scholarly journals Development of a Functional Reporter Gene HTS Assay for the Identification of mGluR7 Modulators

2000 ◽  
Vol 5 (4) ◽  
pp. 255-261 ◽  
Author(s):  
G. C. Terstappen ◽  
A. Giacometti ◽  
E. Ballini ◽  
L. Aldegheri
Keyword(s):  
Reporter Gene ◽  
High Throughput Screening ◽  
Metabotropic Glutamate Receptor ◽  
Signal Transduction Pathway ◽  
Resistant Cell ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
Camp Levels

For the identification of modulators of the metabotropic glutamate receptor mGluR7, a functional cell-based high throughput screening (HTS) assay was developed. This assay utilizes the signal transduction pathway of mGluR7, which is negatively coupled to adenylyl cyclase. A cAMP-responsive luciferase reporter gene and rat mGluR7 cDNA were cotransfected into CHO-K1 cells by electroporation. Stable recombinant cells were selected by resistance to the antibiotic G418. Functional selection was carried out by analyzing the effect of the agonist glutamate to reduce elevated cAMP levels after forskolin stimulation. Out of 83 G418-resistant cell clones, the clone with the best functional characteristics was selected. This clone displayed the strongest reduction of forskolin-stimulated cAMP levels. Glutamate (10 mM) decreased cAMP levels, as monitored by luciferase expression, by about 50%, and the more potent agonist L-2-amino-4-phosphonobutyrate resulted in nearly complete reduction, exhibiting an EC50 of 0-9 mM. The functional response of the clone did not change during cell passages, indicating the stability of this novel recombinant cell line. The luciferase reporter gene assay, which allows easy nonradioactive luminescence detection of mGluR7 activity, was optimized for its application in automated HTS.

Evaluation of a CRE-Directed Luciferase Reporter Gene Assay as an Alternative to Measuring cAMP Accumulation

1997 ◽  
Vol 2 (4) ◽  
pp. 235-240 ◽  
Author(s):  
Samantha E. George ◽  
Peter J. Bungay ◽  
Louise H. Naylor
Keyword(s):  
Reporter Gene ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Functional Assay ◽  
Ionophore A23187 ◽  
Camp Accumulation ◽  
Luciferase Reporter Gene ◽  
Gene System ◽  
Gene Assay ◽  
Reporter Gene System

A CHO reporter cell line expressing the firefly luciferase gene under the control of six cAMP response elements (CREs) was used to investigate the relationship between cAMP accumulation and cAMP dependent reporter gene expression and therefore, to assess the reporter gene system as an alternative functional assay. Timecourse experiments showed that cAMP accumulation preceded luciferase expression and that longer incubations (>2 h) were required to gain results with the reporter gene system. However, forskolin concentration dose-response studies revealed a 100-fold amplification of the signal measured by luciferase expression compared with direct cAMP accumulation, indicating that the reporter gene system afforded greater sensitivity. EC50 values determined for agonist activation of an inhibitory (5-HTlB-like) G-protein-coupled receptor (GPCR) were the same, and for a stimulatory GPCR (calcitonin Cla-like) were 10-fold lower, using the reporter gene system compared to cAMP accumulation assays, indicating the suitability of the reporter system for measuring the activity of receptors differentially coupled to the cAMP pathway. The phorbol ester, PMA, and the Ca2+ ionophore, A23187, were able to potentiate forskolin-stimulated luciferase expression but not cAMP accumulation, suggesting that the former could also be used to monitor cross-talk between different signal transduction pathways at the level of gene transcription.

scholarly journals Measurement of Effects of Antibiotics in Bioluminescent Staphylococcus aureus RN4220

2001 ◽  
Vol 45 (12) ◽  
pp. 3456-3461 ◽  
Author(s):  
Mervi Tenhami ◽  
Kaisa Hakkila ◽  
Matti Karp
Keyword(s):  
Staphylococcus Aureus ◽  
High Throughput Screening ◽  
Pathogenic Bacteria ◽  
Antimicrobial Agents ◽  
Light Emission ◽  
Detection System ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Luciferase Reporter Gene

