• Journal of Microbiology and Biotechnology
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• 제16권3호
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• pp.386-390
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• 2006

A green fluorescent protein-based Pseudomonas putida reporter was successfully constructed and shown to be capable of detecting oxidative stress. In this whole-cell reporter, the promoter of the paraquat-inducible ferredoxin-$NADP^+$ reductase (fpr) was fused to a promoterless gfp gene on a broad-host-range promoter probe vector. Pseudomonas putida KT2440 harboring this reporter plasmid exhibited an increased level of gfp expression in the presence of redox-cycling agents (paraquat and menadione), hydrogen peroxide, and potential environmental pollutant chemicals such as toluene, paint thinner, gasoline, and diesel. Induction of fpr in the presence of these chemicals was confirmed using Northern blot analysis.

• Animal cells and systems
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• 제6권4호
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• pp.317-322
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• 2002

To improve conventional yeast one-hybrid screening, we have developed an efficient mammalian one-hybrid system that allows rapid isolation of com-plementary DNAs which are able to induce human p14$^{ARF}$. tumor suppressor gene. A 1.5 kb promoter region of p14$^{ARF}$ was fused to EGFP to generate ARF promoter-EGFP reporter vector. This reporter plasmid was stably trans-fected into NIH3T3 cells for generation of reporter cell line. When the reporter cell line was infected with E2F-1 together with excess amounts of empty vector, the cells that received the positive modulator were readily identifiable by green fluorescence using FACS. The GFP-positive cells were cloned directly from the cultured cells and expanded in bulk culture. The genomic DNAs from GFP-positive cells were prepared and the CDNA insert in integrated retroviral genome was recovered by PCR using primers annealing to the retroviral vector sequences flanking the insert-cloning site. This system should be useful for efficient screening of expression CDNA libraries in mammalian cells to identify novel upstream regulators for spe-cific genes by one-hybrid interaction.ion.

• Journal of Plant Biotechnology
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• 제2권3호
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• pp.135-142
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• 2000

The activity of promoter sequences was evaluated in garlic cells using the $\beta$-glucuronidase (GUS) gene as a reporter. Histochemical GUS assay indicated transient GUS activity in leaf, callus and root cells 48 hours after particle bombardment transformation. Quantitative fluorometric assays in extracts of transformed leaves demonstrated that the CsVMV promoter induced the highest level of gene expression, which was, on average, ten fold the level induced by CaMV35S and by the Arabidopsis Act2 promoters and two fold the level expression observed with a construct containing a double CaMV35S plus the untranslated leader sequence from AMV. No activity or very low levels were observed when cells were transformed with plasmids rontaining the typical monocot promoters, Actl, from rice or the Ubi-1, from maize. The green fluorescent protein (GFP) was also tested as a marker gene for garlic transformation. Intense fluorescence was observed in leaf, callus and root cells transformed with a construct containing the gfp gene under control of the CaMV35 Promoter. No fluorescence was detected when the gfp was under control of the Ubi-1 promoter.

• Fisheries and Aquatic Sciences
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• 제7권2호
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• pp.70-75
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• 2004

The CMV promoter driven lacZ reporter gene (pcDNA-lacZ) was constructed and used for DNA immunization study. The expression of the lacZ gene was confirmed in vitro using RTG-2 cell line before using for in vivo study in olive flounder (Paralichthys olivaceus). In the dose response study, the maximum expression of the lacZ gene was found in the group injected with 5 ${\mu} g$ of the plasmid DNA. Kinetic study showed a significantly increased expression of $\beta-galactosidase$ gene at 7 days after injection. Effects of DNA vaccine on specific and nonspecific immune responses such as antibody and NO production were studied and the significant effect was found in olive flounder injected with 10 and 15 ${\mu} g$ DNA (sub optimal dose for lacZ gene expression). Two pro inflammatory cytokine genes, $IL-l\beta$ and $TNF-\alpha$, were also found to be up regulated in the muscle injected with the plasmid, suggesting an induction of local inflammatory response.

