Dataset Information

Unique reporter-based sensor platforms to monitor signalling in cells.

ABSTRACT:

Introduction

In recent years much progress has been made in the development of tools for systems biology to study the levels of mRNA and protein, and their interactions within cells. However, few multiplexed methodologies are available to study cell signalling directly at the transcription factor level.

Methods

Here we describe a sensitive, plasmid-based RNA reporter methodology to study transcription factor activation in mammalian cells, and apply this technology to profiling 60 transcription factors in parallel. The methodology uses two robust and easily accessible detection platforms; quantitative real-time PCR for quantitative analysis and DNA microarrays for parallel, higher throughput analysis.

Findings

We test the specificity of the detection platforms with ten inducers and independently validate the transcription factor activation.

Conclusions

We report a methodology for the multiplexed study of transcription factor activation in mammalian cells that is direct and not theoretically limited by the number of available reporters.

SUBMITTER: Jiwaji M

PROVIDER: S-EPMC3510088 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Publications

Unique reporter-based sensor platforms to monitor signalling in cells.

Jiwaji Meesbah M Daly Rónán R Gibriel Abdullah A Barkess Gráinne G McLean Pauline P Yang Jingli J Pansare Kshama K Cumming Sarah S McLauchlan Alisha A Kamola Piotr J PJ Bhutta Musab S MS West Adam G AG West Katherine L KL Kolch Walter W Girolami Mark A MA Pitt Andrew R AR

PloS one 20121129 11

<h4>Introduction</h4>In recent years much progress has been made in the development of tools for systems biology to study the levels of mRNA and protein, and their interactions within cells. However, few multiplexed methodologies are available to study cell signalling directly at the transcription factor level.<h4>Methods</h4>Here we describe a sensitive, plasmid-based RNA reporter methodology to study transcription factor activation in mammalian cells, and apply this technology to profiling 60 ...[more]

PMID: 23209767

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Project description:Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of this include flaviviruses, such as dengue virus and Zika virus, which cause millions of yearly infections around the globe, and coronaviruses, such as SARS-CoV-2, the source of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of research aimed at determining methods to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective treatments. Here, we describe the generation and characterization of a reporter system that can be used to visualize and identify cells infected with dengue virus or SARS-CoV-2. This system is based on viral protease activity that mediates cleavage and nuclear translocation of an engineered fluorescent protein stably expressed in cells. We show the suitability of this system for live cell imaging, for visualization of single infected cells, and for screening and testing of antiviral compounds. With the integrated modular building blocks, this system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility.IMPORTANCE Reporter systems are useful tools for fast and quantitative visualization of virus-infected cells within a host cell population. Here, we describe a reporter system that takes advantage of virus-encoded proteases expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the GFP moiety translocates to the nucleus, allowing for rapid detection of the infected cells. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the Flaviviridae and the Coronaviridae families: dengue virus and SARS-CoV-2. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. This reporter system is not only an invaluable tool for the characterization of viral replication, but also for the discovery and development of antivirals that are urgently needed to halt the spread of these viruses.

Dataset Information

Unique reporter-based sensor platforms to monitor signalling in cells.

Introduction

Methods

Findings

Conclusions

Publications

Unique reporter-based sensor platforms to monitor signalling in cells.

Similar Datasets

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