Fig. 2
Allelic inversion of Xist expression is associated with a reversal of X-inactivation profiles. a Cumulative histograms showing the expression levels of several X-linked genes located within the 129/Pgk1a polymorphic region measured by allelic RT-qPCR in the 8 Xist revertant clones from independent HAT selection experiments (analysis performed at passage 8 or later after clone picking). Standardisation by Rplp0 has been applied. Paternal and maternal expressions are depicted in blue and red above and below the x-axis, respectively. The diagram shows the genomic positions of the genes analysed by RT-qPCR (on the 129/Pgk1a chromosome) and by RNA-FISH (on the 129 chromosome) in panel a and b. For F3 parental female cells, mean and standard deviation have been calculated on the basis of three independent cell cultures. A.U. Arbitrary Units. b Representative images of double RNA-FISH for Xist and for the indicated X-linked gene in F3 parental cells and in cells of clone 1#A. The inactive X-chromosome is identified through Xist RNA accumulation (red). Primary transcription at the indicated X-linked gene is co-detected in green. <1 % of F3 parental TS cells show an Hprt1 signal in agreement with the inactivation of the wild-type, paternal, Hprt1 allele. In contrast, an Hprt1 signal away from the Xist domain is detected in the majority of nuclei of clone 1#A indicating a reactivation of the XP carrying the wild-type Hprt1 gene. Scale bar = 5 μm. c Cumulative histograms of the percentages of nuclei with the depicted expression pattern in F3 TS cells and in nuclei of 3 independent Xist revertant clones (analysis performed at passage 8 or later after clone picking). See Additional file 10 for probe position. No significant difference is observed between any of the clones and F3 parental cells for all X-linked genes except for Hprt1 (χ 2 test p value < 0.05). n > 150