated SCNT (E-SCNT). In particular, expression of ER stress-associated genes and blastomere apoptosis were significantly increased in E-SCNT embryos, which could be alleviated by inhibition of ER stress or by using the SV-mediated fusion method. Taken together, these results strongly suggest that SV is a useful fusion material for improvement of preimplantation development of SCNT embryos through reduction of ER stress-associated apoptosis.

In this study, we examined the effects of porcine granulocyte-macrophage colonystimulating factor (pGM-CSF) on in vitro development of porcine embryos produced by somatic cell nuclear transfer (SCNT) at first time. The objective of present study was to verify effects of pGM-CSF on SCNT-derived blastocyst formation and evaluate gene expressions and qualities of the blastocyst formed after pGM-CSF treatment. Data were analyzed with SPSS 17.0 using Duncan’s multiple range test. A total 522 cloned embryos in 6 replicates were treated with 10 ng/ml concentration of pGM-CSF during in vitro culture (IVC). It was demonstrated that treatment of 10 ng/ml pGM-CSF could increase blastocyst formation and total cell number in blastocyst significantly (p<0.05) compared to the control (12.3% and 41.4 vs. 9.0% and 34.7, respectively). However, there was no any effect on cleavage rate. It was found that the number of cells in the inner cell mass (ICM) and trophectoderm (TE) were significantly increased compared to the control (4.4 and 31.9, respectively) when cloned embryos were cultured with 10 ng/ml pGM-CSF (6.0 and 43.0, respectively). It was also found that treatment of 10 ng/ml pGM-CSF significantly (p<0.05) increased POU5F1 and Cdx2 mRNA expressions in blastocysts. In addition, Bcl-2 mRNA expression was found to be significantly (p< 0.05) up-regulated in blastocysts in the pGM-CSF supplemented group compared to the control. In conclusion, these results suggest that pGM-CSF may improve the quality and developmental viability of porcine cloned embryos by enhancing nuclear reprogramming via regulating transcription factors expression.

This study was conducted to examine the effect of oocyte donor age and micromanipulation medium on the development of mouse cloned embryos receiving cumulus cells. Mouse oocytes were obtained from 6 to 11 week-old mice BDF1 female mice(experiment 1) and cumulus cells were used as donor cells. Micromanipulation procedures for nuclear transfer(NT) were performed in FHM, M2 or Hepes-buffered TCM199(TCM199) medium(experiment 2). After nuclear transfer, the reconstructed oocytes were activated by 10 mM SrCl2 in Ca-free CZB medium in the presence of 5 μg/ml cytochalasin B for 5 h and cultured in KSOM medium for 4 days. In experiment 1, the survival rate of oocytes after injection of cumulus cells were significantly(p<0.05) lower in oocytes from 6～7 week-old mice(53.3%) than in oocytes from 8～9(80.9%) and 10～11 week-old mice(77.1%). In experiment 2, the survival rate of oocytes after cell injection were significantly(p<0.05) higher in FHM and M2 medium(71.7% and 76.9%) than in TCM199 medium (51.2%). The activation rates of cloned embryos were not different among the micromanipulation media. However, the embryos developed to blastocyst stage were significantly(p<0.05) higher in FHM medium(13.9%) than in M2 and TCM199 medium(0.0% and 0.0%). In conclusion, the present study suggest that oocytes from above 8 week-old mice are superior to oocytes from 6～7 week-old mice as a source of recipient cytoplasm and FHM is superior to M2 and TCM199 as a micromanipulation medium for mouse somatic cell cloning.