• Korean Journal of Poultry Science
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• v.27 no.1
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• pp.73-78
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• 2000

Embryonic stem (ES) cells are pluripotent cell lines, which derived from preimplantation embryo. These cells have been used as a vehicle of foreign DNA for production of transgenic mammals. this experiment was performed to examined the possible use of blastodermal cells derived from hen's egg for germline manipulation. Stage X blsdtodermal cells isolated from fertilized eggs were cultured in DMEM containing 15% fetal calf serum. Blastodermal cells wre co-cultured on the chicken embryonic fibroblast (CEF) or mouse embryonic fibroblast(MEF) cells. to examine the effects of growth factors on stem cell growth, bFGF and LIF were added. There was no significant difference in colony formation of putative ES cells between CEF and MEF as a feederlayer, but the addition of growth factors enhanced the proliferation and inhibited differentiation of blastodermal cells. To characterize the cell colonies as a putative ES cells, putative embryonic cell colonies were stained by periodic acid Schiffs (PAS) reagent. The putative ES cell colonies showed intensive positive reaction similar to the property of undifferentiated PGC upto 20days in vitro, but not in other cell types. this result demonstrates that PAS-positive cell colonies may be used for the study of establishment of chicken ES cell lines for the production of transgenic chicken.

• Journal of Life Science
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• v.18 no.4
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• pp.522-529
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• 2008

High-temperature requirement A2(HtrA2) has been known as a human homologue of bacterial HtrA that has a molecular chaperone function. HtrA2 is mitochondrial serine protease that plays a significant role in regulating the apoptosis; however, the physiological function of HtrA2 still remains elusive. To establish experimental system for the investigation of new insights into the function of HtrA2 in mammalian cells, we first obtained $HtrA2^{+/+}$ and $HtrA2^{-/-}$ MEF cells lines and identified those cells based on the expression pattern and subcellular localization of HtrA2, using immunoblot and biochemical assays. Additionally, we observed that the morphological characteristics of $HtrA2^{-/-}$ MEF cells are different form those of $HtrA2^{+/+}$ MEF cells, showing a rounded shape instead of a typical fibroblast-like shape. Growth rate of $HtrA2^{-/-}$ MEF cells was also 1.4-fold higher than that of $HtrA2^{+/+}$ MEF cells at 36 hours. Furthermore, we verified both MEF cell lines induced caspsase-dependent cell death in response to apoptotic stimuli such as heat shock, staurosporine, and rotenone. The relationship between HtrA2 and heat shock-induced cell death is the first demonstration of the research field of HtrA2. Our study suggests that those MEF cell lines are suitable reagents to further investigate the molecular mechanism by which HtrA2 regulates the balance between cell death and survival.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2003.10a
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• pp.109-109
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• 2003

• Development and Reproduction
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• v.20 no.1
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• pp.63-71
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• 2016

Human embryonic stem cells (hESCs) have been routinely cultured on mouse embryonic fibroblast feeder layers with a medium containing animal materials. For clinical application of hESCs, animal-derived products from the animal feeder cells, animal substrates such as gelatin or Matrigel and animal serum are strictly to be eliminated in the culture system. In this study, we performed that SNUhES32 and H1 were cultured on human amniotic fluid cells (hAFCs) with KO-SR XenoFree and a humanized substrate. All of hESCs were relatively well propagated on hAFCs feeders with xeno-free conditions and they expressed pluripotent stem cell markers, alkaline phosphatase, SSEA-4, TRA1-60, TRA1-81, Oct-4, and Nanog like hESCs cultured on STO or human foreskin fibroblast feeders. In addition, we observed the expression of nonhuman N-glycolylneuraminic acid (Neu5GC) molecules by flow cytometry, which was xenotransplantation components of contamination in hESCs cultured on animal feeder conditions, was not detected in this xeno-free condition. In conclusion, SNUhES32 and H1 could be maintained on hAFCs for humanized culture conditions, therefore, we suggested that new xeno-free conditions for clinical grade hESCs culture will be useful data in future clinical studies.

