Abstract
BackgroundHigh-throughput methods based on molecular reporters have greatly advanced our knowledge of cell signaling in mammalian cells. However, their ability to monitor various types of cells is markedly limited by the inefficiency of reporter gene delivery. Recombinant adeno-associated virus (AAV) vectors are efficient tools widely used for delivering and expressing transgenes in diverse animal cells in vitro and in vivo. Here we present the design, construction and validation of a novel AAV-based dual-reporter circuit that can be used to monitor and quantify cell signaling in living human cells.ResultsWe first design and construct the AAV-based reporter system. We then validate the versatility and specificity of this system in monitoring and quantifying two important cell signaling pathways, inflammation (NFκB) and cell growth and differentiation (AP-1), in cultured HEK293 and MCF-7 cells. Our results demonstrate that the AAV reporter system is both specific and versatile, and it can be used in two common experimental protocols including transfection with plasmid DNA and transduction with packaged viruses. Importantly, this system is efficient, with a high signal-to-background noise ratio, and can be easily adapted to monitor other common signaling pathways.ConclusionsThe AAV-based system extends the dual-reporter technology to more cell types, allowing for cost-effective and high throughput applications.
Highlights
High-throughput methods based on molecular reporters have greatly advanced our knowledge of cell signaling in mammalian cells
We generated a genetic circuit that consists of a transcription control unit (TRE and minimal CMV promoter (mCMV)) and a dual-reporter fusion (GFP and Luc) (Fig. 1a, upper panel)
The transcription control unit of Transcription factor response elements (TRE) will respond to the binding of their corresponding activated transcription factors (TFs), resulting in reporter gene expression (Fig. 1a, lower panel)
Summary
We first design and construct the AAV-based reporter system. We validate the versatility and specificity of this system in monitoring and quantifying two important cell signaling pathways, inflammation (NFκB) and cell growth and differentiation (AP-1), in cultured HEK293 and MCF-7 cells. Our results demonstrate that the AAV reporter system is both specific and versatile, and it can be used in two common experimental protocols including transfection with plasmid DNA and transduction with packaged viruses. This system is efficient, with a high signal-to-background noise ratio, and can be adapted to monitor other common signaling pathways
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