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

ulation was used to set a threshold for the maximum background signal from the CytoCy5S substrate, denoted by the dotted line. Cells from the two transduced populations that exceeded this baseline were identified as containing the CRE-NTR reporter molecule. No signal overlap was found in the non-transduced and transduced populations, suggesting that almost all of the cells contained at least one copy of the CRE-NTR reporter molecule. Transduced U2OS cells appear to have a high basal activity in the absence of forskolin. Addition of forskolin to the transduced population increases the average cellular fluorescent signal, shifting the population distribution.

CRE-NTR activity in transduced cells and drug dose-dependence
Figure 4 shows a dose-response curve for forskolin in U2OS cells transduced with the CRE-NTR adenoviral vector and treated with varying doses of forskolin for 5 h and 1-μM CytoCy5S (final concentration) for a further 3 h. Plates were read on a Tecan Ultra and then fixed with 5% formalin solution before being re-read on the Tecan Ultra and imaged on the IN Cell Analyzer. Images obtained on the IN Cell Analyzer 1000 were analyzed using the Nuclear Trafficking Analysis Module. The data show that forskolin response, as measured by the intracellular signal intensity of the red-shifted reduced CytoCy5S substrate, occurs in a dose-dependent manner.

The data in Figure 4 also show that fixing the cells after forskolin treatment did not adversely affect the magnitude of change to the assay signal compared to the non-fixed live-cell assay. This was supported by similar EC50 values obtained from both formats (Table 1).


Conclusion
Adenoviral vector gene delivery can be successfully combined with NTR reporter gene technology to develop a CRE-NTR assay for investigating cAMP-mediated cellular response. Cell-based protocols using adeno
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