Featured Products Adenoviral vector gene delivery system Ad-A-Gene Vectors allow GFP-protein translocation and NTR gene reporter assays to be easily established in a wide range of cell types.

Nitroreductase Gene Reporter System

Reporter gene technology is widely used to monitor the cellular events associated with signal transduction and gene expression. The term reporter gene is used to define a gene with a readily measurable phenotype that can be distinguished easily over a background of endogenous proteins. A reporter gene construct is comprised of an inducible transcriptional control element driving the expression of a reporter gene. Generally, such reporters are selected on the basis of the sensitivity, dynamic range, convenience and reliability of the assay.

To enable flexibility in design of multiparameter single cell fluorescence assays we have developed a novel reporter system based on the use of a bacterial nitroreductase (NTR) and a cell permeant cyanine fluor, CytoCy5S, as its substrate. The assay uses ‘red-shifted’ excitation and emission wavelengths for the reporter enzyme substrate to enable use with other (e.g. EGFP) fluorescent reporters in the same cell.

Nitroreductase (NTR) is an FMN-dependent enzyme isolated from E.coli B. Expression of NTR has been demonstrated in a number of mammalian cells without any reported toxicity. The ability of NTR to reduce nitro groups, has been utilized in the development of this simple signal increase assay. CytoCy5S acts as a membrane permeant substrate and has been optimized such that the fluorescent end-product is retained within the cell

Typically the current reporter gene systems available are invasive and require destruction of the cell in order to measure gene reporter expression (firefly luciferase) or have limited sensitivity (GFP) due to the lack of enzymatic amplification.

To overcome these limitations the NTR gene reporter system has been developed as a non-invasive live cell reporter system using a cell permeable fluorogenic substrate.

Analysis of NTR gene reporter assays on the IN Cell Analyzers allows visualization of gene expression in single living cells.

Thus this live cell approach to gene reporter assays has many potential benefits. As well as being a non-destructive method, NTR offers the potential to screen out non-responding cells, analysis of individual cell responses and measurement of a second fluorescent event occurring within the same cell. Thus, the NTR gene reporter system offers a flexible and convenient format.

Application of NTR Gene Reporter assay:

• Primary screening /secondary screening – functional cell based assays
• Fluorescence activated cell sorting – for selection of biologically responsive clones
• High content screening – combining NTR expression with GFP fusion proteins or receptor internalization
• Investigation of putative regulatory sequences such as gene trap studies – identification of genes responsive to a specific stimulus
• Novel gene function – use of NTR expression in target and lead validation studies.
  

• Red shifted substrate, CytoCy5S, excitation 647nm emission 667nm
• Non-destructive assay protocol
• Compatible with GFP for multiplexing targets on sub-cellular imaging systems

• IN Cell Analyzers
• Fluorescence Activated Cell Sorting (FACS)
• Sub-cellular imaging Platforms
• LEADseeker Multimodality Imaging System
• Fluorimeters such as the Tecan Ultra

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