• Clinical and Experimental Pediatrics
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• v.53 no.8
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• pp.786-789
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• 2010

Induced pluripotent stem (iPS) cells are generated by epigenetic reprogramming of somatic cells through the exogenous expression of transcription factors. Recently, the generation of iPS cells from patients with a variety of genetic diseases was found to likely have a major impact on regenerative medicine, because these cells self-renew indefinitely in culture while retaining the capacity to differentiate into any cell type in the body, thereby enabling disease investigation and drug development. This review focuses on the current state of iPS cell technology and discusses the potential applications of these cells for disease modeling; drug discovery; and eventually, cell replacement therapy.

• Molecules and Cells
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• v.38 no.12
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• pp.1029-1036
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• 2015

Appropriate vessel development and its coordinated function is essential for proper embryogenesis and homeostasis in the adult. Defects in vessels cause birth defects and are an important etiology of diseases such as cardiovascular disease, tumor and diabetes retinopathy. The accumulative data indicate that ETV2, an ETS transcription factor, performs a potent and indispensable function in mediating vessel development. This review discusses the recent progress of the study of ETV2 with special focus on its regulatory mechanisms and cell fate determining role in developing mouse embryos as well as somatic cells.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.67-75
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• 2011

The faulty regulation of imprinting gene lead to the abnormal development of reconstructed embryo after nuclear transfer. However, the correlation between the imprinting status of donor cell and preimplantation stage of embryo development is not yet clear. In this study, to determine this correlation, we used the porcine spermatogonial stem cell (pSSC) and fetal fibroblast (pFF) as donor cells. As the results, the isolated cells with laminin matrix selection strongly expressed the GFR ${\alpha}$-1 and PLZF genes of SSCs specific markers. The pSSCs were maintained to 12 passages and positive for the pluripotent marker including OCT4, SSEA1 and NANOG. The methylation analysis of H19 DMR of pSSCs revealed that the zinc finger protein binding sites CTCF3 of H19 DMRs displayed an androgenic imprinting pattern (92.7%). Also, to investigate the reprogramming potential of pSSCs as donor cell, we compared the development rate and methylation status of H19 gene between the reconstructed embryos from pFF and pSSC. This result showed no significant differences of the development rate between the pFFs ($11.2{\pm}0.8%$) and SSCs ($13.3{\pm}1.1%$). However, interestingly, while the CTCF3 methylation status of pFF-NT blastocyst was decreased (36.3%), and the CTCF3 methylation status of pSSC-NT blastocyst was maintained. Therefore, this result suggested that the genomic imprinting status of pSSCs is more effective than that of normal somatic cells for the normal development because the maintenance of imprinting pattern is very important in early embryo stage.

• Reproductive and Developmental Biology
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• v.38 no.4
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• pp.147-158
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• 2014

Differentiated nuclei can experimentally be returned to an undifferentiated embryonic status after nuclear transfer (NT) to unfertilized metaphase II (MII) oocytes. Nuclear reprogramming is triggered immediately after somatic cell nucleus transfer (SCNT) into recipient cytoplasm and this period is regarded as a key stage for optimizing reprogramming. In a recent study (Dai et al., 2010), use of m-carboxycinnamic acid bishydroxamide (CBHA) as a histone deacetylase inhibitor during the in vitro early culture of murine cloned embryos modifies the acetylation status of somatic nuclei and increases the developmental competence of SCNT embryos. Thus, we examined the effects of CBHA treatment on the in vitro preimplantation development of porcine SCNT embryos and on the acetylated status of histone H3K9 on cloned embryos at the zygote stage. We performed the three groups SCNT: SCNT (NT), CBHA treatment at the porcine fetus fibroblast cells (PFFs) used as donor cells prior to SCNT (CBHA-C) and CBHA treatment at the porcine SCNT embryos during the in vitro early culture after oocyte activation (CBHA-Z). The PFFs were treated with a $15{\mu}M$ of CBHA (8 h) for the early culture and the porcine cloned embryos were treated with a $100{\mu}M$ concentration of CBHA during the in vitro early culture (10 h). Cleavage rates and development to the blastocyst stage were assessed. No significant difference was observed the cleavage rate among the groups (82.6%, 76.4% and 82.2%, respectively). However, the development competence to the blastocyst stage was significantly increased in CBHA-Z embryos (22.7%) as compared to SCNT and CBHA-C embryos (8.6% and 4.1%)(p<0.05). Total cell numbers and viable cell numbers at the blastocyst stage of porcine SCNT embryos were increased in CBHA-Z embryos as compared to those in CBHA-C embryos (p<0.05). Signal level of histone acetylation (H3K9ac) at the zygote stage of SCNT was increased in CBHA-Z embryos as compared to SCNT and CBHA-C embryos. The results of the present study suggested that treatment with CBHA during the in vitro early culture (10 h) had significantly increased the developmental competence and histone acetylation level at the zygote stage.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.62-62
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• 2002

The relationship of Oct-4 to pluripotent cells is suggested by its tightly restricted expression pattern during embryonic development. Just prior to implantation it is limited to pluripotent cells of the inner cell mass (ICM) that will form the embryo proper but is not expressed in the trophectoderm, the structure that will form the extraembryonic tissues. (omitted)

• Journal of Embryo Transfer
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• v.29 no.4
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• pp.345-350
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• 2014

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 $50{\mu}M$ 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 ($32.5{\pm}1.2%$, 78/235) than that of non-treated control SCNT embryos ($22.3{\pm}1.8%$, 50/224). TUNEL assay revealed that the numbers of apoptotic cells in GSH treatment group ($2.3{\pm}0.4%$) were significantly lower (P<0.05) than that of control ($3.8{\pm}0.6%$). Relative caspase-3 activity of GSH treated group was $0.8{\pm}0.06$ fold compared to that of control. DNA methylation status of blastocysts in GSH treatment group ($13.1{\pm}0.5$, pixels/embryo) was significantly lower (P<0.05) than that of control ($17.4{\pm}0.9$, 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.