• BMB Reports
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• v.42 no.3
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• pp.148-153
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• 2009

Pyrrolidine dithiocarbamate (PDTC) is a stable anti-oxidant or pro-oxidant, depending on the situation, and it is widely used to inhibit the activation of NF-${\kappa}B$. We recently reported that PDTC activates the MIP-2 gene in a NF-${\kappa}B$-independent and c-Jun-dependent manner in macrophage cells. In this work, we found that PDTC activates signal transduction pathways in mouse ES cells. Among the three different mitogen-activated protein kinase (MAPK) pathways, including the extracellular-signal-regulated kinase (ERK), p38 MAP kinase, and stress-activated protein kinase (SAPK)/Jun N-terminal kinase (JNK) pathways, only the ERK pathway was significantly activated in mouse ES cells after stimulation with PDTC. Additionally, we observed a synergistic activation of ERK and induction of c-Fos after stimulation with PDTC in the presence of mouse embryonic fibroblast (MEF) conditioned medium. In contrast, another NF-${\kappa}B$ inhibitor, BMS-345541, did not activate the MAP kinase pathways or induce expression of c-Fos. These results suggest that changes in the presence of the NF-${\kappa}B$ inhibitor PDTC should be carefully considered when it used with mouse ES cells.

• Proceedings of the Korean Vacuum Society Conference
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• 2015.08a
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• pp.227.2-227.2
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• 2015

세포를 부착하는 기술은 세포를 배양하기 위한 가장 기초적이며 중요한 기술이다. 세포 부착기술은 대상물과 세포 간의 다양한 생물학적, 물리화학적 연관 관계가 있으나 세포와 부착 대상물 간의 복잡한 상호작용 때문에 완벽히 예측하기는 어렵다. 우리는 이 연구에서 siloxane 성분을 포함하고 있는 전구체인 tetrakis(trimethylsilyloxy)silane과 hydro-carbon을 포함하고 있는 전구체인 cyclohexane을 혼합하여 플라즈마 중합 박막을 만들고 그 박막에서의 mouse embryonic fibroblast cells과 bovine aortic endothelial cell 부착의 정도를 확인하였다. 플라즈마 중합 박막을 제작하기 위해 capacitively coupled plasma chemical vapor deposition system을 사용하였고 carrier gas로는 Ar을 사용하였다. Plasma RF power는 13.56MHz 70W를 사용하였다. Bubbler에서 기화된 전구체를 포함하고 있는 Ar carrier gas가 process chamber에서 혼합되고 두 전구체의 비율을 조절하기 위해 carrier gas를 0 에서 150sccm으로 변화시켜 플라즈마 중합 박막을 제작하였다. 플라즈마 중합 박막의 화학적 조성은 Fourier transform infrared absorption spectroscopy와 X-ray photoelectron spectroscopy를 이용하여 측정하였고, 생물학적 세포 부착 정도는 현미경을 통해 관찰하였다. 또한, 물과 박막의 접촉각(Water contact angle)을 측정함으로써 본 박막과 세포 부착에서의 친, 소수성의 연관성을 확인하였다. Tetrakis(trimethylsilyloxy)silane를 전구체를 사용한 박막에서 세포 부착 억제 표면특성이 관찰되었고, 주입되는 cyclohexane 비율이 늘어날수록 세포부착 가능한 표면 특성을 보였다. 결과적으로, 전구체인 tetrakis(trimethylsilyloxy)silane와 cyclohexane의 비율을 조절함으로써 세포의 부착정도를 제어할 수 있음을 확인하였다.

• Biomedical Science Letters
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• v.18 no.2
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• pp.165-168
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• 2012

Hox proteins containing DNA-binding homedomain act as transcription factors important for anteroposterior body patterning during vertebrate embryogenesis. However, the precise mechanisms by which signal pathways are transduced to regulate the Hox gene expression are not clear. In the course of an attempt to isolate an upstream regulatory factor(s) controlling Hox genes, protein kinase B alpha (Akt1) has been identified as a putative regulator of Hox genes through in silico analysis (GEO profile). In the Gene Expression Omnibus (GEO) dataset GDS1784 at the NCBI (National Center for Biotechnology Information) site, Hox genes were differentially expressed depending on the presence or absence of Akt1. Since it was not well known how Akt1 regulates the specific Hox genes, whose transcription was reported to be regulated by epigenetic modifications such as histone acetylation, methylation etc., the expression of Gcn5, a histone acetyltransferase (HAT), was analyzed in wild type (WT) as well as in $Akt1^{-/-}$ mouse embryonic fibroblast (MEF) cells. RT-PCR analysis revealed that the amount of Gcn5 mRNA was similar in both WT and $Akt1^{-/-}$ MEFs. However, the protein level of Gcn5 was significantly increased in $Akt1^{-/-}$ MEF cells. The half life of Gcn5 was 1 hour in wild type whereas 8 hours in $Akt1^{-/-}$ MEF. These data all together, indicate that Gcn5 is post-transcriptionally down-regulated and the protein stability is negatively regulated by Akt1 in MEF cells.

