• 한국독성학회:학술대회논문집
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• 한국독성학회 2002년도 Molecular and Cellular Response to Toxic Substances
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• pp.166-166
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• 2002

In the present study, estrogenic or antiestrogenic activity of cypermethrin, a pyrethroid insecticide was investigated. We used immature rat uterotrophic assay, estrogen-responsive calbindin-D9k (CaBP-9k) gene expression assay and luciferase reporter gene assay for measure of estrogenic potential of cypermethrin.(omitted)

• 대한한의학회지
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• 제29권3호
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• pp.88-99
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• 2008

Objectives: In order to identify the anti-inflammatory and analgesic properties of Salvia miltiorrhiza (Dan-Sam), widely used in Korean traditional medicine, an in vitro screening system was designed using pGL3, a luciferase reporter vector, and the tumor necrosis factor (TNF)-${\alpha}$ and cyclooxygenase (COX)-2 as target genes. Methods: The promoter regions of each gene were generated by PCR using the human chromosome as template DNA, and inserted into pGL3 vector with Kpn I and Hind III. The final construct was transfected into human myelomonocytic leukemia cells (U-937) that could be differentiated and activated by phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS). Using this system, the anti-inflammatory and analgesic effects of several herbal extracts regarded to have the medicinal effects of diminishing body heat and complementing Qi were tested. The chemicals PD98059 and berberine chloride were used as controls of the transcriptional inhibitors of TNF-${\alpha}$ and COX-2, respectively. Results: Salvia miltiorrhiza (Dan-Sam) demonstrated significant decrease of TNF-${\alpha}$ and COX-2 mRNA in the in vitro assay system. In MTT assay, Salvia miltiorrhiza (Dan-Sam) did not significantly inhibit the survival and proliferation of human myelomonocytic leukemia cells (U-937). Conclusions: Salvia miltiorrhiza (Dan-Sam) was found to exhibit the significant medicinal properties of anti-inflammatory and analgesic effects.

• 한국동물학회지
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• 제39권4호
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• pp.378-386
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• 1996

우리는 두 가지의 새로운 luciferase reporter plasmid를 개발하였는데 그 하나는 이 plasmid에 promoter조각을 삽입한 다음에 그 promoter의 활성을 측정하는 용도로 사용 될수 있고, 다른 하나는 매우 낮은 basal promoter 활성을 갖고 있기 때문에 진핵생물의 전사 조절인자의 연구에 도움이 될 수 있다. 후자의 reporter plasmid에는 17염기쌍의 initator 와 Spl,GAL4그리고 일부 Drosophila homeodomain protein의 결합우뷔에 해당하는 cis elements등을 들어 있다.그리고 tranciption termination을 촉진할 수 있는 signal을 initiator앞서 삽입하여 이런 reporter plasmid에 존재할 수 있는 cryptic promoter에서 시작된 transcript가 luciferase reporter cDNA로 진행되는 것을 방지하는 개선된 reporter plasmid를 제조하여 promoter활성을 Drosophila Schneider line 2 cells을 이용한 transient transfection assay방법으로 측정하였다. 여기에 사용한 termination 촉진 signal은 SV40 polyadenylation signal의 3연속 조각(AAA)과 tenscription termination signal이 포함된 것으로 믿어지는 mouse c-mos 유전자의 일부조각 (UMS)이다. 기대한으로 이 두가지의 signal을 삽입하였을 때 basal promoter활성이 최대한으로 감소하였으며 이 두 가지 signal을 삽입된 reporter plasmid를 사용하여 promoter의 활성을 보다 sensitive하게 측정하였다. 이 reporter plasmid는 Droiophlla melanogaiter뿐만 아니라 포유동물을 포함한 고등생물의 전사 조절인자 연구의 한 도구로 사용될 수 있을 것이다.

