Fig. 2
From: REX1 is the critical target of RNF12 in imprinted X chromosome inactivation in mice
Rex1 knockout mice are viable and fertile. a BAC targeting strategy to generate the Rex1 knockout cas allele in 129:cas ESCs. The Rex1 coding sequence is shown as a blue box. The start site is indicated by a black arrow. LoxP sites, denoted as red triangles, flank the neomycin-resistance gene (neo) as a positive gene selection marker. Primers used to validate gene recombination in ESCs and to genotype are shown as red arrows. b Validation of Rex1 knockout cas allele recombination by XmnI RFLP analysis of WT and Rex1-targeted ESCs (top panel). Pf1M1 RFLP analysis to detect presence of two X chromosomes (bottom panel). c Nuclear extracts of WT, Rex1+/− and Rex1−/− ESCs were immunoblotted with RNF12 and REX1 antibodies. ACTIN was used as a loading control. Uncropped WB images are found in Supplementary Fig. 10b. d Sex and genotype distribution from different matings of Rex1-deficient mice. Number of breedings, number of mice per breeding and total number of mice are indicated below. No significant bias against the birth of female animals was observed (χ2 test, p > 0.05). e Xist RNA-FISH (FITC) analysis on WT, Rex1+/− and Rex1−/− ESCs at day 3 of differentiation. DNA was stained with DAPI (blue). White arrows indicate the presence of two clouds within a nucleus. Scale bar: 20 μm. f Quantification of Xist-positive cells (left panel) and Xist-positive cells with two clouds (right panel) in WT, Rex1+/− and Rex1−/− ESCs at day 0 and day 3 of differentiation. Asterisks indicate p-value <0.05, two-tailed Student's t test (average expression ± s.d., n = 1–3 biological replicates)
