Figure 3

Derivation and analysis of Dicer-deficient XY embryonic stem cell lines. (A) Two approaches used to create Dicer-deficient embryonic stem (ES) cell lines (see Methods for details). (B) PCR genotyping assay to discriminate between Dicer wild-type (wt), floxed and deficient cell lines. 1–3 and 11, Dicer null clones; 4–5 and 7–8, Dicer^lox/lox parental cell lines; 6, a mixed clone with deleted and floxed alleles; 9, wt/Δ heterozygous mouse; 10, wt control. The wt band in Dicer-deficient clones is due to contamination of the ES sample with feeder cells. (C) Northern blot hybridisation of RNA from floxed cell lines (A6 and D3) and Dicer null clones (S5 and S6) with an mi292as probe to assay for Dicer function. Loss of miRNA and gain of pre-miRNA in S5 and S6 clones but not in floxed clones A6 and D3 indicate that Dicer function is abolished in mutant clones. (D) Schematic of the Xist 5'region is shown alongside a restriction map. The grey bar indicates the position of the probe used for Southern blot hybridisation. (E) MSRE analysis of Xist promoter in control and mutant ES cell lines. DNA methylation level in Dicer^Δ/Δ clones is more similar to the hypomethylated XX cell line rather than the methylated XY control or parental XY floxed cell line. (F) Quantification of the degree of hypomethylation of Acl I, Mlu I, and Sac II sites in floxed and Dicer-deficient ES cell lines. The position of the sites relative to the Xist start site is shown in brackets.