Figure 3.

Analysis of BAC RP11–1145H7 shows RPS4X and CITED1 escape XCI at Hprt. (A) Integration of the RPS4X BAC at Hprt; genes on the BAC expected to escape from XCI in green, subject in blue, variable and discordant in yellow, and genes at integration site (both known to be subject to XCI) in grey. Genes with RT-qPCR and DNAm assays are indicated. (B) Description of genotypes, six mice for each. (C) Normalized to Pgk1, RT-qPCR of RPS4X expression in brain shows that the transgene is active on a male X, and escapes inactivation at ∼50% when on the Xi in brain and liver, with a slightly lower level in spleen. An adjacent discordant gene CITED1 also escapes from the Xi in females at similar levels in brain. CITED1 expression was not detected in liver and spleen. Expression from female Xi is shown as percentage of the male X (red text). (D) Average DNAm of skewed knock-in females shows a hypomethylated RPS4X promoter in all tissues, CITED1 is low but different between tissues, and ERCC6L, HPRT and Phf6 are significantly hypermethylated compared to knock-in males in all tissues (Mann–Whitney t-test, significance from hypomethylated males denoted by asterisks; P-value <0.001***, 0.001–0.01**, 0.01–0.05*, >0.05 ns).