Figure 6
FACS analyses of NIH3T3 cells transduced with different PRIG vectors. Cells were analysed using a LSRII cytometer (Becton-Dickinson) 5 to 7 days after exposure to the viral supernatant(s). For each point, 104 events were recorded. A) Non-transduced NIH3T3 cells we analysed for cyan (eCFP*) yellow/green (eYFP/eGFP) or red (Cherry) fluorescences using the lasers and filters described in the Methods section to determine the basal level for each signal. B) Functional test of the pPRIPu eGFP vector. 48 hours after the exposure to a pPRIPu eGFP viral supernatant, cells were plated in a fresh medium containing (right panel) or not (left panel) 10 μg/ml of puromycin, and analysed for eGFP expression after 7 days of culture. All the selected and almost all the non-selected cells strongly express eGFP. Note that at this dose of puromycine, control untransduced NIH3T3 cells died in approximately 24 hours (not shown). C) Functional test of the single color vectors. Cells transduced with the indicated vector (at the bottom of each panel) were analyzed for each fluorescence. Note that under these conditions of transduction and detection, each fluorescent protein (eCFP*, eYFP/eGFP, mCherry) is mainly, if not exclusively, detected in its appropriate channel. D) Feasability of double transductions. Cells were transduced with the indicated (right hand side) mixture of two viral supernatants. In the cases of double transductions, untransduced cells, doubly transduced cells and cells transduced with either one of the two single color vectors can be discriminated from each other. E) Feasability of triple transductions. Cells were transduced with the indicated mixture of the three viral supernatants. Again, because of the minimal overlapping between the three signals, the transduction status of any cell with respect to each vector can be determined. Note however, that for D and E, the quantity of each viral supernatant was not equal in the mixture to correct for either relatively inefficient detection of the signal in our conditions (mCherry) or relatively intrinsic weakness of the protein (eCFP*) (see text). F) Functional test of the doublecolor vectors. Cells were transduced with the indicated vector (at the bottom of the panel). In each case, the vast majority of the cells are positive for the two expected signals.