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type=\u0022text\/css\u0022 rel=\u0022stylesheet\u0022 href=\u0022https:\/\/www.stormoverpacific.com\/sites\/default\/files\/advagg_css\/css__KwIbTmK5u_TU0NIDnj2cXC8qhcT3ubSigju5zweyvGo__wfUCN1ftcRJqvO1mXYEDxSMyoCswJBppiBXHDPwtaqM__MWGPudBpBmsaTs1FKLLZ0RHlWpbgNx0qxDcFgKr833o.css\u0022 media=\u0022all\u0022 \/\u003E\n\u003Clink rel=\u0027stylesheet\u0027 type=\u0027text\/css\u0027 href=\u0027\/sites\/all\/modules\/contrib\/panels\/plugins\/layouts\/onecol\/onecol.css\u0027 \/\u003E\u003C\/head\u003E\u003Cbody\u003E\u003Cdiv class=\u0022panels-ajax-tab-panel panels-ajax-tab-panel-jnl-template-cob-tab-data\u0022\u003E\u003Cdiv class=\u0022panel-display panel-1col clearfix\u0022 \u003E\n \u003Cdiv class=\u0022panel-panel panel-col\u0022\u003E\n \u003Cdiv\u003E\u003Cdiv class=\u0022panel-pane pane-cob-fragment-figures\u0022 \u003E\n \n \u003Ch2 class=\u0022pane-title\u0022\u003EArticle Figures \u0026amp; Tables\u003C\/h2\u003E\n \n \n \u003Cdiv class=\u0022pane-content\u0022\u003E\n \u003Cdiv class=\u0022elements-frag-data highwire-markup\u0022 id=\u0022fig-data\u0022\u003E\u003Cdiv id=\u0022fig-data-figures\u0022 class=\u0022group frag-figures\u0022\u003E\u003Cdiv class=\u0022fig-data-title-jump clearfix\u0022\u003E\u003Ch3 id=\u0022fig-frag-data-title\u0022 class=\u0022fig-data-group-title\u0022\u003EFigures\u003C\/h3\u003E\u003Cdiv class=\u0022fig-data-jump-links\u0022\u003E\u003Cul class=\u0022fig-data-jump-links-list links inline\u0022\u003E\u003Cli class=\u0022tables first last\u0022\u003E\u003Ca href=\u0022#fig-data-tables\u0022 class=\u0022fig-data-jump-link fig-data-jump-link-tables link-icon\u0022\u003E\u003Ci class=\u0022icon-caret-down\u0022\u003E\u003C\/i\u003E \u003Cspan class=\u0022title\u0022\u003ETables\u003C\/span\u003E\u003C\/a\u003E\u003C\/li\u003E\u003C\/ul\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cdiv class=\u0022item-list\u0022\u003E\u003Cul id=\u0022fig-frag-fig\u0022 class=\u0022fig-frag-data-list clearfix\u0022\u003E\u003Cli class=\u0022first\u0022\u003E\u003Cdiv class=\u0022element-fig-frag-data clearfix supplementary-material-caption\u0022\u003E\u003Cdiv class=\u0022highwire-markup\u0022\u003E\u003Cdiv xmlns=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022 id=\u0022content-block-markup\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cdiv class=\u0022fig-expansion \u0022 id=\u0022F1\u0022\u003E\u003Cspan class=\u0022highwire-journal-article-marker-start\u0022\u003E\u003C\/span\u003E\u003Cdiv class=\u0022highwire-figure\u0022\u003E\u003Cdiv class=\u0022fig-inline-img-wrapper\u0022\u003E\u003Cdiv class=\u0022fig-inline-img\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F1.large.jpg?width=800\u0026amp;height=600\u0026amp;carousel=1\u0022 title=\u0022Gene editing to engineer the 521\u0026#x394;T Sgcg point mutation in mice. (A) Schematic of the human SGCG gene on the left and the 521\u0026#x394;T mutation on the right. The 521\u0026#x394;T mutation is the most common LGMD 2C mutation. The single thymine deletion causes a frameshift, the expression of a premature stop codon, and loss of \u0026#x3B3;-sarcoglycan protein expression. (B) Schematic of the genetic locus and composition of the murine Sgcg gene. Both the human and the mouse genes include eight exons, with exons 2-8 encoding protein. Each coding region is 876\u0026#x2005;bp with each individual exon having the same number of coding base pairs. Exon 6 is highlighted in red. (C) Both human and murine exon 6 are 73\u0026#x2005;bp in length, and each has a stretch of five thymines in the same location (highlighted in yellow). Individual base pair mismatches are shown in red. (D) Schematic depicting the CRISPR design strategy to introduce the 521\u0026#x394;T mutation into the mouse genome. A 20\u0026#xA0;nt gRNA sequence (blue line) was used to direct Cas9 nuclease to the specific locus, defined by the presence of a -NGG protospacer adjacent motif (PAM, pink line). The nuclease created a double- strand break 3\u0026#x2005;bp upstream of the PAM (arrowhead). The addition of a 180\u0026#xA0;nt repair template promoted HDR and contained the single base pair deletion (gray lines depict homology arms).