Figure 2.

Reactivation of GFP in Xist mutant fibroblasts. FACS^® analysis of cells. (A) Live cells were gated, and their GFP fluorescence was plotted against autofluorescence. Dots to the right of the diagonal represent cells in which GFP fluorescence is greater than autofluorescence and therefore are considered GFP positive. GFP-negative (+/Y) and GFP-positive (GFP/Y) populations are shown for control. Xist conditional mutant cell populations, before Cre-mediated deletion of Xist (−cre), contained a small number of GFP-positive cells. After Cre-mediated deletion of Xist (+cre), the number of GFP-positive cells increased. (B) Number of GFP-positive cells in primary conditional mutants and in controls with or without adenovirus-Cre infection on day 7 after infection. In Xist conditional mutants, Cre-mediated deletion of Xist led to a twofold increase in GFP-positive cells, whereas controls remained unchanged. (C) Long-term culture of SV-40 T-antigen–transformed conditional mutant cells. FACS^® analysis was performed at various time points after adenovirus-Cre infection. Although initially we observed an increase in the number of GFP-positive cells after Cre-mediated deletion of Xist, the number decreased after another week in culture to reach the level of spontaneous reactivation and remained at that level for the duration of the experiment.