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

ay, the assay plate must be contained within BSL-2 level facilities.


Results
Optimization of MOI in U2OS cells

To ensure good assay performance, functional cellular assays involving adenoviral transduction require initial determination of the optimal MOI, which is achieved by titrating into target cells. Figure 1 shows MOI optimization data for U2OS cells transduced for 24 h at a range of MOI with the Ad-A-Gene CRE-NTR vector. Cells were treated with 10-μM forskolin for 5 h followed by addition of 10-μM CytoCy5S substrate (final concentration 1-μM) for a further 5 h; fluorescence intensity was measured 24 h after transduction on a Tecan Ultra. Optimal virus loading was found to occur between 25 and 75 MOI, based on the highest relative fluorescent unit (RFU) value. Users should choose the MOI that provides the optimum balance between viral load on a cell and acceptable signal. For this assay, 25 MOI was adequate for future experiments using U2OS cells.

CREB-induced NTR expression
Figure 2 shows images of U2OS cells transduced with the CRE-NTR adenovirus vector acquired on the IN Cell Analyzer 1000. Cells were treated with 10-μM forskolin for 5 h followed by addition of 1-μM CytoCy5S substrate for a further 3 h. Minimal red fluorescence was detected in non-transduced cells (Fig 2a) or in transduced cells treated with assay medium alone (Fig 2b). In contrast, images of transduced cells treated with forskolin (Fig 2c) display a marked increase in ‘red’ cellular fluorescence, demonstrating that this system functions as a cell-based reporter assay for CRE that is sensitive to chemical stimulation.

Cell-by-cell population analysis of transduced U2OS cells
Figure 3 shows the response of individual cells for each of the three different populations from a typical Ad-A-Gene CRE-NTR assay. The non-transduced pop
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