ABSTRACT The spread of antibiotic resistance among pathogenic bacteria is a serious threat to humans and animals. Therefore, unnecessary use should be minimized, and new antimicrobial agents with novel mechanisms of action are needed. We have developed an efficient method for measuring the action of antibiotics which is applied to a gram-positive strain,Staphylococcus aureus RN4220. The method utilizes the firefly luciferase reporter gene coupled to the metal-induciblecadA promoter in a plasmid, pTOO24. Correctly timed induction by micromolar concentrations of antimonite rapidly triggers the luciferase gene transcription and translation. This sensitizes the detection system to the action of antibiotics, and especially for transcriptional and translational inhibitors. We show the results for 11 model antibiotics with the present approach and compare them to an analytical setup with a strain where luciferase expression is under the regulation of a constitutive promoter giving only a report of metabolic inhibition. The measurement of light emission from intact living cells is shown to correlate extremely well (r = 0.99) with the conventional overnight growth inhibition measurement. Four of the antibiotics were within a 20% concentration range and four were within a 60% concentration range of the drugs tested. This approach shortens the assay time needed, and it can be performed in 1 to 4 h, depending on the sensitivity needed. Furthermore, the assay can be automatized for high-throughput screening by the pharmaceutical industry.

scholarly journals Identification of Alkaloids from Corydalis yanhusuo W. T. Wang as Dopamine D1 Receptor Antagonists by Using CRE-Luciferase Reporter Gene Assay

Molecules ◽  
2018 ◽  
Vol 23 (10) ◽  
pp. 2585 ◽  
Author(s):  
Lehao Wu ◽  
Weiyue Zhang ◽  
Xin Qiu ◽  
Chaoran Wang ◽  
Yanfang Liu ◽  
...  
Keyword(s):  
Dopamine Receptors ◽  
Reporter Gene ◽  
Reporter Gene Assay ◽  
D1 Receptor ◽  
Assay System ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Antagonistic Effects ◽  
Gene Assay

Corydalis yanhusuo W. T. Wang (C. yanhusuo) has been traditionally used for drug addiction and pain relief in China. In our previous study, we showed that the extract of C. yanhusuo blocks dopamine receptors, demonstrating that its pharmacological activities are mostly due to the antagonistic effects of some of its components at dopamine receptors. As part of our ongoing project on C. yanhusuo, the aim of the present study is to establish a high-throughput and low-cost screening assay system and test the abilities of the isolated alkaloids from C. yanhusuo to inhibit dopamine-induced dopamine D1 receptor activity. By using our established cyclic adenosine monophosphate (cAMP)-response element (CRE)-luciferase reporter gene assay system, we identified eight alkaloids from C. yanhusuo with D1 receptor antagonistic activities. We next validated the activities of these compounds using fluorometric imaging plate reader (FLIPR) assay by measuring the intracellular Ca2+ change. Six out of eight compounds, including tetrahydropalmatine, corydaline, 13-methyldehydrocorydalmine, dehydrocorybubine, dehydrocorydaline, and columbamine, can be confirmed for their inhibitory activities. The dopamine-receptor-antagonistic effects of four compounds, including 13-methyldehydrocorydalmine, dehydrocorydaline, columbamine, and corydaline, are reported for the first time. The present study provides an important pharmacological basis to support the traditional use of C. yanhusuo in China.

scholarly journals eNOS Promoter Activation by Red Wine Polyphenols: an Interaction Study

2013 ◽  
Vol 60 (1) ◽  
pp. 27-33 ◽  
Author(s):  
E. Kurin ◽  
N. Fakhrudin ◽  
M. Nagy
Keyword(s):  
Reporter Gene ◽  
Red Wine ◽  
Interaction Analysis ◽  
Mass Action ◽  
Luciferase Reporter ◽  
Interaction Study ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
Wine Polyphenols ◽  
Red Wine Polyphenols

