A Versatile ΦC31 Based Reporter System for Measuring AP-1 and Nrf2 Signaling in Drosophila and in Tissue Culture

Nirmalya Chatterjee,Dirk Bohmann,Barbara Jennings

doi:10.1371/journal.pone.0034063

Nirmalya Chatterjee, Dirk Bohmann + Show 1 more

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https://doi.org/10.1371/journal.pone.0034063

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Journal: PloS one	Publication Date: Apr 11, 2012
Citations: 220	License type: CC BY 4.0

Affiliation: University of Rochester Medical Center

Abstract

This paper describes the construction and characterization of a system of transcriptional reporter genes for monitoring the activity of signaling pathways and gene regulation mechanisms in intact Drosophila, dissected tissues or cultured cells. Transgenic integration of the reporters into the Drosophila germline was performed in a site-directed manner, using ΦC31 integrase. This strategy avoids variable position effects and assures low base level activity and high signal responsiveness. Defined integration sites furthermore enable the experimenter to compare the activity of different reporters in one organism. The reporter constructs have a modular design to facilitate the combination of promoter elements (synthetic transcription factor binding sites or natural regulatory sequences), reporter genes (eGFP, or DsRed.T4), and genomic integration sites. The system was used to analyze and compare the activity and signal response profiles of two stress inducible transcription factors, AP-1 and Nrf2. To complement the transgenic reporter fly lines, tissue culture assays were developed in which the same synthetic ARE and TRE elements control the expression of firefly luciferase.

Highlights

Signal transduction mechanisms that modulate transcription profiles in response to extracellular inputs control all major biological processes, including development, growth and homeostasis
Reporter genes that can register the activity of transcription factors or other signaling proteins have been designed
Such reporter genes have been used in tissue culture or in transgenic animals [1]

Summary

Introduction

Signal transduction mechanisms that modulate transcription profiles in response to extracellular inputs control all major biological processes, including development, growth and homeostasis. A widespread type of reporter relies on fusion genes in which synthetic DNA elements, that represent defined binding sites for a transcription factor of interest, are inserted upstream of sequences encoding protein products such as fluorescent proteins and enzymes, which are easy to visualize or quantify, are not normally present in the test organism, and for which sensitive assays are available such as chloramphenicol acetyl transferase or luciferase. Such reporter genes have been used in tissue culture or in transgenic animals [1].

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