• 한국독성학회:학술대회논문집
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• 한국독성학회 2002년도 Molecular and Cellular Response to Toxic Substances
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• pp.164-164
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• 2002

The estrogenic activity of 47 plant extracts was assessed by reporter gene assay using MCF-7 breast cancer cell lines stably transfected with luciferase reporter gene. The estrogenic activity of food extracts was expressed as 17${\beta}$-estradiol(E2) equivalent concentration(EEQ), the concentration of E2 that resulted in the same relative luciferase unit(RLU) of the food extract of 0.2mg/$m\ell$.(omitted)

• Molecules and Cells
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• 제42권11호
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• pp.783-793
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• 2019

When endoplasmic reticulum (ER) functions are perturbed, the ER induces several signaling pathways called unfolded protein response to reestablish ER homeostasis through three ER transmembrane proteins: inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Although it is important to measure the activity of ATF6 that can indicate the status of the ER, no specific cell-based reporter assay is currently available. Here, we report a new cell-based method for monitoring ER stress based on the cleavage of $ATF6{\alpha}$ by sequential actions of proteases at the Golgi apparatus during ER stress. A new expressing vector was constructed by using fusion gene of GAL4 DNA binding domain (GAL4DBD) and activation domain derived from herpes simplex virus VP16 protein (VP16AD) followed by a human $ATF6{\alpha}$ N-terminal deletion variant. During ER stress, the GAL4DBD-VP16AD(GV)-$hATF6{\alpha}$ deletion variant was cleaved to liberate active transcription activator encompassing GV-$hATF6{\alpha}$ fragment which could translocate into the nucleus. The translocated GV-$hATF6{\alpha}$ fragment strongly induced the expression of firefly luciferase in HeLa Luciferase Reporter cell line containing a stably integrated 5X GAL4 site-luciferase gene. The established double stable reporter cell line HLR-GV-$hATF6{\alpha}$(333) represents an innovative tool to investigate regulated intramembrane proteolysis of $ATF6{\alpha}$. It can substitute active pATF6(N) binding motif-based reporter cell lines.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2003년도 Annual Meeting of KSAP : International Symposium on Pharmaceutical and Biomedical Sciences on Obesity
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• pp.89-89
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• 2003

Exposure of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a variety of biological and toxicology effects, most of which are mediated by aryl hydrocarbon receptor (AhR). The ligand-bound AhR as a heterodimer with AhR nuclear translocator (ARNT) binds to its specific DNA recognition site, the dioxin-responsive element (DRE), and it results in increased transcription of CYP1A1 gene. Retinoic acid (RA) regulates the transcription of various genes for several essential functions through binding to two classes of nuclear receptors, the retinoic acid receptor (RAR) and retinoid X receptor (RXR). Constitutive androstane receptor (CAR) also regulates the transcription of gene. In this study, we have examined how RAR, RXR and CAR regulated CYP1A1 in rainbow trout hepatoma cell (RTH 149) using luciferase reporter gene assay system. We did transient transfection with CYP1A1 luciferase reporter gene and treated with TCDD, all-trans RA, 9-cis RA and phenobarbital. Treatment of all-trans RA, 9-cis RA or phenobarbital decreased the TCDD induced transcription of CYP1Al. When we did transient cotransfection with CYP1A1 luciferase reporter gene and RXR, as increase of RXR concentration, the TCDD induced transcription of CYP1A1 was decreased. Transfection with CAR also decreased the TCDD induced transcription of CYP1A1 in RTH 149 cells.

• 한국환경독성학회:학술대회논문집
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• 한국환경독성학회 2003년도 추계국제학술대회
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• pp.179-179
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• 2003

