Fig. 2.

Heterozygous female embryos with a maternally transmitted Xist^INV allele exhibit secondary non-random X inactivation. (A) Allele-specific RT-PCR analysis of Xist from X ^INV_mX_p adult female kidneys (females 1-5). Top panel (wt) shows amplification with wild-type Xist allele specific primers (e and l) and the bottom panel presents amplification with Xist^INV specific primers (b and e). Numbers above the top panel show the number of cycles used. Note the six to nine cycle difference in amplification of wild-type versus Xist^INV fragments between different females. (B) Light and GFP microscopy images of E8.5 and E11.5 embryos with a maternally inherited Xist^INV allele and a paternally inherited GFP transgene. The diminishing proportion of green cells indicates secondary non-random X inactivation. (C) Examples of FACS histograms showing relative proportion of GFP-negative (M1) and GFP-positive (M2) cells in X_mX_p ^GFP and X_m ^INVX_p ^GFP primary fibroblast cell lines established from E14.5 females. The x-axis represents the logarithmic level of GFP fluorescence and the y-axis shows the number of cells. 50,000 total events were acquired for each sample. (D) Quantitative analysis of FACS data obtained for two primary control fibroblast cultures (X_mX ^GFP_p) and for four primary fibroblast cultures from mutant heterozygous E14.5 embryos (X_m ^INVX_p ^GFP). (E) RT-PCR analysis of the Xist transcripts produced by fibroblast cell lines expressing wild-type Xist, Xist^Δex4 or Xist^INV. Top panel, primers amplifying a sequence upstream of the inversion in exon 1 (a and h); second panel, primers situated in exon 3 and exon 5, respectively (e and m); third panel, primers flanking the inversion break-point that only amplify from the Xist^INV allele (b and e); bottom panel, β-actin loading control. + and − indicate reactions set up with and without reverse transcriptase, respectively. Positions of all Xist primers used are shown in Fig. S1 in the supplementary material.