• 한국발생생물학회지:발생과생식
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• 제14권2호
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• pp.123-129
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• 2010

배아 줄기세포는 미분화상태에서 자가 재생을 유지할 수 있다. 자가 재생은 OCT4, SOX2와 NANOG와 같은 많은 인자들이 작용한다. 생쥐 배아 줄기세포에서 OCT4와 SOX2가 Nanog 프로모터에 결합하여 Nanog 유전자의 발현을 촉진한다는 사실은 생쥐 promoter에 관한 정밀분석으로 알려져 있다. 본 연구에서는 인간 Nanog promoter를 정밀 분석하기 위해 연속적인 결손 돌연변이를 가진 promoter-reporter construct를 제조하였다. Promoter의 최대 활성은 0.6 kb(-253/+365) promoter-reporter construct에서 발견되었으며, 이 construct에는 OCT4 및 SOX2의 결합부위가 포함된다. OCT4와 SOX2의 기여도를 확인하기 위하여 OCT4 및 SOX2의 결합부위에 자리 특이적 돌연변이를 유도하고 promoter 활성에 미치는 영향을 조사한 결과, OCT4나 SOX2 어느 한 군데라도 돌연변이가 존재하면 promoter 활성이 현저히 저해되었다. 본 연구 결과를 통해 인간 Nanog 유전자 발현에 있어 OCT4 및 SOX2가 필수적임을 직접적으로 확인할 수 있었다.

• 한국환경성돌연변이발암원학회지
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• 제23권2호
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• pp.45-50
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• 2003

Biological activities of PAHs are not known although PAHs are considered as carcinogens. Recent industrial society has human widely exposed to PAHs (polynuclear aromatic hydrocarbons) that are comming from the incomplete combustion of organic material as wider spread environmental contaminants. Our laboratory have been studied the effect of PAHs in the human breast cancer MCF-7 cells. In this study, we examined the human breast cancer MCF-7 cells as a new system to evaluate bioactivity of PAHs. We have selected 13 PAHs to examine bioassay using CYP1A1-luciferase reporter gene expression system where CYP1A1 1.6 Kb 5flanking region DNA was cloned in front of luciferase reporter gene and this plasmid was transfected into MCF-7 cells transiently. This cells then used for the study to observe the effect of PAHs. We demonstrated that PAHs induced the CYP1A1 promoter, CYP1A1 mRNA and 7-ethoxyresolufin O-deethylase (EROD) activities in a concentration-dependant manner. None of PAHs that we have tested showed stronger stimulatory effect on CYP1 gene expression than TCDD. Benz(a)anthracene and benzo(b)fluoranthene were weak responders to CYP1A1 promoter activity stimulation, CYP1A1 mRNA and EROD induction in MCF-7 cells and these chemicals seemed to respond less either CYP1A1 mRNA or EROD than CYP1A1 promoter activity. Benzo(k)fluoranthene, chrysene, and dibenzo(a, h)anthracene showed strong response to CYP1A1 promoter activity stimulation, CYP1A1 mRNA increase and also EROD induction in MCF-7 cells. Results of dose response study suggested that two strong responding PAHs, such as benzo(k)fluoranthene and dibenzo(a, h)anthracene might be mediated through Aryl hydrocarbon receptors system in MCF-7 cells.

• 한국환경성돌연변이발암원학회지
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• 제23권1호
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• pp.30-34
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• 2003

