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Performance of Ad-A-Gene Cyclic AMP Response Element-Nitroreductase (CRE-NTR): an adenoviral vector gene delivery system

Key words: adenoviral vector ● viral transduction ● NTR ● IN Cell Analyzer ● gene delivery


Biological assays developed in cultured and primary cells can greatly aid secondary screening as well as lead and target validation, but their development presents several challenges. Establishing stable cell systems to express target genes of interest at detectable levels takes time. In addition, cellular signaling is complex and some data can be difficult to interpret reproducibly.

Adenoviral vectors mitigate these challenges by providing a way to deliver genes and to generate transient expression of key gene targets in cells. The Ad-A-Gene adenoviral vector gene delivery system uses viral transduction to rapidly deliver a range of signal pathway ‘sensors’ into a variety of target cells (1, 2). Ad-A-Gene Vectors comprise a panel of key cellular genes, each of which is fused either to enhanced green fluorescent protein (EGFP) or emerald fluorescent protein (eFP), or a response element controlling the expression of the E. coli B nitroreductase (NTR) reporter gene (3–8). The genes become transiently expressed within transduced target cells, allowing for the development of cellular assays that exploit the signal readouts from the sensor gene markers.

This application note describes the development of an NTR reporter assay for the cyclic adenosine monophosphate response element (CRE). CRE acts as a molecular docking site for two endogeneous transcription factors (cAMP binding protein, or CREB, and cAMP-dependent transcription factor, or ATF) and is modulated by several key signal transduction pathways regulating cAMP. We have exploited the binding of CREB to CRE through an adenoviral construct containing four repeats of CRE upstream of a minimal promoter and NTR (CRE-NTR). Upon adenoviral vector transduction of target cells, the CRE-NTR gene becomes tran
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DeStreak Reagent from GE Healthcare, formerly Amersham Biosciences
Cytotoxicity Fluorometric (green / red) Assay Kit, LIVE/DEAD® from Molecular Probes (Invitrogen)
SlowFade Antifade Kit with DAPI from Molecular Probes (Invitrogen)
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