• 대한생식의학회:학술대회논문집
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• 2007.11a
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• pp.113-114
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• 2007

• 대한생식의학회:학술대회논문집
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• 2005.06a
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• pp.113-113
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• 2005

• 대한생식의학회:학술대회논문집
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• 2002.11a
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• pp.103-103
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• 2002

• 대한생식의학회:학술대회논문집
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• 2002.05a
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• pp.89.2-90
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• 2002

• Reproductive and Developmental Biology
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• v.30 no.2
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• pp.143-156
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• 2006

Major characteristics of embryonic stem cells (ESCs) are sustaining of sternness and pluripotency by self-renewal. In this report, transcriptional profiles of the molecules in the developmentally important signaling pathways including Wnt, BMP4, $TGF-\beta$, RTK, Hh, Notch, and JAK/STAT signaling pathways were investigated to understand the self-renewal of mouse ESCs (mESCs), J1 line, and compared with the NIH3T3 cell line and mouse embryonic fibroblast (MEF) cells as controls. In the Wnt signaling pathway, the expression of Wnt3 was seen widely in mESCs, suggesting that the ligand may be an important regulator for self-renewal in mESCs. In the Hh signaling pathway, the expression of Gli and N-myc were observed extensively in mESCs, whereas the expression levels of in a Shh was low, suggesting that intracellular molecules may be essential for the self-renewal of mESCs. IGF-I, IGF-II, IGF-IR and IGF-IIR of RTK signaling showed a lower expression in mESCs, these molecules related to embryo development may be restrained in mESCs. The expression levels of the Delta and HESS in Notch signaling were enriched in mESCs. The expression of the molecules related to BMP and JAK-STAT signaling pathways were similar or at a slightly lower level in mESCs compared to those in MEF and NIH3T3 cells. It is suggested that the observed differences in gene expression profiles among the signaling pathways may contribute to the self-renewal and differentiation of mESCs in a signaling-specific manner.

• Proceedings of the Korean Society of Toxicology Conference
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• 2005.05a
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• pp.159-159
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• 2005

• Development and Reproduction
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• v.11 no.2
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• pp.111-119
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• 2007

Differentiation of cells can be induced through modulation of endogenous regulators using exogenous factors. Useful transfection systems to transport a specific exogenous regulator into cell have been tried but still there are many obstacles to overcome. In this study, we examined the transfection efficiency of cell permeable peptides (CPPs) in mouse embryonic stem cell under the various conditions. To identify the CPP-mediated translocation of a protein, we employed recombinant CPP-enhanced green fluorescent protein (EGFP). Viability of R1 cells was different between experimental groups depending on the kind of CPP and the concentration of CPP-EGFP. Translocation of CPP-EGFPs into the R1 cells was not detected until 30 min after CPP-EGFPs treatment in all groups. After 1 hr, translocation of pEP-1-EGFP-N was detected, but it could not be detected in the other group. Transfection of pEP-1EGFP-N was independent on its concentration. The time course did not show saturation even after 24 hr in pEP-1-EGFP-N. These results showed that the permeability depended on the kind of CPP and the location of His-tag in the case of examined CPPs, and did not need biological energy. On summary, the efficiency of transfection of CPP-EGFP depends on the CPP sequences but the culture time is not a key factor in transfection for the mouse embryonic stem cell. For the future studies to improve the efficiency of translocation of protein into embryonic stem cells, it is needed to develop modified CPP or mediator. The studies would be very useful to induce the differentiation of embryonic stem cells.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.121-121
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• 2003

The standard protocol for the production of transgenic mouse from ES-injected embryo has to process via chimera producing and several times breeding steps, In contrast, tetraploid-ES cell complementation method allows the immediate generation of targeted murine mutants from genetically modified ES cell clones. The advantage of this advanced technique is a simple and efficient without chimeric intermediates. Recently, this method has been significantly improved through the discovery that ES cells derived from hybrid strains support the development of viable ES mice more efficiently than inbred ES cells do. Therefore, the objective of this study was to generate transgenic mice overexpressing human resistin gene by using tetrapioid-ES cell complementation method. Human resistin gene was amplified from human fetal liver cDNA library by PCR and cloned into pCR 2.1 TOPO T-vector and constructed in pCMV-Tag4C vector. Human resistin mammalian expression plasmid was transfected into D3-GL ES cells by lipofectamine 2000, and then after 8~10 days of transfection, the human resistin-expressing cells were selected with G418. In order to produce tetraploid embryos, blastomeres of diploid embryos at the two-cell stage were fused with two times of electric pulse using 60 V 30 $\mu$sec. (fusion rate : 93.5%) and cultured upto the blastocyst stage (development rate : 94.6%). The 15~20 previously G418-selected ES cells were injected into tetraploid blastocysts, and then transferred into the uterus of E2.5d pseudopregnant recipient mice. To investigate the gestation progress, two El9.5d fetus were recovered by Casarean section and one fetus was confirmed to contain human resistin gene by genomic DNA-PCR. Therefore, this finding demonstrates that tetraploid-ES mouse technology can be considered as a useful tool to produce transgenic mouse for the rapid analysis of gene function in vivo.

• Development and Reproduction
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• v.14 no.4
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• pp.215-223
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• 2010

The construction of transgenic mouse using embryonic stem (ES) cells has been crucial in the functional studies of gene on mouse genome. Gene knockout mice have been powerful for elucidating the function of genes as well as a research model for human diseases. Gene targeting and gene trapping mathods have been the representative technologies for making the knockout mice by using ES cells. Since the gene targeting and the gene trapping methods were independently developed about 20 years ago, it's efficiency and productivity has been improved with a advance of molecular biology. Conventional gene targeting method has been changes to high throughput conditional gene targeting. The combination of the advantage of gene targeting and gene tapping elements allows to extend a spectrum of gene trapping and to improve the efficiency of gene targeting. These advance should be able to produce the mutant with various phenotype to target a certain gene, and in postgenome era they have served as crucial research tools in understanding the functional study of whole genome in mouse.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.3
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• pp.335-343
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• 2018

Objective: Remarkable difference in cellular activity was found between early and late subpassaged embryonic stem cell (ESCs) lines, which can be created by subtle changes in cell manipulation protocol. This study subsequently examined whether post-thaw subculture of early subpassaged ESC lines could further affect the activity of the ESCs. Methods: Fresh (as a control treatment) or cryopreserved F1 hybrid (B6CBAF1) early ESC lines (C57BL/6xCBA) of the 4 (P4) or the 19 passage (P19) were subcultured once, twice or six times under the same condition. The post-thaw survival of the ESCs was monitored after the post-treatment subculture and the ability of cell proliferation, reactive oxygen species (ROS) generation, apoptosis and mitochondrial ATP synthesis was subsequently examined. Results: Regardless of the subculture number, P19 ESCs showed better (p<0.05) doubling time and less ATP production than P4 ESCs and such difference was not influenced by fresh or cryopreservation. The difference between P4 and P19 ESC lines became decreased as the post-treatment subculture was increased and the six times subculture eliminated such difference. Similarly, transient but prominent difference in ROS production and apoptotic cell number was detected between P4 and P19 ESCs only at the 1st subculture after treatment, but no statistical differences between two ESC lines was detected in other observations. Conclusion: The results of this study suggest that post-thaw subculture of ESCs under the same environment is recommended for standardizing their cellular activity. The activity of cell proliferation ability and ATP synthesis can be used as parameters for quality control of ESCs.