Escape from X inactivation is directly modulated by Xist non-coding RNA [in vivo]

X-chromosome inactivation silences one copy of the X-chromosome in female cells, but a number of genes escapes XCI. We asked whether increased levels of Xist RNA, the molecular actor driving X-inactivation, impacts expression levels of escapees. We tested whether dox-mediated Xist-overexpression affected the expression levels of escapees during mouse early embryonic development. Overall design: To investigate the effect of Xist overexpression on escape levels in early embryos, Xist was overexpressed via doxycycline induction in early mouse embryos carrying an inducible Xist allele on the inactive X. Embryos were derived from natural mating between C57BL/6 TX males Xptet/Y; R26rtTA/WT with wild type JF1 females. Embryos were harvested at E2.5 and E3.5 and cultured in vitro in the presence of Doxycycline for 24 hours. Embryos were harvested for RNA-Sequencing using the Smart-Seq2 protocol and allele-specific expression was determined for X-linked genes.