Exposure of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a variety of biological and toxicology effects, most of which are mediated by aryl hydrocarbon receptor (AhR). The ligand-bound AhR as a heterodimer with AhR nuclear translocator (ARNT) binds to its specific DNA recognition site, the dioxin-responsive element (DRE), and it results in increased transcription of CYP1A1 gene. Retinoic acid (RA) regulates the transcription of various genes for several essential functions through binding to two classes of nuclear receptors, the retinoic acid receptor (RAR) and retinoid X receptor (RXR). Constitutive androstane receptor (CAR) also regulates the transcription of gene. In this study, we have examined how RAR, RXR and CAR regulated CYP1A1 in rainbow trout hepatoma cell (RTH 149) using luciferase reporter gene assay system. We did transient transfection with CYP1A1 luciferase reporter gene and treated with TCDD, all-trans RA, 9-cis RA and phenobarbital. Treatment of all-trans RA, 9-cis RA or phenobarbital decreased the TCDD induced transcription of CYP1A1. When we did transient cotransfection with CYP1A1 luciferase reporter gene and RXR, as increase of RXR concentration, the TCDD induced transcription of CYP1A1 was decreased. Transfection with CAR also decreased the TCDD induced transcription of CYP1A1 in RTH 149 cells.

• 농업과학연구
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• 제48권2호
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• pp.353-365
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• 2021

Previously, we reported that tissue factor (Tf) was included in the list of differentially expressed genes as an upregulated gene in a rejected porcine heart after xenotransplantation into monkey. In this study, we analyzed that expression of Tf in aortic endothelial cells (pAEC) isolated from alpha 1,3-galactosyltransferase knockout pig in response to allogeneic porcine serum and xenogeneic human serum. The consequence was significant upregulation of Tf expression by responding to human serum compared with porcine serum. To analyze the function of Tf gene as a promoter, we constructed reporter vectors for expression of luciferase linked to 1,246 and 787 base pairs of porcine Tf (pTF1246 and pTF787), and 535 base pairs of human TF (hTF535) sequences including putative promoter regions and AP-1 biding site at the 5' end. The reporter vectors were transfected into pAEC including cytomegalovirus enhancer/chicken β-actin (CAG)-luciferase vector as a control. Luciferase assay showed that all of the promoters were insufficient to express luciferase compared with CAG promoter in basic culture conditions. Notably, pTF1246, pTF787, and hTF535 led to a significant increase of luciferase expression in response to human serum compared with porcine serum while no change of CAG. pTF1246 and pTF787 showed higher expression than hTF535. Taken together, our findings suggest that pTF1246 and pTF787 promoters could mediate target gene expression specifically at xenogeneic stress condition.

• Parasites, Hosts and Diseases
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• 제53권4호
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• pp.385-394
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• 2015

Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain. Next, we evaluated the sensitivity of these parasites to amphotericin B (AmB) as a standard drug in 2 parasitic phases, promastigote and amastigote. This comparison was made by MTT and nitric oxide (NO) assay and by quantifying the specific signals derived from reporter genes like EGFP intensity and luciferase activity. To study the amastigote form, both B10R and THP-1 macrophage cell lines were infected in the stationary phase and were exposed to AmB at different time points. Our results clearly revealed that the 3 parasite lines had similar in vitro infectivity rates with comparable parasite-induced levels of NO following interferon-${\gamma}$/lipopolysaccharide induction. Based on our results we proposed the more reporter gene, the faster and more sensitive evaluation of the drug efficiency.

• Journal of Microbiology and Biotechnology
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• 제16권10호
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• pp.1646-1649
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• 2006

Protein synthesis is the ultimate outcome of gene expression which, in turn, is regulated by several translation factors. We attempted to identify substances that can inhibit the translation process in vitro when the outcome protein is luciferase. To this end, we developed a sensitive cell-free protein synthesis assay using luciferase as the reporter. The synthesis of luciferase increased proportionately as mRNA was added to a $15-{\mu}l$reaction medium in concentrations raging from 5 ng to 500 ng. The maximum amount of luciferase was synthesized when the media were incubated at $25^{\circ}C$ for 40 min. The concentration of each compound that inhibited luciferase production by 50% ($IC_{50}$) was calculated. Hygromycin, puromycin, and cycloheximide yielded an $IC_{50}$ of 0.008, 0.8, and $0.7{\mu}g/ml$, respectively. A filtrate of Streptomyces spp. isolates inhibited protein synthesis up to S-fold when added to the in vitro translation assay mixture.