Analysis of Xist expression in Dicer-deficient XY embryonic stem cells. (A) RNA FISH analysis in the undifferentiated wt XY ES cell line (129/1), wt XX ES cell line (Pgk12.1) and Dicer-deficient XY ES clone (S5) using full length DIG labelled Xist probe. The probe is detected with a FITC-coupled antibody (green) and DNA is counterstained with DAPI. Merged colour images are shown in the right-hand panels. The majority of Dicer mutant ES cells show one pinpoint signal per cell corresponding to Xist and Tsix transcripts, similar to the XY control cell line. A proportion of mutant cells demonstrate an elevated level of Xist signal (arrow) which either accumulates tightly along the chromosome, similar to XX cells (compare the two middle panels), or shows more dispersed and scattered localisation in the vicinity of the X chromosome (arrow, bottom panel). Occasional accumulation of Xist in undifferentiated Pgk12.1 XX ES cultures is attributable to a small proportion of differentiatingcells. (B) Quantitative RT-PCR analysis of Xist expression in Dicerlox/lox and deficient XY ES cells. The three panels show three groups of Dicer null clones with corresponding floxed parental controls. The right-hand panel shows relative level of Xist expression in 129/1 XY and Pgk12.1 XX ES cells. All data is normalised to β-actin transcript levels and presented relative to the 129/1 Xist RNA level. Accumulation of Xist RNA detected by RNA FISH correlates with elevated level of Xist transcript determined by quantitative RT-PCR.