• Development and Reproduction
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• v.14 no.2
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• pp.123-129
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• 2010

Embryonic stem (ES) cells can self-renew maintaining the undifferentiated state. Self-renewal requires many factors such as OCT4, SOX2, and NANOG. It is previously known that OCT4 and SOX2 can bind to NANOG promoter and support Nanog gene expression in mouse ES cells by the detailed studies using the mouse Nanog promoter. Here, we constructed serial deletion mutant promoter-reporter constructs to investigate the human Nanog gene promoter in detail. The highest promoter activity was obtained in the 0.6 kb (-253/+365) promoter-reporter construct which includes the binding sites of OCT4 and SOX2. To further confirm contribution of OCT4 and SOX2 in Nanog gene expression, we introduced site- directed mutation(s) in the OCT4 and/or SOX2 binding sites of the human Nanog promoter 0.6 kb (-253/+365) and checked the influence of the mutation on the promoter activity using human EC cell line NCCIT. Mutation either in OCT4 binding site or SOX2 binding site significantly reduced the activity of Nanog promoter which directly confirmed that OCT4 and SOX2 binding is essential in human Nanog gene expression.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.3
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• pp.449-456
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• 2018

Objective: Nanog, a homeodomain protein, has been investigated in humans and mice using embryonic stem cells (ESCs). Because of the limited availability of ESCs, few studies have reported the function and role of Nanog in porcine ESCs. Therefore, in this study, we investigated the location of the porcine Nanog chromosome and its basal promoter activity, which might have potential applications in development of ESCs specific marker as well as understanding its operating systems in the porcine. Methods: To characterize the porcine Nanog promoter, the 5'-flanking region of Nanog was isolated from cells of mini-pig ears. BLAST database search showed that there are two porcine Nanog genomic loci, chromosome 1 and 5, both of which contain an exon with a start codon. Deletion mutants from the 5'-flanking region of both loci were measured using the Dual-Luciferase Reporter Assay System, and a fluorescence marker, green fluorescence protein. Results: Promoter activity was detected in the sequences of chromosome 5, but not in those of chromosome 1. We identified the sequences from -99 to +194 that possessed promoter activity and contained transcription factor binding sites from deletion fragment analysis. Among the transcription factor binding sites, a Sp1 was found to play a crucial role in basal promoter activity, and point mutation of this site abolished its activity, confirming its role in promoter activity. Furthermore, gel shift analysis and chromatin immunoprecipitation analysis confirmed that Sp1 transcription factor binds to the Sp1 binding site in the porcine Nanog promoter. Taken together, these results show that Sp1 transcription factor is an essential element for porcine Nanog basal activity the same as in human and mouse. Conclusion: We showed that the porcine Nanog gene is located on porcine chromosome 5 and its basal transcriptional activity is controlled by Sp1 transcription factor.

• Laboraroty Animal Research
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• v.34 no.4
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• pp.257-263
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• 2018

Trefoil factor 1 (TFF1, also known as pS2) is strongly expressed in the gastrointestinal mucosa and plays a critical role in the differentiation of gastric glands. Since approximately 50% of all human gastric cancers are associated with decreased TFF1 expression, it is considered a tumor suppressor gene. Tff1 deficiency in mice results in histological changes in the antral and pyloric gastric mucosa, with severe hyperplasia and dysplasia of epithelial cells, resulting in the development of antropyloric adenoma. Here, we generated Tff1-knockout (KO) mice, without a neomycin resistant ($Neo^R$) cassette, using the clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRSIPR/Cas9) system. Though our Tff1-KO mice showed phenotypes very similar to the previous embryonic stem (ES)-cell-based KO mice, they differed from the previous reports in that a reduction in body weight was observed in males. These results demonstrate that these newly established Tff1-KO mice are useful tools for investigating genetic and environmental factors influencing gastric cancer, without the effects of artificial gene insertion. Furthermore, these findings suggest a novel hypothesis that Tff1 expression influences gender differences.

