Jpx transgenes induce Xist expression in mESCs using both trans and cis mechanisms.
(A) Map of the X-inactivation center (Xic) with 90kb Tg(Jpx) and 120kb Tg(Jpx, Xist) transgenes and the probes used for Fluorescence in situ Hybridization (FISH). (B) Combined RNA-DNA FISH for Xist (green, FITC) and Xpct (red, Cy3) on female mESC at differentiation days 0, 2, and 4. Top: Control female ESCs transfected with Tg(pSKYneo+), a plasmid that does not contain X-chromosome sequence but provides the same neomycin resistance as Tg(Jpx, Xist); bottom: Tg(Jpx, Xist) transgenic female mESC line #7, which has a single-copy Tg(Jpx, Xist) integrated in an autosome; right: schematic of Xist expression at Day 4. Green wavy line, Xist RNA; Red dot, Xpct (X-Chr.) DNA locus; Open arrowhead: Tg(Jpx, Xist) transgenic site. Scale bar: 2μm. (C) Xist cloud frequency throughout differentiation. **, P<0.01; ***, P<0.001, from a chi-square test comparing total Xist cloud counts in wildtype and transgenic cells at each differentiation day. Sample sizes and additional Tg(Jpx, Xist) mESC lines are included in S1 Fig.