• Journal of Life Science
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• v.20 no.9
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• pp.1314-1323
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• 2010

In this study, we constructed various kinds of binary vectors with the pPZP backbone for co-overexpression, tissue- or development-specific expression and stress-inducible expression, and validated them for ectopic expression of target genes. Using a modified CaMV 35S promoter, a binary vector was generated for co-overexpression of two different genes and was confirmed to be efficient for overexpressing two different target genes at the same time and place. Binary vectors containing At2S3, KNAT1 or LFY promoters were constructed for tissue-specific or development-specific gene expression, and the binary vectors were suited for embryo/young seedling stage-, shoot apical meristem- or leaf primordia-specific expressions. Furthermore, the binary vectors containing RD29A or AtNCED3 promoters were validated as suitable vectors for gene expression induced by abiotic stresses such as high salt, ABA, MV and low temperature. Taken together, the binary vectors constructed in this study would be very useful for analyzing the biological functions of target genes and molecular mechanisms through ectopic expression.

• Biomolecules & Therapeutics
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• v.22 no.6
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• pp.497-502
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• 2014

In the present study, we found that the natural compound arctigenin inhibited hydrogen peroxide-induced reactive oxygen species (ROS) production in rat primary astrocytes. Since hemeoxygenase-1 (HO-1) plays a critical role as an antioxidant defense factor in the brain, we examined the effect of arctigenin on HO-1 expression in rat primary astrocytes. We found that arctigenin increased HO-1 mRNA and protein levels. Arctigenin also increases the nuclear translocation and DNA binding of Nrf2/c-Jun to the antioxidant response element (ARE) on HO-1 promoter. In addition, arctigenin increased ARE-mediated transcriptional activities in rat primary astrocytes. Further mechanistic studies revealed that arctigenin increased the phosphorylation of AKT, a downstream substrate of phosphatidylinositol 3-kinase (PI3K). Treatment of cells with a PI3K-specific inhibitor, LY294002, suppressed the HO-1 expression, Nrf2 DNA binding and ARE-mediated transcriptional activities in arctigenin-treated astrocyte cells. The results collectively suggest that PI3K/AKT signaling pathway is at least partly involved in HO-1 expression by arctigenin via modulation of Nrf2/ARE axis in rat primary astrocytes.

• Molecules and Cells
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• v.19 no.1
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• pp.81-87
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• 2005

Phytochelatins play an important role in heavy metal detoxification in plants as well as in other organisms. The Arabidopsis thaliana mutant cad1-3 does not produce detectable levels of phytochelatins in response to cadmium stress. The hypersensitivity of cad1-3 to cadmium stress is attributed to a mutation in the phytochelatin synthase 1 (AtPCS1) gene. However, A. thaliana also contains a functional phytochelatin synthase 2 (AtPCS2). In this study, we investigated why the cad1-3 mutant is hypersensitive to cadmium stress despite the presence of AtPCS2. Northern and Western blot analyses showed that expression of AtPCS2 is weak compared to AtPCS1 in both roots and shoots of transgenic Arabidopsis. The lower level of AtPCS2 expression was confirmed by RT-PCR analysis of wild type Arabidopsis. Moreover, no tissue-specific expression of AtPCS2 was observed. Even when AtPCS2 was under the control of the AtPCS1 promoter or of the cauliflower mosaic virus 35S promoter (CaMV 35S) it was not capable of fully complementing the cad1-3 mutant for cadmium resistance.