• Korean Journal of Poultry Science
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• v.36 no.2
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• pp.177-186
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• 2009

It is well-known that unregulated over-expression of foreign gene may have unwanted physiological or toxic effects in transgenic animals. To circumvent these problems, we constructed retrovirus vector designed to express the foreign gene under the control of the tetracycline-inducible promoter. However, gene expressions in the tetracycline-inducible expression system (Tet system) are not completely regulated but a little leaky due to the inherent defects in conventional Tet-based systems. A more tightly controllable regulatory system can be achieved when the advanced versions ($rtTA2^SM2$) of rtTA and a minimal promoter in responsive components (pTRE-tight) are used in combination therein. In this study, we tried to produce human thrombopoietin (hTPO) from various target cells and transgenic chickens using the retrovirus vector combined with Tet system. hTPO is the primary regulator of platelet production and has an important role in the survival and expansion of hematopoietic stem cells. In a preliminary experiment in vitro, higher hTPO expression and tighter expression control were observed in chicken embryonic fibroblast (CEF) cells. We also measured the biological activity of the hTPO using Mo7e cells whose proliferation is dependant on hTPO. The biological activity of the recombinant hTPO from CEF was higher than both its commercial counterpart and hTPO from other target cells. The recombinant retrovirus was injected beneath the blastoderm of non-incubated chicken embryos (stage X). Out of 138 injected eggs, 15 chicks hatched after 21 days of incubation. Among them, 8 hatched chicks were hTPO positive. When the Go transgenic chicken was fed doxycycline (0.5 mg per 1 gram of feed), a tetracycline derivative, hTPO concentration of the transgenic chicken blood was 200 ng/mL. Germline transmission of the transgene was confirmed in sperm of the Go transgenic roosters. These results are informative to establish transgenic chickens as bioreactors for the mass production of commercially valuable and biological active human cytokine proteins.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.16
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• pp.6813-6823
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• 2015

Glioblastoma, also known as glioblastoma multiforme (GBM), is the most aggressive of human brain tumors and has a stunning progression with a mean survival of one year from the date of diagnosis. High cell proliferation, angiogenesis and/or necrosis are histopathological features of this cancer, which has no efficient curative therapy. This aggressiveness is associated with particular heterogeneity of the tumor featuring multiple genetic and epigenetic alterations, but also with implications of aberrant signaling driven by growth factors. The transforming growth factor ${\beta}$ ($TGF{\beta}$) superfamily is a large group of structurally related proteins including $TGF{\beta}$ subfamily members Nodal, Activin, Lefty, bone morphogenetic proteins (BMPs) and growth and differentiation factor (GDF). It is involved in important biological functions including morphogenesis, embryonic development, adult stem cell differentiation, immune regulation, wound healing and inflammation. This superfamily is also considered to impact on cancer biology including that of GBM, with various effects depending on the member. The $TGF{\beta}$ subfamily, in particular, is overexpressed in some GBM types which exhibit aggressive phenotypes. This subfamily impairs anti-cancer immune responses in several ways, including immune cells inhibition and major histocompatibility (MHC) class I and II abolishment. It promotes GBM angiogenesis by inducing angiogenic factors such as vascular endothelial growth factor (VEGF), plasminogen activator inhibitor (PAI-I) and insulinlike growth factor-binding protein 7 (IGFBP7), contributes to GBM progression by inducing metalloproteinases (MMPs), "pro-neoplastic" integrins (${\alpha}v{\beta}3$, ${\alpha}5{\beta}1$) and GBM initiating cells (GICs) as well as inducing a GBM mesenchymal phenotype. Equally, Nodal promotes GICs, induces cancer metabolic switch and supports GBM cell proliferation, but is negatively regulated by Lefty. Activin promotes GBM cell proliferation while GDF yields immune-escape function. On the other hand, BMPs target GICS and induce differentiation and sensitivity to chemotherapy. This multifaceted involvement of this superfamily in GBM necessitates different strategies in anti-cancer therapy. While suppressing the $TGF{\beta}$ subfamily yields advantageous results, enhancing BMPs production is also beneficial.