Figure 7

Fluorescence microscopy analyses of cells transfected or transduced with PRIG vectors. A. pPRIPu confers puro-resistance. HEK293T cells were transfected with either peGFP-N1 + pPRIPu (left) or peGFP-N1 (Clontech) + pRK5 (irrelevant carrier DNA) (right). 24 hours after transfections, cells were further grown for 30 hours in the presence of 10 μg/ml of puromycin. eGFP serves as a control for transfection efficiency. pPRIPu + peGFP-N1 transfected cells are living while most mock-transfected cells are dying. B. Single color PRIGs are expressed in HEK293T transfected cells. HEK293T cells were transfected with the indicated single color PRIG vector. Fluorescence microscopic analyses were performed 24 hours later. The signal emitted by each fluorescent protein was observed and photographed using the appropriate filter. Note that the mCherry signal is relatively weak probably due to the suboptimal excitation light delivered by our filter (546/12 nm). Moreover, a weak portion of the eGFP signal is detectable in the cyan filter, and conversely a residual portion of the eCFP* signal is detectable in the green filter. By contrast, the eYFP (green filter) and mCherry (red filter) signals are only detected in their appropriate filter. C. Single color PRIGs are expressed in transduced rat primary osteoblasts. Rat primary osteoblasts were transduced by the indicated single color PRIG vector and observed using a fluorescent microscope 48 hours later. Signals were photographed using the appropriate filter for each fluorescent protein. D. mCherry localization in HEK293T transfected cells. Left: HEK293T cells were transfected with pPRIGp mChHA. The red signal appears to be predominantly cytoplasmic, which is not observed in pPRIChp transfected cells, where mCherry shows an uniform distribution (see B). Right: HEK293T cells were transfected with a pPRIGp mChHA derivative containing an in-frame deletion of the 5' part of the MCS (PvuII/StuI). This deletion completely restores a normal (uniform) distribution of mCherry. Note that in cells transfected with pPRIGp mChHA (left) and with its PvuII/StuI deleted derivative (right), the eGFP (green, encoded by the 3' cistron) appears normally and uniformly distributed throughout the cell. The regions of interest were enlarged for a better visualization.