• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2005.10a
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• pp.89-90
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• 2005

• Journal of Embryo Transfer
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• v.27 no.2
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• pp.113-119
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• 2012

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.

• Journal of Embryo Transfer
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• v.26 no.1
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• pp.85-89
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• 2011

Somatic cell nuclear transfer (SCNT) is a useful tool for reproducing genetically identical animals or producing transgenic animals. Many reports have demonstrated that the efficiency of animal cloning by SCNT requires reprogramming of the somatic nucleus to a totipotent like-state. The SCNT-related reprogramming might mimic the natural reprogramming process that occurs during normal mammalian development. However, recent evidence indicates that the reprogramming event by SCNT is incomplete. In this study, the traditional SCNT procedure (TNT) was modified by injecting donor nuclei into recipient cytoplasm prior to the enucleation process to expose the donor nucleus before removing the karyoplast containing the chromosomes of the oocytes which might possess additional reprogramming factors, and this modified technique was named as reversing the usual order of SCNT (RONT). Other procedures including activation and in vitro culture were the same as TNT. Contrary to expectations, the rate of blastocyst development was not different significantly between RONT and TNT (8.6% and 7.9%, respectively). However, duration of micromanipulation performed by the same technician and equipments was remarkably reduced because the ruptured oocytes after nuclear injection were excluded from the enucleation process. This study suggests that RONT, a simplified SCNT protocol, shortens the duration of SCNT procedure and this less time-costing protocol may enable the researchers to perform murine SCNT easier.

• Journal of Embryo Transfer
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• v.30 no.1
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• pp.33-43
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• 2015

Successful somatic cell nuclear transfer (SCNT) has been reported across a range of species using a range of recipient cells including enucleated metaphase II (MII) arrested oocytes, enucleated activated MII oocytes, and mitotic zygotes. However, the frequency of development to term varies significantly, not only between different cytoplast recipients but also within what is thought to be a homogenous population of cytoplasts. One of the major differences between cytoplasts is the activities of the cell cycle regulated protein kinases, maturation promoting factor (MPF) and mitogen activated protein kinase (MAPK). Dependent upon their activity, exposure of the donor nucleus to these kinases can have both positive and negative effects on subsequent development. Co-ordination of cell cycle stage of the donor nucleus with the activities of MPF and MAPK in the cytoplast is essential to avoid DNA damage and maintain correct ploidy. However, recent information suggests that these kinases may also effect reprogramming of the somatic nucleus and preimplantation embryo development by other mechanisms. This article will summarise the differences between cytoplast recipients, their effects on development and discuss the potential role/s of MPF and or MAPK in nuclear reprogramming.

• Reproductive and Developmental Biology
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• v.32 no.4
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• pp.249-253
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• 2008

Interspecies somatic cell nuclear transfer (iSCNT) is a valuable tool for studying the interactions between an oocyte and somatic nucleus. The object of this study was to investigate the developmental competence of in vitro-matured porcine oocytes after transfer of the somatic cell nuclei of 2 different species (goat and rabbit). Porcine cumulus oocytes were obtained from the follicles of ovaries and matured in TCM-199. The reconstructed embryos were electrically fused with 2 DC pulses of 1.1kV/cm for $30{\mu}s$ 0.3M mannitol medium. The activated cloned embryos were cultured in porcine zygote medium-3 (PZM-3), mSOF or RDH medium for 7 days. The blastocyst formation rate of the embryos reconstructed from goat or rabbit fetal fibroblasts was significantly lower than that of the embryos reconstructed from porcine fetal fibroblast cells. However, a significantly higher number of embryos reconstructed from goat or rabbit fetal fibroblasts cultured in mSOF or RDH, respectively, developed to the morular stage than those cultured in PZM-3. These results suggest that goat and bovine fetal fibroblasts were less efficacious than porcine-porcine cloned embryos and that culture condition could be an important factor in iSCNT. The lower developmental potential of goat-porcine and porcine-bovine cloned embryos may be due to incompatibility between the porcine oocyte cytoplasm and goat and bovine somatic nuclei.

• Journal of Embryo Transfer
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• v.23 no.1
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• pp.31-36
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• 2008

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.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.1
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• pp.21-27
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• 2020

Somatic cell nuclear transfer derived embryonic stem cells (NT-ESCs) have significant advantages in various fields such as genetics, embryology, stem cell science, and regenerative medicine. However, the poor establishment of NT-ESCs hinders various research. Here, we applied fasudil, a Rho-associated kinase (ROCK) inhibitor, to develop somatic cell nuclear transfer (SCNT) embryos and establish NT-ESCs. In the study, MII oocytes were isolated from female B6D2F1 mice and performed SCNT with mouse embryonic fibroblasts (MEFs). The reconstructed NT-oocytes were activated artificially, and cultured to blastocysts in KSOM supplemented with 10 μM fasudil. Further, the blastocysts were seeded on inactivated MEFs in embryonic stem cell medium supplemented with 10 μM fasudil. A total of 26% of embryos formed into blastocysts in the fasudil treated group, while this ratio was 44% in the fasudil free control group. On the other hand, 30% of blastocysts were established NT-ESCs after exposure of fasudil, which was significantly higher than the control group (10%). The results suggest that fasudil reduced blastocyst development after SCNT due to inhibition of 2 cell cleavage while improved the establishment of NT-ESCs through the anti-apoptotic pathway.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2005.10a
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• pp.16-22
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• 2005

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2000.11a
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• pp.27-33
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• 2000

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.66-66
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• 2001

Since the first birth of pig derived from embryonic cells by nuclear transfer, many researches to produce cloned pig have been carried out. Recently, two reports about the birth of somatic cell cloned pigs using in vivo oocytes and also Betthauser et al. (2000) reported the birth of somatic cell cloned pigs using in vitro oocytes. So here we investigated the effect of activation method and culture medium on in vitro development of porcine nuclear transfer embryo using fetal fibroblast. Oocytes derived from slaughter house obtained ovaries were matured for 42 to 44 h in TCM 199. Matured oocytes were denuded using 0.1% hyaluronidase and then Oocytes with the first polar body were used for enucleation by aspirating the first polar body and adjacent cytoplasm in TCM 199 supplemented with 7.5 $\mu\textrm{g}$ cytochalasin B. Petal fibroblast cells were prepared from 35 days old fetus. To be used as donor cells, fetal fibroblast cells were serum starved for 3 to 5 days and then isolated into single co:1 by trypsinization. Nuclear transfer embryos were fused using 2 times 1.25㎸ for 30$mutextrm{s}$. Fused NT embryos were activated with calcium ionophore (CI) and 6-dimethyl-aminopurine (6-DMAP). Activated oocytes were cultured in NCSU 23 or BECM 3 for 6 days. There was no significant difference between chemical activation and no chemical activation for blastocyst development rate(11.6 vs. 14.8%). However, cell number was significantly higher when NT embryos were activated with CI and 6-DMAP (31.2 vs. 22.6). When NT embryos were cultured in NCSU 23 or BECM 3, blastocyst development rate was 16.4 and 13.2%, respectively, and cell number was 31.5 and 24.1, respectively. These results suggest that chemical activation after fusion and culture in NCSU 23 could increase cell number of porcine NT embryos.