Dystrophin expression in <i>mdx-Xist</i>^Δhs mice.

<p><b>A:</b> Representative Western blot of <i>mdx-Xist</i>^Δhs brains revealed that mice expressed low levels of full-length dystrophin in their brain. Levels varied between individuals but were below 20% in all mice. Post Synaptic Density Protein 95 (Psdp95) served as a loading control. Wildtype samples for the concentration series were diluted in <i>mdx</i> protein lysate to ensure equal protein loading of all samples. <b>B:</b> mRNA expression levels of neither full-length nor Dp71 differed between <i>mdx</i> and BL10 mice. However, expression of Dp427c was higher in BL10 compared to <i>mdx</i>-<i>Xist</i>^Δhs mice. Error bars represents the standard deviation. Asterisk indicates <i>P</i><0.05.</p

The volumes of 22 segmented brain structures in <i>mdx</i>, BL10, <i>mdx</i>-<i>Xist</i>^Δhs and BL10-<i>Xist</i>^Δhs mice were compared within each genetic background (<i>mdx vs</i> BL10 and <i>mdx</i>-<i>Xist</i>^Δhs <i>vs</i> BL10-<i>Xist</i>^Δhs).

<p>The brain structures found significantly different in volume after false discovery rate correction are shown and consisted of both grey and white matter structures for both comparisons. Coronal, sagittal and axial planes are shown indicated by white lines. <b>A:</b> Comparison between <i>mdx</i> and BL10 showed four different brain structures: the hippocampus, globus pallidus, caudate putamen and hypothalamus. <b>B:</b> Comparison between <i>mdx</i>-<i>Xist</i>^Δhs and BL10-<i>Xist</i>^Δhs showed 13 different structures.</p

Volumes (in mm³) of 22 brain structures in mouse models of Duchenne muscular dystrophy.

<p>The volumes of 22 segmented brain structures in <i>mdx</i>, BL10, <i>mdx</i>-<i>Xist</i>^Δhs and BL10-<i>Xist</i>^Δhs mice are presented in mm³. Body mass was measured just before sacrificing the mice, except for two <i>mdx-Xist</i>^Δhs mice of which body mass was not obtained. The volumes of these 22 structures were normalized to whole brain volume and compared using Welch’s T-tests within each genetic background (<i>mdx vs</i> BL10 and <i>mdx</i>-<i>Xist</i>^Δhs <i>vs</i> BL10-<i>Xist</i>^Δhs) and corrected for multiple comparisons using the false discovery rate. The corresponding <i>P</i>-values are presented and structures which are significantly different in volume are written in bold. The hippocampus, globus pallidus, caudate putamen and hypothalamus were different between <i>mdx</i> and BL10 mice. Thirteen structures were different between <i>mdx</i>-<i>Xist</i>^Δhs and BL10-<i>Xist</i>^Δhs mice, consisting of a mixture of both grey and white matter structures.</p

Dystrophin expression pattern and morphological examination of del52hDMD/<i>mdx</i> mouse lines.

<p><b>A</b>) Western blot analyses of heart and quadriceps, incubated with either GTX (human and mouse specific) or Mandys106 (human specific). Wild type expression levels of human dystrophin were observed in hDMD/<i>mdx</i> mice. Notably, del52hDMD/<i>mdx</i>#37 mice expressed traces of human dystrophin, in both cardiac and skeletal muscle, while this was not observed in del52hDMD/<i>mdx</i>#35 and <i>mdx</i>(BL6) mice. <b>B</b>) Sections of the heart and quadriceps stained with human specific dystrophin antibodies. Expression of human dystrophin is at wild type level in hDMD/<i>mdx</i> mice as anticipated. Both C57BL/6J, <i>mdx</i>(BL6) and del52hDMD/<i>mdx</i>#35 mice did not express human dystrophin. Interestingly, in most fibers of del52hDMD/<i>mdx</i>#37 mice, human dystrophin was expressed at low levels. Haematoxylin and eosin staining revealed signs of degeneration and regeneration in the quadriceps of both del52hDMD/<i>mdx</i> strains, as evident by variation in fiber size, centralized nuclei and patches of fibrosis and inflammation. Overall pathology appeared to be slightly less extensive in del52hDMD/<i>mdx</i>#37 mice compared to <i>mdx</i>(BL6) and del52hDMD/<i>mdx</i>#35 mice. <b>C</b>) Almost no centralized nuclei were found in wild type mice, while half of the myofibers in <i>mdx</i>(BL6) and del52hDMD/<i>mdx</i>#35 mice had centrally located nuclei. The percentage in del52hDMD/<i>mdx</i>#37 mice was with 26% significantly lower. Data were based on manual counts of 5 randomly taken pictures of 2 males and 2 females per genotype. Asterisks indicate <i>P</i><0.01.</p

Confirmational analysis of offspring from blastocyst transplanted mice.