\u0022 class=\u0022highwire-fragment fragment-images colorbox-load\u0022 rel=\u0022gallery-fragment-images-363810696\u0022 data-figure-caption=\u0022\u0026lt;div class=\u0026quot;highwire-markup\u0026quot;\u0026gt;\u0026lt;div xmlns=\u0026quot;http:\/\/www.w3.org\/1999\/xhtml\u0026quot;\u0026gt;\u0026lt;strong\u0026gt;Gene editing to engineer the 521\u0026#x394;T \u0026lt;em\u0026gt;Sgcg\u0026lt;\/em\u0026gt; point mutation in mice.\u0026lt;\/strong\u0026gt; (A) Schematic of the human \u0026lt;em\u0026gt;SGCG\u0026lt;\/em\u0026gt; gene on the left and the 521\u0026#x394;T mutation on the right. The 521\u0026#x394;T mutation is the most common LGMD 2C mutation. The single thymine deletion causes a frameshift, the expression of a premature stop codon, and loss of \u0026#x3B3;-sarcoglycan protein expression. (B) Schematic of the genetic locus and composition of the murine \u0026lt;em\u0026gt;Sgcg\u0026lt;\/em\u0026gt; gene. Both the human and the mouse genes include eight exons, with exons 2-8 encoding protein. Each coding region is 876\u0026#x2005;bp with each individual exon having the same number of coding base pairs. Exon 6 is highlighted in red. (C) Both human and murine exon 6 are 73\u0026#x2005;bp in length, and each has a stretch of five thymines in the same location (highlighted in yellow). Individual base pair mismatches are shown in red. (D) Schematic depicting the CRISPR design strategy to introduce the 521\u0026#x394;T mutation into the mouse genome. A 20\u0026#xA0;nt gRNA sequence (blue line) was used to direct Cas9 nuclease to the specific locus, defined by the presence of a -NGG protospacer adjacent motif (PAM, pink line). The nuclease created a double- strand break 3\u0026#x2005;bp upstream of the PAM (arrowhead). The addition of a 180\u0026#xA0;nt repair template promoted HDR and contained the single base pair deletion (gray lines depict homology arms).\u0026lt;\/div\u0026gt;\u0026lt;\/div\u0026gt;\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003E\u003Cspan class=\u0022hw-responsive-img\u0022\u003E\u003Cimg class=\u0022highwire-fragment fragment-image lazyload\u0022 alt=\u0022Fig. 1.\u0022 src=\u0022data:image\/gif;base64,R0lGODlhAQABAIAAAAAAAP\/\/\/yH5BAEAAAAALAAAAAABAAEAAAIBRAA7\u0022 data-src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F1.medium.gif\u0022 width=\u0022440\u0022 height=\u0022375\u0022\/\u003E\u003Cnoscript\u003E\u003Cimg class=\u0022highwire-fragment fragment-image\u0022 alt=\u0022Fig. 1.\u0022 src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F1.medium.gif\u0022 width=\u0022440\u0022 height=\u0022375\u0022\/\u003E\u003C\/noscript\u003E\u003C\/span\u003E\u003C\/a\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cul class=\u0022highwire-figure-links inline\u0022\u003E\u003Cli class=\u0022download-fig first\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F1.large.jpg?download=true\u0022 class=\u0022highwire-figure-link highwire-figure-link-download\u0022 title=\u0022Download Fig. 1.\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload figure\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022new-tab\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F1.large.jpg\u0022 class=\u0022highwire-figure-link highwire-figure-link-newtab\u0022 target=\u0022_blank\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EOpen in new tab\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022download-ppt last\u0022\u003E\u003Ca href=\u0022\/highwire\/powerpoint\/1339098\u0022 class=\u0022highwire-figure-link highwire-figure-link-ppt\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload powerpoint\u003C\/a\u003E\u003C\/li\u003E\u003C\/ul\u003E\u003C\/div\u003E\u003Cdiv class=\u0022fig-caption\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cspan class=\u0022fig-label\u0022\u003EFig. 1.