Figure 4

Simultaneous deletion of the Tsix CpG island and Xist intron 1 further increases transgenic Xist activation in undifferentiated XY ES cell lines. (A) RNA FISH analysis of Xist and Tsix expression in undifferentiated XY ES clones carrying P1 construct with deletion of the Tsix promoter (ΔCpG) and Xist intron 1 (Δint2.1, clone L8F1). (B) Graph showing proportional representation of four patterns of Xist expression in XY ES clones carrying P1 transgenes. Average data for 12 clones of each genotype are shown. Individual clone data are shown in Figure 3A. (C) A graph showing the percentage of clones with upregulated Xist. (D) qRT-PCR analysis of Xist expression in XY ES clones carrying either wt P1, P1 with deletion of Tsix promoter (ΔCpG), P1 with 2.1 kb (Δint2.1) deletion in Xist intron 1 or simultaneous deletion of Tsix promoter (ΔCpG) and Xist intron 1 (Δint2.1). 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. See Figure 2 for detailed annotation. ES: embryonic stem; FISH: fluorescent in situ hybridisation; qRT-PCR: quantitative reverse transcription polymerase chain reaction; wt: wild type.