Beneficial effects of red wine polyphenols on cardiovascular health are well known. The aim of our research was an interaction study of four red wine polyphenols – resveratrol (R), quercetin (Q), kaempferol (KF) and isorhamnetin (IR) of their ability to activate endothelial NO synthase (eNOS) promoter when used alone and in equimolar mixtures. To determine their activity, we performed a luciferase reporter gene assay on EA.hy926 cells stably transfected with a luciferase reporter gene construct containing eNOS promoter. The Bradford assay was also performed to account the cytotoxicity and/or the cell number differences. The median effect equation, as an interaction analysis evaluating synergy or antagonism of the combinations was done according to mass-action law principle. Isobolographic method was performed on selected double mixtures and dose reduction index was calculated for all mixtures. All single polyphenols activated eNOS promoter. The EC50 values were in micromolar concentrations ranging from 3.44 μM (R2 = 0.96) for kaempferol to 9.89 μM for isorhamnetin (R2 = 0.94). All mixtures activated eNOS promoter, but their interactions varied from synergy (Q+R, Q+IR+KF, Q+R+KF and Q+R+IR+KF), through additive (R+IR+KF) to antagonistic interaction (R+IR, R+KF, Q+IR, Q+KF, IR+KF and R+Q+IR). In this study, we show for the first time that red wine polyphenols activated eNOS promoter when used alone and in mixtures with different type of interactions.

scholarly journals Use of Leishmania donovani Field Isolates Expressing the Luciferase Reporter Gene in In Vitro Drug Screening

2005 ◽  
Vol 49 (9) ◽  
pp. 3776-3783 ◽  
Author(s):  
Ashutosh ◽  
Suman Gupta ◽  
Ramesh ◽  
Shyam Sundar ◽  
Neena Goyal
Keyword(s):  
Reporter Gene ◽  
High Throughput ◽  
High Throughput Screening ◽  
Leishmania Donovani ◽  
Luciferase Activity ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Field Isolates

ABSTRACT Currently available primary screens for the selection of candidate antileishmanial compounds are not ideal. These techniques are time-consuming, laborious, and difficult to scale and require macrophages, which limit their use for high-throughput screening. We have developed Leishmania donovani field isolates that constitutively express the firefly luciferase reporter gene (luc) as a part of an episomal vector. An excellent correlation between parasite number and luciferase activity was observed. luc expression was stable, even in the absence of drug selection, for 4 weeks. The transfectants were infective to macrophages, and intracellular amastigotes exhibited luciferase activity. The suitability of these recombinant field isolates for in vitro screening of antileishmanial drugs was established. The luciferase-expressing sodium stibogluconate-resistant cell lines offer a model for the screening of compounds for resistance. The system is in routine use at the Central Drug Research Institute, Lucknow, India, for high-throughput screening of newly synthesized compounds.

scholarly journals Oxymatrine inhibits proliferation and migration of breast cancer cells by inhibiting miRNA-188 and upregulating its target gene, PTEN

2021 ◽  
Vol 20 (11) ◽  
pp. 2267-2272
Author(s):  
Xiaoying Ma ◽  
Zijiang Sang ◽  
Qinghua Zhang ◽  
Wenbiao Ma
Keyword(s):  
Breast Cancer ◽  
Reporter Gene ◽  
Target Gene ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Pten Gene ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
And Migration ◽  
Regulatory Loop

Purpose: To explore the potential biological functions of oxymatrine on breast cancer (BCa) cells and the underlying molecular mechanism.Methods: Relative levels of microRNA-188 (miRNA-188) and PTEN (gene of phosphate and tension homology deleted on chromosome ten) in BCa cells, MDA-MB-231 and TB549, were determined. The influence of oxymatrine treatment, miRNA-188 and PTEN on proliferative and migratory abilities in BCa cells were assessed by 3-(4,5-imethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), cell counting kit-8 (CCK-8) and Transwell assay, respectively. The binding relationship between miRNA-188 and PTEN was evaluated by dual-luciferase reporter gene assay.Results: Oxymatrine downregulated miRNA-188 and upregulated PTEN in BCa cells. Proliferative and migratory activities in BCa were inhibited by treatment of oxymatrine (p < 0.05). Dual-luciferase reporter gene assay results indicated that PTEN was the target gene of miRNA-188. Furthermore, rescue experiments demonstrated that the regulatory loop, oxymatrine/miRNA-188/PTEN, was involved in the regulation of the migration and proliferation of BCa.Conclusion: Oxymatrine treatment inhibits BCa progression by downregulating miRNA-188, leading to the upregulation of PTEN. The results of the current study may provide new insight into the diagnosis and treatment of BCa.

scholarly journals Circ_0000005 Facilitates Proliferation, Apoptosis, Migration and Invasion of Acute Myeloid Leukemia Cells via Modulating miR-139-5p/Tspan3 Axis