<p>Pups were analysed for chimerism by multiplex PCR and MCA. <b>A</b>) Four of the pups derived after transplantation of blastocysts injected with ES cells of clone 9B4 showed presence of the exon 52 deleted <i>hDMD</i> gene (lines 1, 4, 8 and 11). <b>B</b>) Melting curve analysis revealed that all male pups were also chimeric for the <i>mdx</i> point mutation.</p

ViM treatment results in exon skipping and subsequent dystrophin restoration.

<p><b>A</b>) Nested PCR revealing exon skipping upon local 51ViM or 53ViM treatment in right and left gastrocnemius and triceps muscle (respectively GR, GL and TR, TL) of two del52hDMD<i>/mdx</i>#35 mice. We confirmed by Sanger sequencing that the upper band in the untreated del52hDMD<i>/mdx</i> samples involves a cryptic splicing event that is occasionally observed in untreated mice of this strain. It contains exon 51, part of intron 51, the last 101 nucleotides of exon 52, exon 53 and multiple stop codons. The arrows indicate the expected heights of fragments lacking exon 51 (700 bp) or exon 53 (721 bp). <b>B</b>) Murine specific nested PCR confirmed that the exon 51 ViM induced low levels of mouse exon 51 skipping. The exon 53 ViM only resulted in exon 53 skipping in the human transcript as no skipping band was seen in the PCR performed with mouse-specific primers (expected size 483 bp). Sanger sequence confirmed that the smaller fragment obtained in the ViM exon 51 treated muscles contained the boundary of exon 50–52. Human ctrl; healthy human control sample. The arrow indicates the expected height of fragments lacking exon 51 (463 bp). <b>C-D</b>) Exon skipping resulted in the restored dystrophin expression in gastrocnemius (C) and triceps (D) of 51ViM and 53ViM treated mice.</p

PCR analysis of targeted clones and confirmation of deletion exon 52 on RNA level.

<p>Single ES clones were cultured in 96-well plates and DNA was isolated and used as template in a multiplex PCR. Here the exons 46, 51 and 52 of the <i>hDMD</i> gene were analysed where exon 46 and 51 are positive controls and exon 52 the target to be deleted. <b>A</b>) An example is shown where candidate samples 2 and 5 are of interest because they are negative for exon 52 but positive for the control exons. <b>B</b>) For a large number of clones additional fragments were found for exon 52, suggesting non-homologues end joining (NHEJ) of TALEN induced double stranded breaks <b>C</b>) Representative image of LR-PCR performed on DNA of sub-clones of four exon 52 negative clones (9B4, 10H2, 11C9 and 11E7). LR-PCR was performed with primers targeting intron 51 (outside the targeting arm) and blasticidin (only present after homologous recombination), to rule out loss of PCR primer recognition sites by NHEJ and to confirm true targeting. <b>D</b>) RT-PCR was performed for RNA isolated from embryoid bodies of selected clones. The different fragments were isolated, purified and Sanger sequence analysed. In the wild type situation exon 52 was present, whereas in the properly targeted clones exon 52 was not present. This confirmed the exon 52 deletion on RNA level.</p

Functional performance is impaired in del52hDMD/<i>mdx</i> mice.

<p><b>A</b>) Forelimb grip strength was impaired in <i>mdx</i>(BL6) and del52hDMD<i>/mdx</i> mice. <b>B</b>) hDMD/<i>mdx</i> mice were resistant against fatigue, while muscles of <i>mdx</i>(BL6) and del52hDMD<i>/mdx</i> mice were fatigued at the end of the grip strength protocol. Performance in hanging tests starting with two <b>C</b>) and four limbs <b>D</b>) was impaired in <i>mdx</i>(BL6) and del52hDMD<i>/mdx</i> mice. Overall, del52hDMD<i>/mdx</i>#37 mice outperformed <i>mdx</i>(BL6) and del52hDMD<i>/mdx</i>#35 mice. <b>E</b>) Creatine kinase levels were elevated in both del52hDMD<i>/mdx</i> and <i>mdx</i>(BL6) strains compared to hDMD/<i>mdx</i> mice. Asterisk indicates <i>P</i><0.05, data are represented as the mean ± SEM. hDMD/<i>mdx</i>, <i>mdx</i>(BL6) and del52hDMD<i>/mdx</i>#35 groups consisted of n = 3 males and n = 2 females (one male <i>mdx</i>(BL6) mouse died), while the del52hDMD<i>/mdx</i>#37 group consisted of n = 2 males and n = 3 females.</p