Fig. 4.

Failure of X-linked gene silencing in female embryos carrying a paternally inherited Xist^INV allele. (A,B) Light and GFP microscopy images of E3.5 (A) and E6.5 (B) female embryos with a paternally inherited Xist^INV allele and XGFP transgene. GFP expression in blastocysts (E3.5) and extra-embryonic tissue at E6.5 indicates that Xist^INV cannot establish or maintain appropriate levels of X-linked gene silencing. The arrowhead marks a single blastocyst with a maternally inherited GFP transgene to show the intensity of full activation of the transgene. Scale bars: 100 μm in A, 500 μm in B. emb, embryonic part; ex, extra-embryonic part; wt, wild type. (C) Allele-specific gene expression analysis of three X-linked genes: Pgk-1, Gla and Smc1l1. E3.5 embryos were obtained from crosses between either wild-type or Xist^INV-carrying males and PGK females. Polymorphisms arising between the PGK strain and the targeted 129 strain allowed expression from X_m (PGK) and X_p (129) alleles to be distinguished by single nucleotide primer extension (SNuPE). Female embryos were identified by GFP expression and pooled together for analysis. At least six embryos were included in each pool and at least four pools were analysed. Examples show duplicate loading of RT-PCR reactions detecting the presence of expression from X_p (129) and X_m (PGK) alleles. PGK × 129 F1 genomic DNA was also included in the analysis as a control. (D) The percentage of total expression coming from the X_p allele in wild-type and Xist^INV-carrying embryos. PGK × 129 F1 genomic PCR fragments were used to normalise the data. X_p, paternally inherited X chromosome; X_m, maternally inherited X chromosome. Error bars indicate s.d.