• Development and Reproduction
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• v.17 no.4
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• pp.379-387
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• 2013

Embryonic stem (ES) cells can self-renew and differentiate to various cells depending on the culture condition. Although ES cells are a good model for cell type specification and can be useful for application in clinics in the future, studies on ES cells have many experimental restraints including low transfection efficiency and transgene expression. Here, we observed that transgene expression after transfection was enhanced by treatment with histone deacetylse (HDAC) inhibitors such as trichostatin A, sodium butyrate, and valproic acid. Transfection was performed using conventional transfection reagents with a retroviral vector encoding GFP under the control of CMV promoter as a reporter. Treatment of ES cells with HDAC inhibitors after transfection increased population of GFP positive cells up to 180% compared with untreated control. ES cells showed normal expression of stem cell markers after treatment with HDAC inhibitors. Transgene expression was further enhanced by modifying transfection procedure. GFP positive cells selected after transfection were proved to have the stem cell properties. Our improved protocol for enhanced gene delivery and expression in mouse ES cells without hampering ES cell properties will be useful for study and application of ES cells.

• Molecules and Cells
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• v.46 no.4
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• pp.209-218
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• 2023

In induced pluripotent stem cells (iPSCs), pluripotency is induced artificially by introducing the transcription factors Oct4, Sox2, Klf4, and c-Myc. When a transgene is introduced using a viral vector, the transgene may be integrated into the host genome and cause a mutation and cancer. No integration occurs when an episomal vector is used, but this method has a limitation in that remnants of the virus or vector remain in the cell, which limits the use of such iPSCs in therapeutic applications. Chemical reprogramming, which relies on treatment with small-molecule compounds to induce pluripotency, can overcome this problem. In this method, reprogramming is induced according to the gene expression pattern of extra-embryonic endoderm (XEN) cells, which are used as an intermediate stage in pluripotency induction. Therefore, iPSCs can be induced only from established XEN cells. We induced XEN cells using small molecules that modulate a signaling pathway and affect epigenetic modifications, and devised a culture method which can produce homogeneous XEN cells. At least 4 passages were required to establish morphologically homogeneous chemically induced XEN (CiXEN) cells, whose properties were similar to those of XEN cells, as revealed through cellular and molecular characterization. Chemically iPSCs derived from CiXEN cells showed characteristics similar to those of mouse embryonic stem cells. Our results show that the homogeneity of CiXEN cells is critical for the efficient induction of pluripotency by chemicals.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.20-20
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• 2002

This study was to establish the use of parthenogenetic mouse ES (P-mES02) cells as a reproducible differentiation system for mouse cardiomyocytes. To induce differentiation, P-mES02 cells were dispersed by dissociation and the formation of ES cell aggregates in differentiation medium. After 7 days in differentiation culture, the embryoid bodies (EBs) were plated onto gelatin-coated dish. Cultures were observed daily using an inverted light microscope to determine the day of contraction onset and total duration of continuous contractile activity for each contracting focus. (omitted)

• 대한생식의학회:학술대회논문집
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• 2001.06a
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• pp.49.2-50
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• 2001

• 대한생식의학회:학술대회논문집
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• 2007.11a
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• pp.117-117
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• 2007

• BMB Reports
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• v.54 no.12
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• pp.626-631
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• 2021

Janus kinase 2 (JAK2), a non-receptor tyrosine kinase, is a critical component of cytokine and growth factor signaling pathways regulating hematopoietic cell proliferation. JAK2 mutations are associated with multiple myeloproliferative neoplasms. Although physiological and pathological functions of JAK2 in hematopoietic tissues are well-known, such functions of JAK2 in the nervous system are not well studied yet. The present study demonstrated that JAK2 could negatively regulate neuronal differentiation of mouse embryonic stem cells (ESCs). Depletion of JAK2 stimulated neuronal differentiation of mouse ESCs and activated glycogen synthase kinase 3β, Fyn, and cyclin-dependent kinase 5. Knockdown of JAK2 resulted in accumulation of GTP-bound Rac1, a Rho GTPase implicated in the regulation of cytoskeletal dynamics. These findings suggest that JAK2 might negatively regulate neuronal differentiation by suppressing the GSK-3β/Fyn/CDK5 signaling pathway responsible for morphological maturation.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.6
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• pp.910-927
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• 2003

Transgenesis is a very powerful tool not only to help understanding the basics of life science but also to improve the efficiency of animal production. Since the first transgenic mouse was born in 1980, rapid development and wide application of this technique have been made in laboratory animals as well as in domestic animals. Although pronuclear injection is the most widely used method and nuclear transfer using somatic cells broadens the choice of making transgenic domestic animals, the demand for precise manipulation of the genome leads to the utilization of gene targeting. To make this technique possible, a pluripotent embryonic cell line such as embryonic stem (ES) cell is required to carry genetic mutation to further generations. However, ES cell, well established in mice, is not available in domestic animals even though many attempt to establish the cell line. An alternate source of pluripotent cells is embryonic germ (EG) cells derived from primordial germ cells (PGCs). To make gene targeting feasible in this cell line, a better culture system would help to minimize the unnecessary loss of cells in vitro. In this review, general methods to produce transgenic domestic animals will be mentioned. Also, it will focus on germ cell engineering and methods to improve the establishment of pluripotent embryonic cell lines in domestic animals.

