Figure 2

Deletion of NOS binding region within Xist intron 1 causes moderate upregulation of transgenic Xist expression in undifferentiated XY ES cell lines. (A) schematic representation of X inactivation centre region cloned into bacteriophage clone P1. Xist and Tsix exons are indicated as black and grey rectangles, respectively. The first three exons of Enox/Jpx are also shown. The direction of transcription for each locus is indicated by arrows. An enlarged region spanning Xist exons 1 to 3 is shown underneath the main schematic. Blue horizontal bars underneath indicate the position of homology arms used for recombineering. 0.3 kb (Δint0.3) and 2.1 kb (Δint2.1) sequences within intron 1 deleted from P1 clone by recombineering both encompass NOS-binding region. Blue lines above the main schematic indicate the position of the homology arms and the deleted region of the Tsix promoter (ΔCpG). (B) RNA FISH analysis of Xist and Tsix expression in undifferentiated XY ES clones carrying wt P1 construct (clone L5E2), P1 construct with small (Δint0.3, clone L9D7) or large deletion (Δint2.1, clone L7B2). Bar, 10 μm. (C) A graph showing proportional representation of four patterns of Xist expression in XY ES clones carrying P1 transgenes: light grey, no detectable Xist expression; red, upregulated Xist cloud; grey, two punctate Xist signals; dark grey, one punctate Xist signal. Average data for 12 clones of each genotype are shown. Individual clone data are shown in Figure 3A. (D) Graph showing a percentage of clones with upregulated Xist. (E) qRT-PCR analysis of Xist expression in XY ES clones carrying either wt P1 or P1 with 0.3 kb (Δint0.3) or 2.1 kb (Δint2.1) deletions in Xist intron 1. All data is normalised to β-actin transcript levels and presented relative to the wt XY ES (129/1) Xist RNA level. Average data for 12 clones of each genotype are shown. Individual clone data are shown in Figure 3B. ES: embryonic stem; FISH: fluorescent in situ hybridisation; NOS: Nanog/Oct4/Sox2; qRT-PCR: quantitative reverse transcription polymerase chain reaction; wt: wild type.