scholarly journals Cell-Based High-Throughput Luciferase Reporter Gene Assays for Identifying and Profiling Chemical Modulators of Endoplasmic Reticulum Signaling Protein, IRE1

2015 ◽  
Vol 20 (10) ◽  
pp. 1232-1245 ◽  
Author(s):  
Juan Rong ◽  
Ian Pass ◽  
Paul W. Diaz ◽  
Tram A. Ngo ◽  
Michelle Sauer ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Reporter Gene ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fine Tuning ◽  
Luciferase Reporter ◽  
Signaling Cascade ◽  
Specific Chemical ◽  
Reporter Gene Assays

Endoplasmic reticulum (ER) stress activates three distinct signal transducers on the ER membrane. Inositol-requiring protein 1 (IRE1), the most conserved signal transducer, plays a key role in ER stress-mediated signaling. During ER stress, IRE1 initiates two discrete signaling cascades: the “adaptive” signaling cascade mediated by the XBP1 pathway and the “alarm” signaling cascade mediated by stress-activated protein kinase pathways. Fine-tuning of the balance between the adaptive and alarm signals contributes significantly to cellular fate under ER stress. Thus, we propose that the design of high-throughput screening (HTS) assays to selectively monitor IRE1 mediated-signaling would be desirable for drug discovery. To this end, we report the generation of stable human neural cell lines and development of cell-based HTS luciferase (Luc) reporter gene assays for the identification of pathway-specific chemical modulators of IRE1. We implemented a cell-based Luc assay using a chimeric CHOP-Gal4 transcription factor in 384-well format for monitoring IRE1 kinase-mediated p38MAPK activation and an unfolded response pathway element (URPE)–Luc cell-based assay in 1536-well format for monitoring IRE1’s RNase-mediated activation of XBP1. Chemical library screening was successfully conducted with both the CHOP/Gal4-Luc cells and UPRE-Luc engineered cells. The studies demonstrate the feasibility of using these HTS assays for discovery of pathway-selective modulators of IRE1.

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.

Baseline Toxicity and Volatility Cutoff in Reporter Gene Assays Used for High-Throughput Screening

2019 ◽  
Vol 32 (8) ◽  
pp. 1646-1655 ◽  
Author(s):  
Beate I. Escher ◽  
Lisa Glauch ◽  
Maria König ◽  
Philipp Mayer ◽  
Rita Schlichting
Keyword(s):  
Reporter Gene ◽  
High Throughput ◽  
High Throughput Screening ◽  
Reporter Gene Assays

scholarly journals Multiplexed Reporter Gene Assays: Monitoring the Cell Viability and the Compound Kinetics on Luciferase Activity

2011 ◽  
Vol 16 (7) ◽  
pp. 786-793 ◽  
Author(s):  
Marie-Cecile Didiot ◽  
Sergio Serafini ◽  
Martin J. Pfeifer ◽  
Frederick J. King ◽  
Christian N. Parker
Keyword(s):  
Cell Viability ◽  
Reporter Gene ◽  
High Throughput Screening ◽  
Time Course ◽  
Technical Note ◽  
Live Cells ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Multiple Parameters ◽  
Reporter Gene Assays

High-throughput screening assays with multiple readouts enable one to monitor multiple assay parameters. By capturing as much information about the underlying biology as possible, the detection of true actives can be improved. This report describes an extension to standard luciferase reporter gene assays that enables multiple parameters to be monitored from each sample. The report describes multiplexing luciferase assays with an orthogonal readout monitoring cell viability using reduction of resazurin. In addition, this technical note shows that by using the luciferin substrate in live cells, an assay time course can be recorded. This enables the identification of nonactive or unspecific compounds that act by inhibiting luciferase, as well as compounds altering gene expression or cell growth.

scholarly journals Luciferase Measurements in High Throughput Screening

1996 ◽  
Vol 1 (2) ◽  
pp. 85-88 ◽  
Author(s):  
Alfred J. Kolb ◽  
Kenneth Neumann
Keyword(s):  
Reporter Gene ◽  
High Throughput ◽  
High Throughput Screening ◽  
Culture Media ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Cell Culture Media ◽  
Luminescence Signal ◽  
Temperature Controlled