• Korean Journal of Poultry Science
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• v.41 no.3
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• pp.217-225
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• 2014

Telomeres are the ends of the eukaryotic chromosomes and consist of a tandem repetitive DNA sequence and shelterin protein complex. The function of telomere is to protect chromosome. Telomere length in somatic cells tends to decrease with organismal age due to the end replication problem. However, several factors at the genetic, epigenetic and environmental level affect telomere length. In this study, we estimated heritability of telomere length and investigated inheritance of telomeres in a chicken. Telomere length of lymphocytes was analyzed by semi-quantitative polymerase chain reaction using telomere primer and quantitative fluorescence in situ hybridization using telomeric DNA probe. In results, heritability of telomere length was estimated 0.9 at birth by offspring-parent regression analysis and was estimated 0.03 and 0.04 at 10 and 30 weeks old, respectively, by parental variance analysis. There was a significant positive correlation in telomere length between father and their offspring (r=0.348), and mother and their offspring (r=0.380). In inheritance patterns of telomere length, the influence of paternal and maternal effect on their offspring was similar. The influence of inherited telomeres on male and female progeny was also roughly alike. These results implicated that imprinting of parental telomere length was regulated by autosomal genes, not sex linked genes. In addition, telomere length of offspring at birth did not differ along with their maternal age. Thus, maternal age does not affects telomere length in their offspring at birth owing to cellular reprogramming at early embryonic stage.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.81-81
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• 2003

DNA methylation is a covalent modification of DNA that can modulate gene expression and is now recognized as a major component of the epigenome. During evolution, the dinucleotide CpG has been progressively eliminated from the genome of higher eukaryotes and is present at only 5% to 10% of its predicted frequency. Approxymately 80% of the remaining CpG sites contain methylated cytosines in most vertebrates and they are distributed in a pattern that is unique in each tissue and is inversely correlated with gene expression. The pattern of methylation is faithfully maintained during cell division by the enzyme Dnmt1, the maintenance DNA methyltransferase, which catalyzes the transfer of a methyl group from S-adenosyl-methionine to the 5'-position of the cytosine ring. We have been identified bovine Dnmt1 cDNA full-length recently (AY173048) Little is known on the functions of Dnmt1 in bovine preimplantation embryos. Thus, we analyzed the specific pattern of Dnmt1 in in vitro derived/nuclear transfer bovine and in vivo derived mouse embryos to monitor the epigenetic reprogramming process. We investigated these process by using indirect immunofluresence with an antibody to Dnmt1. According to other studies, Dnmt1 accumulates in nuclei of early growing oocytes but is sequestered in the cytoplasm of mature oocytes. In 2-cell and 4-cell embryos, Dnmt1 is cytoplasmic, but at the 8-cell stage, it is present only in the nucleus. By the blastocyst stage, Dnmt1o is again found only in the cytoplasm. Thus, nuclear localization of Dnmt1o in preimplantation embryos is limited to the 8-cell stages After implantation, Dnmt1 is localized in the nucleus in mouse. However, we have found different patterns of Dnmt1 nuclear localization. Though we used the common antibody, immune-localization data revealed that Dnmt1 antibody have been detected at the nucleus in 1-cell to blastocyst embryos. Therefore, maybe we think that the functions of Dnmt1 between bovine and mice are different. In order to Identify the mechanisms that regulate DNA methylation in bovine preimplantation embryo, we have plans on using bovine oocyte and somatic specific Dnmt1 antibodies.

• Reproductive and Developmental Biology
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• v.36 no.4
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• pp.261-267
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• 2012

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.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.4
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• pp.487-495
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• 2007

The objectives of the present study were to initiate cloning of Korean native goat by somatic cell nuclear transfer (NT) and to examine whether unovulated (follicular) oocytes can support the same developmental ability of NT embryos as ovulated (oviductal) oocytes after hCG injection in stimulated cycles of the goat. The in vivo-matured and immature oocytes were collected from the oviducts and follicles of superovulated does, respectively, and the immature oocytes were maturated in vitro. Ear skin fibroblasts derived from a 3-yr-old female Korean native goat were used as the donors of nuclei or karyoplasts. Following fusion, activation and in vitro culture to a 2- to 4-cell stage, 49 in vitro-derived and 105 in vivo-derived embryos were transferred to 6 and 17 recipient does, respectively. One doe and three does of the respective groups were identified as pregnant by ultrasonography on day 30 after embryo transfer. However, only one doe, which had received in vivo-derived embryos, delivered a normal female kid of 1.9 kg on d 149. The cloned kid gained more weight than her age-matched females as much as 87% during the first 4 mo after birth (17.7 vs. $9.4{\pm}0.8$ kg) and reached puberty at 6-mo age a few months earlier than normal female does. The telomere length of the kid, which was similar to that of the donor fibroblast at 2-mo age, decreased 8% between 2- and 7-mo ages. Moreover, at 7-mo age, she had 21% shorter telomere than her age-matched goats. To our knowledge, this is the first case in which a cloned animal born with a normal weight exhibited accelerated growth and development. The unusually rapid growth and development of the cloned goat may have resulted from SCNT-associated epigenetic reprogramming involving telomere shortening.