• BMB Reports
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• v.47 no.2
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• pp.86-91
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• 2014

Tissue-specific gene expression is regulated by epigenetic modification involving trans-acting factors. Here, we identified that the human MAGEB16 gene and its mouse homolog, Mageb16, are only expressed in the testis. To investigate the mechanism governing their expression, the promoter methylation status of these genes was examined in different samples. Two CpG islands (CGIs) in the 5' upstream region of MAGEB16 were highly demethylated in human testes, whereas they were methylated in cells without MAGEB16 expression. Similarly, the CGI in Mageb16 was hypomethylated in mouse testes but hypermethylated in other tissues and cells without Mageb16 expression. Additionally, the expression of these genes could be activated by treatment with the demethylation agent 5'-aza-2'-deoxycytidine (5'-aza-CdR). Luciferase assays revealed that both gene promoter activities were inhibited by methylation of the CGI regions. Therefore, we propose that the testis-specific expression of MAGEB16 and Mageb16 is regulated by the methylation status of their promoter regions.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.6
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• pp.2799-2806
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• 2012

The aim of this study was to elucidate the role of the integrin-linked kinase (ILK) gene in development of human bladder transitional cell carcinoma (BTCC). Expression of ILK protein and ILK mRNA in 56 cases of human BTCC tissue and in 30 cases of adjacent normal bladder tissue was detected by immunohistochemistry S-P and reverse transcription polymerase chain reaction (RT-PCR), respectively. Four specific miRNA RNAi vectors targeting human ILK were synthesized and transfected into BIU-87 cells by liposome to obtain stable expression cell strains. The influence of ILK on proliferation of BTCC was detected by MTT, FCM on athymic mouse tumorigenesis. The positive rate of ILK protein in BTCC tissue (53.6%) was much higher than adjacent normal bladder tissue (10.0%) (p<0.05). Similarly, expression of ILK mRNA in BTCC tissue ($0.540{\pm}0.083$) was significantly higher than in adjacent normal bladder tissue ($0.492{\pm}0.070$) (p<0.05). MTT showed that the proliferation ability of miRNA-ILK transfected group was clearly decreased (p<0.05), the cell cycle being arrested in G0/G1-S, an tumorigenesis in vivo was also significantly reduced (p<0.05). ILK gene transcription and protein expression may be involved in the development of BTCC, so that ILK might be the new marker for early diagnosis and the new target for gene treatment.

• Korean Journal of Ichthyology
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• v.9 no.1
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• pp.91-98
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• 1997

The transgene, pFV4CAT, containing CAT reporter gene regulated by carp $\beta$-actin promoter, was expressed in independent transgenic mud loach germ lines, determined by reverse transcriptase-PCR (RT-PCR) and enzyme-linked immunosorbant assay (ELISA). Expression of the transmitted transgene was found to be tissue-specific in F1 and F2 generations. Tissue specificity of the expression was dependent on each transgenic line with reproducible patterns. Liver and spleen did express the transgene more frequently than other tissues tested, and muscle and heart revealed the higher amount of CAT than other tissues, while testes showed the lowest expression level. The highest level of CAT expression in muscle from a transgenic F1 line was corresponding to 68-fold compared to the basal levels of controls.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.4
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• pp.1557-1561
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• 2012

Objective: To investigate the promoter methylation status of the E-cadherin gene in non-small cell lung cancer (NSCLC) and its association with clinical pathological parameters, and to explore the relationship between downregulation of E-cadherin gene expression and the methylation status of its promoter region. Methods: Nested methylation-specific PCR was performed to examine CpG methylation within the 5' CpG island of the E-cadherin gene in lung cancer and para-cancerous tissue from 37 patients with primary non-small cell lung cancer. Quantitative real-time PCR was performed to measure the level of E-cadherin mRNA. Results: Of thirty-seven cases, 12 (32.4%) samples showed aberrant CpG methylation in tumor tissues compared with the corresponding normal tissues. In addition, a reduction in E-cadherin mRNA levels was observed in 11 of the 12 (91.7%) tumor tissues carrying a methylated E-cadherin gene. However, only 10 (43.5%) cases displayed reduced mRNA levels in tumor tissues from the remaining 23 cases (excluding 2 samples from which mRNA was unavailable) without methylation events. Downregulation of E-cadherin gene expression significantly correlated with the promoter methylation status of this gene. Conclusion: These results provide strong evidence that the methylation status of E-cadherin gene contributes to a reduction in the expression of E-cadherin mRNA, and may play a role in the development and progression of NSCLC.

