Fig. 2.

Tet-induced Xist mediates gene silencing in male Dicer-deficient ES cells. (A) FISH staining for Xist RNA using a single-stranded RNA probe (green) and coimmunostaining of the elongating form of RNA-polymeraseII (pol II phospho-Ser-2) (red). Exclusion from the Xist RNA-coated territory is indicated by arrows. (B) FISH for Pgk1 nascent primary transcripts (red) and Xist (green) in uninduced (−dox, Left images) and induced ES cells (+dox, Right images) using double stranded DNA probes. The Xist pinpoint signal seen is the result of Tsix RNA because we did not observe this signal when strand specific Xist RNA probes were used. Nuclei were counterstained with Hoechst 33342 (blue). (C) Graphs depict quantitation of the percentage of total cells which display a focus of Pgk1 nascent transcripts (blue bars) or Xist RNA coating (red bars) before (−tet) and after (+tet) dox addition. (D) qRT-PCR analysis of Pgk1 (blue bars) and Hprt (red bars) mRNA levels before (−tet) and after (+tet) dox addition (E) qRT-PCR analysis of spliced (blue bars) or unspliced Xist transcripts (intron3; red bars) before and after dox addition. Middle and Right images in C through (E) represent 2 different Dicer-deficient clones.