Embryonic stem (ES) cell lines carrying autosomal-inducible Xist transgenes. Three independent ES cell lines carrying inducible Xist transgenes on chromosomes 3 (8A), 12 (12B), and 17 (3E), were characterised in detail. (a) Panels illustrate example of DNA fluorescence in situ hybridisation (FISH) mapping of Xist transgene (green, arrow), and chromosome specific bacterial artificial chromosome (BAC) probe (red, arrowhead). DNA was counterstained with 4',6-diamidino-2-phenylindole (DAPI; blue). Maps illustrate fine mapping of each transgene (Tg) by DNA FISH, indicating position of nearest BAC probes in Mb. (b) RNA FISH analysis illustrating Xist RNA domains (red) in cells differentiated for 3 days in the presence of doxycycline. DNA was counterstained with DAPI (blue). Graphs illustrate percentage of cells with Xist RNA domains at different number of days (d) of differentiation. Error bars indicate variation from 2 independent determinations, scoring at least 100 cells in each case. (c) Example of RNA FISH of iXist in a mitotic cell from differentiating 3E cells (chromosome 17) illustrating Xist RNA bands along the length of the chromosome (right panels). (d) Immunofluorescence analysis of mitotic 3E cells illustrates chromosome-wide enrichment of Xist RNA dependent histone modifications H2AK119ub1 and H3K27 me3.