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 siently expressed within those target cells at basal levels. When activated, CREB binds to the CRE sequences within the CRE-NTR gene, leading to promoter-induced up-regulation of NTR production. NTR is detected using the pro-fluorescent substrate, CytoCy5S™ (9). Quantitation of the NTR signal indicates the strength of the cellular response to the physical or chemical stimuli being investigated.
The CRE-NTR gene reporter assay described here was performed in live- and fixed-cell format in microplates. Assay plates were imaged on IN Cell Analyzer 1000 and read on a Tecan™ Ultra fluorimeter.
Reagents and instrumentation
Ad-A-Gene CRE-NTR GDS 40001
IN Cell Analyzer 1000 25-8010-26
IN Cell Analyzer 1000 Seat License* 25-8098-22
Object Intensity Analysis Module
(for IN Analyzer Cell 1000)† 25-8010-56
Nuclear Trafficking Analysis Module
(for IN Cell Analyzer 1000)† 25-8010-31
CytoCy5S, 0.2 mg PA76140
* A seat license is a cost-effective single-user or server license that gives access to all ready-to-use Image Analysis Modules provided with IN Cell Analyzer. License holders have access to all appropriate analysis software and more licenses can be purchased as the number of users grows.
† Available to seat license holders only.
Additional materials required
Tecan Ultra fluorimeter (Tecan group Ltd)
Culture medium: McCoy’s 5A (Sigma) with 10% fetal bovine serum (F BS) (Sigma), 2-mM L-glutamine (Sigma), 100-μg/ml penicillin, and 100-μg/ml streptomycin (Sigma)
Assay medium: DMEM (Sigma) without phenol red or FBS, containing 2-mM L-glutamine (Sigma), 100-μg/ml penicillin, and 100-μg/ml streptomycin (Sigma)
Dulbecco’s (GIBCO BRL)
Formalin solution (10%), neutral-buffered, 4% (w/v) formaldehyde, (Sigma)
Hoechst™ 33342 nuclear dye (Molecular Probes)
96-well microplates (Greiner Bio One)
U2OS cell lines (ATCC)
Assay method in 96-well microplate format
On the day before the assay, suitable target cells in log-phase growth were detached by treatment with trypsin. Cell concentrations were adjusted to suitable levels with culture medium. Ad-A-Gene CRE-NTR vector was thawed by placing the tube on ice, added to the cell suspension, and mixed to provide the appropriate multiplicity of infection (MOI) for the assay*. The cell and virus suspension was dispensed at 200 μl/well into microplates and incubated for 24 h at 37 °C, 5% CO2.
Note: All adenoviral vector preparations are handled as BSL-2 category reagents. Local safety assessments are made before using the reagent. The product pack literature contains detailed instructions for product handling.
*An MOI of 25 ifu/cell in U2OS cells has been calculated as optimum for the CRE-NTR assay. Other assay conditions will vary and can be optimized by users.
Assay protocol (fixed-cell format)
On the day of the assay, test compound (10-μM forskolin) was prepared from stocks in assay buffer†. Medium was removed from the cells, and the cells were washed with PBS. Test wells were treated with 90 μl of forskolin in assay medium; 90 μl of medium alone was added to control wells. Pla tes were incubated at 37 ºC for 5 h, after which 10-μM CytoCy5S substrate in assay medium (10 μl) was added to give a final concentration of 1 μM. The solution was incubated for an additional 3 h at 37 ºC.
Solutions were decanted from the wells and fixing solution was added (100 μl/well), followed by 15-min incubation at room temperature. Fixing solution was decanted from all wells, and the cells were washed with PBS (200 μl/well). Finally, Hoechst nuclear dye (2.5 μM) in PBS was added (100 μl/well), and the wells were incubated for 20 min at room temperature (the plates can be stored at 2–8 °C at this stage if imaging is not performed immediately).
Plates were imaged on IN Cell Analyzer 1000 using 620/60-nm excitation and 700/75-nm emission filters (CytoCy5S). Images were analyzed using the Object Intensity Analysis Module. Plates were read on a Tecan Ultra using 610/20-nm excitation and 680/30-nm emission filters (CytoCy5S).
†Time-controlled dispensing of compound solutions to the cells can be avoided by completing the assay using live cells and then fixing the cells prior to imaging. Cells should be fixed at the peak induction time point. Fixing of cells reduces the handling requirements for post-assay material to BSL-1 categorization.
Assay protocol (kinetic live-cell)
Test compound (micromolar) was prepared from stocks in assay buffer, medium was removed from the cells, and the cells were washed with PBS. Test wells were treated with 90 μl of forskolin in assay medium; 90 μl of medium alone was added to control wells. Plates were incubated at 37 ºC for 5 h, after which 10-μM CytoCy5S substrate in assay medium (10 μl) was added to give a final concentration of 1 μM. The solution was incubated for an additional 3 h at 37 ºC followed by reading on a Tecan Ultra.
Note: At all stages of the live-cell ass ay, the assay plate must be contained within BSL-2 level facilities.
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 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).
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 viral gene delivery are relatively simple compared to plasmid-based transfection techniques and data can be obtained more rapidly. Transfection efficiency is efficent and uniform, with each target cell carrying at least one copy of the CRE-NTR gene under the conditions employed. In addition, this work suggests that the sequential process of viral transduction, exposure of cells to reagents, and data acquisition can be transcribed into suitable automated and integrated workflow systems with standard liquid-handling dispensing units as well as image capture and data analysis workstations.
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