• The Korean Journal of Zoology
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• v.39 no.3
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• pp.239-247
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• 1996

An embryonic stem (ES) cell-based in vitro model system was examined to determine whether a simple differentiation of embryoid bodies (EB) in the suspension medium is useful to dissect early erythropoiesis. Characteristics of the differentiating EBs were monitored for their differentiation potential to generate hematopoietic cell types by general morphology, benzidine staining and two-step colony assays, and expressivity of several erythroid marker genes by the RT-PCR analysis for total cellular RNA prepared from the differentiating EBs. Every ematopoietic lineage cells were generated from the differentiating EBs with reproducible frequencies, similar to the other sophisticated differentiation protocols. Furthermore, the globin gene switching in differentiating ES cells paralleled the sequence of events found in the mouse embryo, and such that their expression was activated by at least 12 hrs later than those of erythroid-specific transcription factors, GATA-1 and Tal-1 The erythropoietic differentiation program initiated reproducibly and efficiently in this simple differentiation system in a suspension culture, such that this system may be useful for dissection of the molecular events of early erythropoiesis.

• Development and Reproduction
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• v.9 no.1
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• pp.33-41
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• 2005

Identification of differentially expressed genes at blastocyst stage embryos would provide insights into early development and differentiation. Here, we applied a new differential display reverse transcription polymerase chain reaction(DD RT-PCR) technology, called annealing control primers(ACP) system to identify the genes that are specifically or prominently expressed in mouse blastocysts compared to embryonic stem(ES) cells. Using 100 ACPs, 26 clones were perceived as differentially expressed genes in mouse blastocysts. A BLAST search revealed that cloned genes had significant sequence similarities with known genes in the GenBank/EMBL data base. Among them, 15 genes were selected and conformed by RT-PCR. This analysis suggests that the ACP system is a practical method for the identification of stage-specific genes using small numbers of mouse embryos.

• Journal of Embryo Transfer
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• v.14 no.1
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• pp.6-15
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• 1999

ES cell의 수립으로 특히 mouse를 중심으로 한 발생학, 유전학 연구의 획기적 발전과 형질변환 동물의 생산 및 동물 체내에서 유전자 기능의 탐구에 매우 큰 변혁을 가져오게 되었다. 또한 ES cell과 embryoid body는 체외 분화능의 연구에 있어 새로운 cytokine의 발견 및 세포 수준에서의 유전자 기능 해석의 강력한 연구수단으로서 폭 넓게 이용되어 질 수 있는 가능성을 시사하고 있다. 이는 ES cell line이 지닌 두 가지 장점, 즉, 유전자 조작의 용이함과, 거의 모든 종류의 성체 구성세포로 분화할 수 있는 성질 때문이다. 이러한 ES cell technology를 실제로 제반 학문과 특히, 인간에게 적용하기 위해서는 반드시 해결해야 할 중요한 문제점이 있다. 첫째로, ES cell을 대상으로 하는 형질변환 방법의 편의성 및 효율개선이 이루어 wu야 하며, 두 번째로 인간의 유전자 및 세포 이식 치료 등을 비롯한 제반 연구에 직접 적용 가능한 ES cell line의 수립과 체외에서 목적으로 하는 분화 세포를 얻기 위한 배양조건이 확립되어져야 한다. 이러한 목표를 달성하기 위해 ES cell의 발생, 분화과정에 있어서의 분자조절기구, 세포 특이적 promotor, 유도 signal등에 대한 연구가 활발히 진행되어져야 할 것이다.

• BMB Reports
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• v.45 no.12
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• pp.686-692
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• 2012

Phenotypic analysis of gene-specific knockout (KO) mice has revolutionized our understanding of in vivo gene functions. As the use of mouse embryonic stem (ES) cells is inevitable for conventional gene targeting, the generation of knockout mice remains a very time-consuming and expensive process. To accelerate the large-scale production and phenotype analyses of KO mice, international efforts have organized global consortia such as the International Knockout Mouse Consortium (IKMC) and International Mouse Phenotype Consortium (IMPC), and they are persistently expanding the KO mouse catalogue that is publicly available for the researches studying specific genes of interests in vivo. However, new technologies, adopting zinc-finger nucleases (ZFNs) or Transcription Activator-Like Effector (TALE) Nucleases (TALENs) to edit the mouse genome, are now emerging as valuable and effective shortcuts alternative for the conventional gene targeting using ES cells. Here, we introduce the recent achievement of IKMC, and evaluate the significance of ZFN/TALEN technology in mouse genetics.

• Reproductive and Developmental Biology
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• v.35 no.4
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• pp.449-455
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• 2011

Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by etopic expression of transcription factors. iPS cells are indistinguishable from ES cells in terms of morphology and stem cell marker expression. Moreover, mouse iPS cells give rise to chimeric mice that are competent for germline transmission. However, mice derived from iPS cells often develop tumors. Furthermore, the low efficiency of iPS cell generation is a big disadvantage for mechanistic studies. Nonviral plasmid.based vectors are free of many of the drawbacks that constrain viral vectors. The histone deacetylase inhibitor valproic acid (VPA) has been shown to improve the efficiency of mouse and human iPS cell generation, and vitamin C (Vc) accelerates gene expression changes and establishment of the fully reprogrammed state. The MEK inhibitor PD0325901 (Stemgent) has been shown to increase the efficiency of the reprogramming of human primary fibroblasts into iPS cells. In this report, we described the generation of mouse iPS cells devoid of exogenous DNA by the simple transient transfection of a nonviral vector carrying 2A-peptide-linked reprogramming factors. We used VPA, Vc, and the MEK inhibitor PD0325901 to increase the reprogramming efficiency. The reprogrammed somatic cells expressed pluripotency markers and formed EBs.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.60-60
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• 2003

