Fig. 2. Splicing products of Tsix are eliminated in ES cells. (A) The structure of the respective mutant allele is delineated with respect to the position of Tsix exons shown in gray. A proximal part of Tsix exon 4 shown in light gray indicates the region missing in the Tsix^SA allele. Tsix on X^dc chromosome is truncated by the insertion of IRES ßgeo (Sado et al., 2005). Positions of primers used for PCR and real-time PCR are also shown below the exons of Tsix. (R) and (Pm) are the EcoRI and PmaCI sites destroyed in the cloning process. (B) The absence of the splicing products of Tsix was confirmed by RT-PCR using cDNA prepared by random priming. The spliced form was detected using primer pair Xist1175F and 21b80F. The presence of the antisense transcription was monitored using primer pairs Tsix2F/Tsix2R and 8111F/8420R located in exon 2 and 4, respectively. (C) Antisense activity of Tsix was analyzed in each undifferentiated ES cell line by real-time PCR using primer sets Tsix2F/Tsix2R (exon 2) and 8111F/8420R (exon 4). (D) Northern blot analysis of polyA RNA isolated from ES cells carrying the respective mutation. Hybridization was serially performed using an RNA probe specific to Tsix (left) and Xist (middle), respectively, and a cDNA fragment of Gapd (right). The absence of the major splicing products is evident in Tsix^SA/Y ES cells. (E) DNaseI hypersensitive site assay using nuclei isolated from each ES cell line. Purified DNA was digested with HindIII and probed with Af0.4 (see Fig. 6). Neither of the known DNaseI hypersensitive sites (HS1 and HS5) found on the transcriptionally active Xist allele in somatic cells was observed in ES cells regardless of whether they harbor the mutation or not. A DNaseI hypersensitive site HS3 shown by an asterisk, which is known to be common to both the active and inactive X in somatic cells, was more prominent in X^dcY than others. (F) Real-time PCR analysis of the transcripts from the Tsix/Xist locus in undifferentiated ES cells and 12-day embryoid bodies using primers R700P2 and Xist6(-)20 on strandspecifically prepared cDNA.