2020 ◽  
Author(s):  
Juan Tong ◽  
Huilan Liu ◽  
Changcheng Zheng ◽  
Xiaoyu Zhu ◽  
Xiang Wan ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Reporter Gene ◽  
Reporter Gene Assay ◽  
Luciferase Reporter ◽  
Regulatory Function ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Qrt Pcr ◽  
Gene Assay

Abstract Background: Accumulating circular RNAs (circRNAs) are reported to be abnormally expressed in diverse cancers, hematologic malignancies included. This study aimed to investigate the biological function and underlying mechanisms of circ_0000005 in acute myeloid leukemia (AML).Materials and methods: Bone marrow samples were enrolled from AML patients with normal samples as controls. Circ_0000005, miR-139-5p and tetraspanin 3 (Tspan3) were detected by qRT-PCR and Western blot, respectively. AML cell lines (KG1 and HL60) were used as cell models. CCK-8, Transwell and flow cytometry assays were adopted to study the biological functions of circ_0000005 on AML cells in vitro. The interrelation between circ_0000005 and miR-139-5p was detected by bioinformatics, qRT-PCR, luciferase reporter gene assay, RNA pull-down assay, and RNA-binding protein immunoprecipitation (RIP) assays. Ultimately, Western blot, qRT-PCR, luciferase reporter gene assay were adopted to corroborate the interrelation between miR-139-5p and its target Tspan3. Results: Circ_0000005 was demonstrably elevated in both AML clinical samples and cell lines. Circ_0000005 overexpression promoted the viability, migration and invasion of AML cells, and repressed the apoptosis of AML cells, while silencing circ_0000005 showed opposite biological effects. Circ_0000005 interacted with miR-139-5p and repressed its expression, and Tspan3 was proved to be negatively regulated by miR-139-5p. Circ_0000005 could promote the expression of Tspan3 via repressing miR-139-5p, and the oncogenic functions of circ_0000005 were dependent on its regulatory function on miR-139-5p/Tspan3 axis.Conclusion: Circ_0000005 facilitates the malignant phenotypes of AML cells via miR-139-5p/Tspan3 axis. Circ_0000005 may serve as a potential therapeutic target in AML.

scholarly journals A Potential miRNA-mRNA Network for Dementia and Hernia Crosstalk

2021 ◽  
Vol 2021 ◽  
pp. 1-14
Author(s):  
De-jian Chen ◽  
Da-peng Li
Keyword(s):  
Reporter Gene ◽  
Expression Profiles ◽  
Differentially Expressed ◽  
Luciferase Reporter ◽  
Clinical Samples ◽  
Luciferase Reporter Gene ◽  
Tissue Samples ◽  
Regulatory Pathways ◽  
Gene Assay ◽  
Potential Link

Background. It has been reported that there may be a potential link between hernia and dementia. However, the exact mechanisms of their association have not been established. This study is aimed at constructing miRNA-mRNA networks to elucidate on the potential link between dementia and hernia. Methods. Gene expression profiles for dementia, herniation, and skeletal muscle were downloaded from the GEO database after which differentially expressed mRNAs and miRNAs were obtained. In addition, fascia tissue samples were obtained during surgery. A total of 41 patients were recruited in this study, and expression levels of candidate genes were examined using quantitative RT-PCR. Luciferase reporter gene assays were used to identify potential miRNA-mRNA regulatory pathways. Results. Differentially expressed mRNAs and miRNAs were screened. A potential miRNA-mRNA network revealing the crosstalk mechanism between herniation and dementia was identified. Single cell analysis revealed that PI16 was highly enriched in adipose tissues, skeletal muscles, and in the skin. GSEA enrichment analysis showed that PI16 is involved in adipose metabolism, muscle functions, and energy metabolism. In clinical samples, PI16 was found to be upregulated in hernia, while miR-4451 was found to be downregulated. The luciferase reporter gene assay revealed that downregulation of circulating miR-4451 may be responsible for the upregulated PI16 expression in hernia sacs. Conclusions. We constructed an miRNA-mRNA network that shows the potential association between dementia and hernia. We also found that miR-4451 regulates the PI16 expression, which may be a key target and biomarker for hernia pathogenesis and dementia crosstalk.

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