Embryonic stem (ES) cells have property of self-renewal and can differentiate into the cells of all three primary germ layers. Recently, many growth factors, alteration of culture condition and gene modifications have been used to differentiate mouse and human ES cells into specific cell types. This study was performed to evaluate the differentiation protocol for human ES cells to the endodermal lineage cells. Human ES cells (Miz-hESl ) were cultured on STO feeder layer mitotically inactivated with mitemycin C, and embryoid bodies (EBs) were formed by suspension culture. Differentiation protocol of EBs consisted of three steps: stage I, culture of EBs for 6 days with ITSFn medium; stage II, culture of stage I cells for 8 days with N2 medium ; stage III, culture of stage II cells for 22 days with N2 medium. mRNA levels of the endodermal lineage differentiation genes were analyzed by semi- quantitative RT-PCR. The Oct-4 expression, a marker of the pluripotent state, was detected in undifferentiated human ES cells but progressively decreased after EBs formation. Differentiating human ES cells expressed marker genes of endodermal differentiation and pancreatic islet cells. GATA4, a-fetoprotein, Glut-2, and Ngn3 were expressed in all stages. However, albumin and insulin were expressed in only stage III cells. The human ES cells can be differentiated into endodermal lineage cells by multiple step culture system using various supplements. We are developing the more effective protocols for guided differentiation of human ES cells.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2003.10a
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• pp.100-100
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• 2003

• Animal cells and systems
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• v.1 no.1
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• pp.143-150
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• 1997

Transcription factor CP2 was identified initially to bind the promoter region of the murine a-globin gene and its activity was shown to increase 2 to 3 fold during the induced differentiation of murine erythroleukemia (MEL) cells. To get further insight into the role of CP2 during development and differentiation, steady-state levels of CP2 message were monitored by using reverse transcriptase (RT)-PCR and in situ hybridization assays in the cultured MEL cells and differentiating embryonic stem (ES) cells in vitro, and in fetal and adult mouse tissues. The amount of CP2 messages increased 3 to 5 fold during induced differentiation of MEL cells, suggesting that the increment of CP2 activity during induced differentiation of MEL cells is originated from the increase of transcription initiation. On the other hand, CP2 expression is not restricted to the erythroid lineage cells; CP2 expressed ubiquitously from the undifferentiated ES cells to adult tissue cells. CP2 transcript was observed even in the undifferentiated ES cells and the level of expression increased from day 8 of the differentiating embryoid bodies. RT-PCR assay in the total RNAs prepared from several tissues of the adult mouse also showed ubiquitous expression profile, although the levels of expression were variable among tissues. When non-radioactive in situ hybridization assay was performed to the paraffin-sectioned whole body mouse embryos at days 11.5, 13.5, and 16.5 after fertilization, variable amounts of positive signals were also detected in different tissues.

• Korean Journal of Animal Reproduction
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• v.18 no.4
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• pp.299-307
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• 1995

The goal of cell stem cell technology is to produce a viable and genetically normal animal. To achieve this goal various laboratories have followed 2 different pathways beginning with either the culture of 1) single or pooled ICMs grown with or without a feeder layer or 2) single or pooled 16-20 cell stage embryos grown with a feeder layer. Also, thus far embryonic cell cultures or lines have been established by several methods including loose suspension culture for short-term cultures and more commonly murine or bovine fibroblast feeder layers for long-term culture. Pluripotent lines have been derived from 16-cell through blastocyst inner cell mass stages. The efficiency of establishing cell lines and cell proliferation apper to be affected by the number of cells or embryos starting the line. Most attempts to produce offspring from long term STO cell feeder layer cultured ICM or morulae derived ES cells have resulted in pregnancy failure in the first trimester when ES cells were used in cuclear transfer or have failed to retain ES cells in the progeny produced by chimerization. The exception is 1 chimeric fetus from use of morula ES cells in the chimerization with early embryonic cells. There is much to be learned yet about ES cell culture requirements for maintenance of totipotency. If bovine ES cell lines loose imprinting pattern and totipotency with long-term culture and passage as suggested for mouse ES cells, we may be limited to the use of short-term cultures for multiplication of embryos and efficient production of transgenic animals. No bovine ES cell system has yet met all of the criteria indicated for a totipotent ES cell line.

• Molecules and Cells
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• v.26 no.4
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• pp.338-343
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• 2008

An embryonic stem cell is a powerful tool for investigation of early development in vitro. The study of embryonic stem cell mediated neuronal differentiation allows for improved understanding of the mechanisms involved in embryonic neuronal development. We investigated expression profile changes using time course cDNA microarray to identify clues for the signaling network of neuronal differentiation. For the short time course microarray data, pattern analysis based on the quadratic regression method is an effective approach for identification and classification of a variety of expressed genes that have biological relevance. We studied the expression patterns, at each of 5 stages, after neuronal induction at the mRNA level of embryonic stem cells using the quadratic regression method for pattern analysis. As a result, a total of 316 genes (3.1%) including 166 (1.7%) informative genes in 8 possible expression patterns were identified by pattern analysis. Among the selected genes associated with neurological system, all three genes showing linearly increasing pattern over time, and one gene showing decreasing pattern over time, were verified by RT-PCR. Therefore, an increase in gene expression over time, in a linear pattern, may be associated with embryonic development. The genes: Tcfap2c, Ttr, Wnt3a, Btg2 and Foxk1 detected by pattern analysis, and verified by RT-PCR simultaneously, may be candidate markers associated with the development of the nervous system. Our study shows that pattern analysis, using the quadratic regression method, is very useful for investigation of time course cDNA microarray data. The pattern analysis used in this study has biological significance for the study of embryonic stem cells.