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Fig. 4. The Xist locus is ectopically activated on the active X^pA. (A) Total RNA prepared from E13.5 embryos was analyzed by northern blotting. Ectopic expression of Xist was evident from the single active X in X^pAY male embryos. (B) Allelic expression of Xist was analyzed by RT-PCR in the embryo proper and the visceral endoderm. Females heterozygous for either Tsix^pA or Xist^IVS were crossed with JF1 males (X^JF1Y), and RNA prepared from the embryo proper and visceral endoderm at E13.5 was subjected to RT-PCR on cDNA primed by Xist7(-)20 in a strand-specific manner. PvuII digestion of the amplified product revealed that in spite of the fact that X^pA stayed active in essentially every cell in X^pAX^JF1 female embryos, Xist was expressed from the active X^pA as well as the inactivated X^JF1 in both the embryo proper and the visceral endoderm (upper panel). Similar activation of the Xist locus on X^pA was also seen in both tissues of X^pAY males. Such ectopic activation was never observed on X^IVS in either tissue in either sex (lower panel). cDNA synthesis was carried out in the presence (+) or absence (-) of reverse transcriptase. (C) The levels of ectopic expression of Xist in the embryo proper and the visceral endoderm of X^pAY males (X^pAY-1 and X^pAY-2) were compared with those of Xist in wild-type XX and XY embryos by real-time PCR using cDNA synthesized in B. Xist and Gapd sequences were amplified using primer set R700P2/Xist6(-)20 and GapdF/Gapdr2, respectively, and the expression level was normalized by the value for Gapd. Values are means ± s.d.

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