• Food Science and Biotechnology
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• v.18 no.4
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• pp.965-970
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• 2009

Oleuropein content of olive leaf extracts (OLE; ethanol extract) was evaluated by high performance liquid chromatography analysis. Oleuropein contents were $4.21{\pm}0.57$, $3.92{\pm}0.43$, $0.32{\pm}0.03$, $5.76{\pm}0.32$, and $32.47{\pm}0.25$ mg/100 g for ethanol extract, and hexane, chloroform, ethyl acetate, and butanol fraction, respectively. The removal of DPPH free radical increased in OLE and all 5 fractions of OLE in a concentration dependent manner. In order to investigate the antioxidant effect of OLE in vitro, 80%(v/v) ethanol OLE, $H_2O_2$, or combined treatment of 80%(v/v) ethanol OLE and $H_2O_2$ were applied on mouse embryonic fibroblast (MEF) cells. Cells were damaged by oxidative stress decreased their viability followed by increasing concentration of $H_2O_2$, but co-treatment of OLE and $H_2O_2$ showed an increase in cell growth about 20% compare to the cells treated with $H_2O_2$. OLE suppresses cytotoxicity induced by $H_2O_2$ in dose dependent manner. OLE treatment on MEF cells was also examined by analyzing cell cycle and apoptotic rate using flow cytometry. Apoptotic and necrotic cell accumulation was decreased in addition of OLE to $H_2O_2$ compare to the oxidative damaged cells. Taken together, these results demonstrated that OLE suppresses cytotoxicity induced by $H_2O_2$ and protect cells against oxidative stress on MEF cells.

• Animal cells and systems
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• v.9 no.2
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• pp.79-85
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• 2005

Oxidized abasic residues arise as a major class of DNA damage by a variety of agents involving free radical attack and oxidation of deoxyribose sugar components. 2-deoxyribonolactone (dL) is a C1'-oxidized abasic lesion implicated in DNA strand scission, mutagenesis, and covalent DNA-protein cross-link (DPC). We show here that mammalian cell-free extract give rise to stable DPC formation that is specifically mediated by dL residue. When a duplex DNA containing dL at the site-specific position was incubated with cell-free extracts of Po ${\beta}-proficient$ and -deficient mouse embryonic fibroblast cells, the formation of major dL-mediated DPC was dependent on the presence of DNA polymerase (Pol) ${\beta}$. Formation of dL-specific DPC was also observed with histones and FEN1 nuclease, although the reactivity in forming dL-mediated DPC was significantly higher with Pol ${\beta}$ than with histones or FEN1. DNA repair assay with a defined DPC revealed that the dL lesion once cross-linked with Pol ${\beta}$ was resistant to nucleotide excision repair activity of cell-free extract. Analysis of nucleotide excision repair utilizing a model DNA substrate containing a (6-4) photoproduct suggested that excision process for DPC was inhibited because of DNA single-strand incision at 5' of the lesion. Consequently DPC mediated by dL lesion may not be readily repaired by DNA excision repair pathway but instead function as unusual DNA damage causing a prolonged DNA strand break and trapping of the major base excision repair enzyme.

• Journal of Embryo Transfer
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• v.27 no.3
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• pp.171-174
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• 2012

Various small molecules can be used to control major signaling pathways to enhance stemness and inhibit differentiation in murine embryonic stem cell (mESC) culture. Small molecules inhibiting the fibroblast growth factor (FGF)/ERK pathway can preserve pluripotent cells from stimulation of differentiation. In this study, we aimed to evaluate the effect of pluripotin (SC-1), an inhibitor of the FGF/ERK pathway, on the colony formation of outgrowing presumptive mESCs. After plating the zona pellucida-free blastocyst on the feeder layer, attached cell clumps was cultured with SC-1 until the endpoint of the experiment at passage 10. In this experiment, when the number of colonies was counted at passage 3, SC-1-treated group showed 3.4 fold more mESC colonies when compared with control group. However, after passage 4, there was no stimulating effect of SC-1 on the colony formation. In conclusion, SC-1 treatment can be used to promote mESC generation by increasing the number of early mESC colonies.