Luminescence assays are becoming more popular in high throughput screening (HTS) laboratories with the luciferase reporter gene being the most common. As with other assays that are adapted to HTS, improvements have been made to the luciferase assay to make it better suited to the requirements of HTS. For the luciferase reporter gene, these improvements included stabilization of the enzyme, increasing the half-life of the luminescence signal to 5 h, and eliminating separation steps (centrifugation and aliquot transfer) after cell lysis. The improved assay, LucLite, is homogeneous and is measured directly in the cell culture media. In addition to reagent improvements, a temperature-controlled, multidetector microplate counter, TopCount, can quickly and accurately measure luminescence signals.

scholarly journals Development of a Facile Method for High Throughput Screening with Reporter Gene Assays

2000 ◽  
Vol 5 (5) ◽  
pp. 377-384 ◽  
Author(s):  
Aaron S. Goetz ◽  
John L. Andrews ◽  
Thomas R. Littleton ◽  
Diane M. Ignar
Keyword(s):  
Cell Line ◽  
Reporter Gene ◽  
High Throughput ◽  
High Throughput Screening ◽  
Adherent Cell ◽  
Cho Cell ◽  
Cho Cell Line ◽  
Automation Systems ◽  
Hands On ◽  
Reporter Gene Assays

This report describes a facile methodology for high throughput screening with stable mammalian cell reporter gene assays. We have adapted a 96-well adherent cell method to an assay in which cells propagated in suspension are dispensed into 96- or 384-well plates containing test compounds in 100% DMSO. The validation of a stable CHO cell line that expresses 6xCRE-luciferase for use as a reporter gene host cell line is described. The reporter gene, when expressed in this particular CHO cell line, appears to respond specifically to modulation of cAMP levels, thus the cell line is appropriate for screening and pharmacological analysis of Gas- and Gas-coupled seven-transmem-brane receptors. The development of the new suspension cell assay in both 96- and 384-well formats was performed using a derivative of the CHO host reporter cell line that was stably transfected with human melanocortin-1 receptor. The response of this cell line to NDP-α-melanocyte-stimulating hormone and forskolin was nearly identical between the adherent and suspension methods. The new method offers improvements in cost, throughput, cell culture effort, compound stability, accuracy of compound delivery, and hands-on time. The 384-well assay can be performed at high capacity in any laboratory without the use of expensive automation systems such that a single person can screen 100 plates per day with 3.54 h hands-on time. Although the system has been validated using Gas-coupled receptor-mediated activation of a cAMP response element, the method can be applied to other types of targets and/or transcriptional response elements.

Influence of chlorophyll and tannins in plant extracts on cell-based luciferase reporter gene assays

Planta Medica ◽  
2009 ◽  
Vol 75 (09) ◽  
Author(s):  
S Vogl ◽  
P Picker ◽  
N Fakhrudin ◽  
A Atanasov ◽  
E Heiß ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Plant Extracts ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Reporter Gene Assays

scholarly journals Affordable Luciferase Reporter Assay for Cell-Based High-Throughput Screening

2012 ◽  
Vol 18 (4) ◽  
pp. 453-461 ◽  
Author(s):  
Ellen Siebring-van Olst ◽  
Christie Vermeulen ◽  
Renee X. de Menezes ◽  
Michael Howell ◽  
Egbert F. Smit ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Firefly Luciferase ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Chemical Components ◽  
Simple Liquid ◽  
Luciferase Reporter Construct ◽  
Reagent Cost ◽  
Firefly Luciferase Gene

The firefly luciferase gene is commonly used in cell-based reporter assays. Convenient luciferase assay reagents for use in high-throughput screening (HTS) are commercially available. However, the high cost of these reagents is not within the means of some academic laboratories. Therefore, we set out to develop an affordable luciferase assay reagent applicable in an HTS format using simple liquid-handling steps. The reagent was homemade from individual chemical components and optimized for luminescence intensity and stability. We determined the minimal concentrations of the most expensive components, dithiothreitol (DTT) and D-luciferin, resulting in a total assay reagent cost of less than 1 cent per sample. Signal stability was maximized by omission of coenzyme A and reduction of DTT concentration. The assay was validated in a high-throughput setting using two cancer cell lines carrying a p53-dependent luciferase reporter construct and siRNAs modulating p53 transcriptional activity. Induction of p53 activity by silencing PPM1D or SYVN1 and reduction of p53 activity by silencing p53 remained constant over a 2-h measurement period, with good assay quality (Z′ factors mostly above 0.5). Hence, the luciferase assay described herein can be used for affordable reporter readout in cell-based HTS.

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