• Development and Reproduction
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• v.13 no.3
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• pp.191-198
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• 2009

In an attempt to simultaneously produce two human proteins, hGH and hG-CSF, in the milk of transgenic mice, we constructed goat $\beta$-casein-directed hGH and hG-CSF expression cassettes individually and generated transgenic mice by co-injecting them into mouse zygotes. Out of 33 transgenic mice, 29 were identified as double transgenic harboring both transgenes on their genome. All analyzed double transgenic females secreted both hGH and hG-CSF in their milks. Concentrations ranged from 2.1 to $12.4\;mg/m{\ell}$ for hGH and from 0.04 to $0.13\;mg/m{\ell}$ for hG-CSF. hG-CSF level was much lower than hGH level but very similar to that of single hG-CSF mice, which were introduced with hG-CSF cassette alone. In order to address the causes of concentration difference between hGH and hG-CSF in milk, we examined mRNA level of hGH and hG-CSF in the mammary glands of double transgenic mice and tissue specificity of hG-CSF mRNA expression in both double and single transgenic mice. Likewise protein levels in milk, hGH mRNA level was much higher than hG-CSF mRNA, and hG-CSF mRNA expression was definitely specific to the mammary glands of both double and single transgenic mice. These results demonstrated that two transgenes have distinct transcriptional potentials without interaction each other in double transgenic mice although two transgenes co-integrated into same genomic sites and their expressions were directed by the same goat $\beta$-casein promoter. Therefore goat $\beta$-casein promoter is very useful for the multiple production of human proteins in the milk of transgenic animals.

• Journal of Sericultural and Entomological Science
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• v.53 no.1
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• pp.44-49
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• 2015

We characterized tissue specific-expressed genes in the posterior silk gland of Bombyx mori using by the Annealing Control Primer based differential display-PCR manner. In this study, we isolated 34 differentially expressed PCR amplicons, which one of these was identified as a novel transcript named as ACP-16 (366 bp), its expression was observed only in the posterior silk glands by Northern blot analysis. To determine promoter region of the ACP-16, we isolated and analyzed a phage DNA having 1.7 kb-long genome DNA including the open reading flame and 5'- upstream untranslated region of the ACP-16 gene from a genomic DNA library. We have estimated a promoter region of the ACP-16 gene by a web promoter prediction engine, which locates -750 ~ -165 from translation initiation site (ATG, +1). ACP-16 gene is necessary to more studies about critical biological role in order to apply the silkworm's transgenic system.

• Journal of Life Science
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• v.18 no.10
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• pp.1348-1354
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• 2008

mi transcription factor (MITF) is important in regulating the differentiation of mast cells. In particular, MITF regulates the transcription of the mouse mast cell-specific serine protease (mMCP)-6 gene, which is generally expressed by the connective tissue-type of mast cells. In this study, we investigated alternative isoforms of MITF that regulate transcription of the mMCP-6 gene in bone marrow-derived cultured mast cells in mice. The expression of MITF isoforms was examined by RT-PCR. We observed that MITF-A, -E, -H and -Mc were expressed by mucosal-type mast cells cultured in the presence of IL-3, whereas the connective tissue-type mast cells cultured in the presence of stem cell factor (SCF) expressed MITF-A. Overexpression of MITF isoforms increased luciferase activity through the mMCP-6 promoter in NIH-3T3 cells and elevated the level of mMCP-6 expression in the MC/9 mast cell line. Moreover, mMCP-6 expression in mast cells was significantly inhibited by the depletion of MITF. The transcriptional activity and DNA binding of MITF-A was comparable to that of MITF isoforms, including MITF-E, -H, and -Mc. Our results therefore suggest that MITF-A may be an important isoform of MITF in regulating the transcription of mMCP-6 in mouse connective tissue mast cells.