Recent industrial society has human widely exposed to PAHs (polynuclear aromatic hydrocarbons) that are comming from the incomplete combustion of organic material as wider spread environmental contaminants. Biological activities of PAHs are not known although PAHs are considered as carcinogens. Our laboratory have been studied the effect of PAHs in the mouse liver hepa 1 cells. In this study, we examined the mouse liver hepa-l cells as a new bioassay system to evaluate bioactivity of PAHs. We have selected 13 PAHs to examine bioassay using cyp1a1-luciferase reporter gene expression system where cyp1a1 1.6 Kb 5flanking region DNA was cloned in front of luciferase reporter gene and this plasmid was transfected into hepa 1 cells transiently. This cells then used for the study to observe the effect of PAHs. We demonstrated that PAHs induced the CYP1A1 promoter and 7-ethoxyresolufin O-deethylase (EROD) activities in a concentration-dependant manner. Some of PAHs showed stronger stimulatory effect on CYP1 gene expression than TCDD. Acenaphthene, anthracene, fluorine, naphthalene, pyrene, phenanthrene, carbazole were weak responders to cyp1a1 promoter activity stimulation and EROD induction in hepa 1 cells and these chemicals seemed to respond less to EROD than cyp1a1 promoter activity. Benz(a)anthracene, benzo(b)fluoranthene, benzo(k)fluoranthene, chrysene, and dibenzo(a,h)anthracene showed strong response to cyp1a1 promoter activity stimulation and also EROD induction in hepa 1cells. Results of dose response study suggested that four strong responding PAHs, such as benzo(a)anthracene benzo(k)fluoranthene, chrysene, and dibenzo(a, h)anthracene might be mediated through arylhydrocarbon receptor system in hepa1 cells.

• 대한의생명과학회지
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• 제10권2호
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• pp.137-142
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• 2004

The peroxisome proliferator-activated receptor $\gamma$ (PPAR${\gamma}$) is the molecular target for a class of drugs, the antidiabetic thiazolidnediones (TZDs). The heterodimer of PPAR${\gamma}$ with retinoid X receptor (RXR) plays a central role in the regulation of adipogenesis and insulin sensitization. We synthesized two chemicals, DANA87 and DANA88, sharing structural characteristics with TZDs. Given this structural similarity, it was hypothesized that DANA87 and DANA88 may act as PPAR$\gamma$ ligands. In transient transfection assays, DANA87 and DANA88 caused slight increases in the endogenous expression of a luciferase reporter gene containing the PPAR responsive element in 3T3-L1 preadipocytes. However, DANA87 and DANA88 significantly inhibited troglitazone-induced reporter gene activation when cells were treated with a combination of DANA87 or DANA 88 and troglitazone, one of the TZDs that activate PPAR$\gamma$. These results suggest that DANA87 and DANA88 are not only weak agonists of PPAR${\gamma}$ transactivation, but also competitively antagonize troglitazone-induced PPAR$\gamma$ reporter activity.

• 대한의생명과학회지
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• 제15권3호
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• pp.179-185
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• 2009

In our previous report, we showed that $PPAR{\gamma}$ does not influence adipogenesis in females with functioning ovaries, indicating that $PPAR{\gamma}$ activity on adipogenesis is associated with sex-related factors. Among the sex-related factors, estrogen has been recognized as a major factor in inhibiting adiposgenesis in females. Thus, we hypothensized that $17{\beta}$-estradiol (E) inhibits 3T3-L1 cell adipogenesis by preventing $PPAR{\gamma}$ activity. E decreased triglyceirde accumulation in differentiated 3T3-L1 cells compared with control group. E also decreased the expression of $PPAR{\gamma}$ mRNA as well as $PPAR{\gamma}$ dependent adipocyte-specific genes, such as adipocyte fatty acid binding protein and tumor necrosis factor $\alpha$. In addition, E not only decreased luciferase reporter activity by $PPAR{\gamma}$, but also transfection of estrogen receptor $\alpha$ ($ER{\alpha}$) or $ER{\beta}$ led to decreases in $PPAR{\gamma}$ reporter gene activation. Moreover, E-activated ERs significantly decreased the luciferase reporter gene activation induced by $PPAR{\gamma}$ transfection, suggesting that estrogen-activated ERs inhibit $PPAR{\gamma}$-dependent transactivation. Accordingly, our results demonstrate that E inhibits the action of $PPAR{\gamma}$ on adipogenesis through E activated ER, providing evidence that lack of estrogen may potentiate $PPAR{\gamma}$ action on adipogenesis.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2005년도 제5회 발생공학 국제심포지엄 및 학술대회
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• pp.118-118
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• 2005