DEV008490 Supplementary Material

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• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. Generation of the Xist^IVS allele. (A) Genomic structure of the Xist^IVS allele is shown below the overall structure of the Xist/Tsix loci. The IVS2 sequence was simply introduced at the XhoI site (107228-107233 in GenBank Acc. No. AJ421479) in the same manner as in the case of Tsix^pA. D, splicing donor; A, splicing acceptor. (B) Targeting scheme for generating the Xist^IVS allele. Positions of the recognition sites of the relevant restriction enzymes and the probes used in C and D are shown. B, BamHI; E, EcoRI; H, HindIII; P, PvuII; X, XhoI. (C) Homologous recombination in ES cells was confirmed by Southern blot analysis in the same manner as described in the legend for Fig. 1. (D) Excision of the puromycin-resistance cassette by cre recombinase was confirmed by Southern blotting using tail DNA in the same manner as described in the legend for Fig.1.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Methylation level of CpG sites in the 5′ region of Xist is unaffected on X^IVS . (A) Genomic structure of the 5′ region of Xist on the wild-type X and X^IVS is depicted along with recognition sites of the methylation-sensitive restriction enzymes and positions of probes used in this assay. (B-E) Methylation level in the 5′ region of Xist was examined in the embryo proper and the visceral endoderm of E13.5 embryos recovered from X^IVSX females crossed with wild-type males by Southern blot analysis in the same manner as described in the legend of Fig. 5.