• Clinical and Experimental Reproductive Medicine
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• v.32 no.2
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• pp.133-147
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• 2005

Objectives: This study was carried out to establish human embryonic stem cells derived from frozen-thawed embryos using mouse embryonic fibroblasts (mEFs), human fetal skin and muscle fibroblasts as feeder cells, and to identify the characteristic of embryonic stem cells. Methods: When primary mEFs, human fetal skin and muscle fibroblasts were prepared, passaging on 4 days from replating could have effective trypsinization and clear feeder layers. Eight of 23 frozenthawed 4~8 cell stage embryos donated from consenting couples developed to blastocysts. Inner cell mass (ICM) was isolated by immunosurgery. ICM was co-cultured on mEFs, human fetal skin or muscle fibroblasts. The ICM colonies grown on mEFs, human fetal skin or muscle fibroblasts were tested the expression of stage specific embryonic antigen-3, -4 (SSEA-3, -4), octamer binding transcription factor-4 mRNA (Oct-4) and alkaline phosphatase surface marker. Results: We obtained 1 ICM colony from 2 ICM co-cultured on mEFs as feeder cells and did not obtain any ICM colony from 6 ICM clumps co-cultured on human fetal skin or muscle fibroblasts. The colony formed on mEFs could be passaged 30 times every 5 days with sustaining undifferentiated colony appearance. When the colonies cultured on mEFs were grown on human fetal skin or muscle fibroblasts, the colonies could be passaged 15 times every 9 days with sustaining undifferentiated colony appearance. The colonies grown on mEFs and human fetal fibroblasts expressed SSEA-4 and alkaline phosphatase surface markers and positive for the expression of Oct-4 by reverse transcription-polymerase chain reaction (RT-PCR). The produced embryoid body differentiated spontaneously to neural progenitorlike cells, neuron-like cells and beating cardiomyocyte-like cells, and frozen-thawed embryonic stem cells displayed normal 46,XX karyotype. Conclusions: The human embryonic stem cells can be established by using mEFs and human fetal fibroblasts produced in laboratory as feeder cells.

• Reproductive and Developmental Biology
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• v.35 no.4
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• pp.427-434
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• 2011

Mitochondria diseases have been reported to involve structural and functional defects of complex I-V. Especially, many of these diseases are known to be related to dysfunction of mitochondrial proton-translocating NADH-ubiquinone oxidoreductase (complex I). The dysfunction of mitochondria complex I is associated with neurodegenerative disorders, such as Parkinson's disease, Huntington's disease, and Leber's hereditary optic neuropathy (LHON). Mammalian mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I) is largest and consists of at least 46 different subunits. In contrast, the NDI1 gene of Saccharomyces cerevisiae is a single subunit rotenone-insensitive NADH-quinone oxidoreductase that is located on the matrix side of the inner mitochondrial membrane. The Saccharomyces cerevisiae NDI1 gene using a recombinant adeno-associated virus vector (rAAV-NDI1) was successfully expressed in AML12 mouse liver hepatocytes and the NDI1-transduced cells were able to grow in media containing rotenone. In contrast, control cells that did not receive the NDI1 gene failed to survive. The expressed Ndi1 enzyme was recognized to be localized in mitochondria by confocal immunofluorescence microscopic analyses and immunoblotting. Using digitonin-permeabilized cells, it was shown that the NADH oxidase activity of the NDI1-transduced cells was not affected by rotenone which is inhibitor of complex I, but was inhibited by antimycin A. Furthermore, these results indicate that Ndi1 can be functionally expressed in the AML12 mouse liver hepatocytes. It is conceivable that the NDI1 gene is powerful tool for gene therapy of mitochondrial diseases caused by complex I deficiency. In the future, we will attempt to functionally express the NDI1 gene in mouse embryonic stem (mES) cell.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.83-83
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• 2002

This study was to compare the characteristics of parthenogenetic mES (P-mES) cells and in vitro fertilization mES cells. Mouse oocytes were recovered from superovulated 4wks hybrid F1 (C57BL/6xCBA/N) female mice. The oocytes were treated with 7% ethanol for 5 min and 5 ㎍/㎖ cytochalasin-B for 4 h. For IVF, the oocytes were inseminated with epididymal sperm of hybrid Fl male mice (1×10/sup 6//㎖). IVF and parthenogenetic embryos were cultured in M16 medium for 4 days. Cell number count in blastocysts was carried out differential labelling using propidium iodide (red) and bisbenzimide(blue). (omitted)