Mdx-XistΔhs mice.

A. To breed mice with low dystrophin levels, female XistΔhs mice, carrying a mutation in the Xist promoter which coordinates X-inactivation, were crossed with dystrophin negative mdx males. During embryogenesis, the maternal X-chromosome encoding a functional dystrophin gene will be preferentially (60–90%) inactivated as a result of the mutated Xist promoter. The XistΔhs mice were a kind gift from N. Brockdorff (MRC Clinical Sciences Center, London, UK, current affiliation Department of Biochemistry, University of Oxford, UK). B. Picture of a representative Western blot. The percentage of dystrophin was determined for the quadriceps of all mdx-XistΔhs mice by Western blot (2–9 technical replicates per mouse). The percentage of individual mice was determined using a concentration curve made from wild type samples. Myosin was used as a loading control. C. Skewed X-inactivation resulted in dystrophin levels of 3–47% (mean 22.7, stdev 12.1, n = 24) (as determined by Western blot) in the female mdx-XistΔhs offspring. Each bar represents the dystrophin level of an individual mouse. The dystrophin levels of the individual mice belonging to the three dystrophin groups can be appreciated from this graph. D. Dystrophin levels determined by Western blot and manual counting of dystrophin positive fibers demonstrate a strong correlation (R = 0.97). E. Longitudinal sections of a quadriceps stained with dystrophin (green) and spectrin (red). From the pictures it can be appreciated that dystrophin expression is not homogeneously expressed across the fiber but rather confined to certain nuclear domains.