Multiple switching of the binary expression vector. a, b, the clonal population carrying a single insertion of the binary expression vector (Clone S, Figure 2) by bright field and under fluorescence, respectively. c, d, the same population as in panels A and B after introduction of Cre recombinase, and FACS cell sorting to obtain an EGFP-positive population. e, FACs analysis of the EGFP-positive population in panel D, with fluorescence plotted logarithmically on the vertical axis and pulse width (particle size) plotted logarithmically on the horizontal axis. f, Cre recombinase was introduced into the cell population in panel E, and analyzed by FACS. The major peak at top center (green arrows) represents GFP-positive cells, while the region at lower center is the expected peak location for GFP-negative cells (white arrows). The signal in the lower left quadrant of the FACS profile represents cellular debris. Since the binary vector used to establish Clone S (EGFP OFF) had been switched from ON to OFF in vitro prior to introduction into cells (see Materials and Methods), these data establish at least three consecutive switching events are possible.