\u003C\/span\u003E \u003Cp id=\u0022p-11\u0022\u003E\u003Cstrong\u003EGene editing to engineer the 521\u0394T \u003Cem\u003ESgcg\u003C\/em\u003E point mutation in mice.\u003C\/strong\u003E (A) Schematic of the human \u003Cem\u003ESGCG\u003C\/em\u003E gene on the left and the 521\u0394T mutation on the right. The 521\u0394T mutation is the most common LGMD 2C mutation. The single thymine deletion causes a frameshift, the expression of a premature stop codon, and loss of \u03b3-sarcoglycan protein expression. (B) Schematic of the genetic locus and composition of the murine \u003Cem\u003ESgcg\u003C\/em\u003E gene. Both the human and the mouse genes include eight exons, with exons 2-8 encoding protein. Each coding region is 876\u2005bp with each individual exon having the same number of coding base pairs. Exon 6 is highlighted in red. (C) Both human and murine exon 6 are 73\u2005bp in length, and each has a stretch of five thymines in the same location (highlighted in yellow). Individual base pair mismatches are shown in red. (D) Schematic depicting the CRISPR design strategy to introduce the 521\u0394T mutation into the mouse genome. A 20\u00a0nt gRNA sequence (blue line) was used to direct Cas9 nuclease to the specific locus, defined by the presence of a -NGG protospacer adjacent motif (PAM, pink line). The nuclease created a double- strand break 3\u2005bp upstream of the PAM (arrowhead). The addition of a 180\u00a0nt repair template promoted HDR and contained the single base pair deletion (gray lines depict homology arms).\u003C\/p\u003E\u003Cdiv class=\u0022sb-div caption-clear\u0022\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cspan class=\u0022highwire-journal-article-marker-end\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003Cspan id=\u0022related-urls\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/li\u003E\u003Cli\u003E\u003Cdiv class=\u0022element-fig-frag-data clearfix supplementary-material-caption\u0022\u003E\u003Cdiv class=\u0022highwire-markup\u0022\u003E\u003Cdiv xmlns=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022 id=\u0022content-block-markup\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cdiv class=\u0022fig-expansion \u0022 id=\u0022F2\u0022\u003E\u003Cspan class=\u0022highwire-journal-article-marker-start\u0022\u003E\u003C\/span\u003E\u003Cdiv class=\u0022highwire-figure\u0022\u003E\u003Cdiv class=\u0022fig-inline-img-wrapper\u0022\u003E\u003Cdiv class=\u0022fig-inline-img\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F2.large.jpg?width=800\u0026amp;height=600\u0026amp;carousel=1\u0022 title=\u0022Reduction of Sgcg transcript and loss of \u0026#x3B3;-sarcoglycan protein expression in Sgcg 521\u0026#x394;T muscle. (A) RT-PCR illustrating the presence of Sgcg (715\u0026#x2005;bp) and Sgcg 521\u0026#x394;T (714\u0026#x2005;bp) transcripts. (B) Sequence chromatograms of the 5T region in the Sgcg and Sgcg 521\u0026#x394;T transcripts illustrating the deletion of one thymine in exon 6 (red box) of the Sgcg 521\u0026#x394;T transcript. (C) \u0026#x3B3;-Sarcoglycan protein (red) was readily detected in WT muscle, but not in 521\u0026#x394;T muscle. Hoechst (blue) marked nuclei. (D) Immunoblot analysis revealed the presence of \u0026#x3B3;-sarcoglycan protein in WT muscle lysates, whereas \u0026#x3B3;-sarcoglycan was not detected in 521\u0026#x394;T muscle lysates. Data are mean\u0026#xB1;s.e.m. *P\u0022 class=\u0022highwire-fragment fragment-images colorbox-load\u0022 rel=\u0022gallery-fragment-images-363810696\u0022 data-figure-caption=\u0022\u0026lt;div class=\u0026quot;highwire-markup\u0026quot;\u0026gt;\u0026lt;div xmlns=\u0026quot;http:\/\/www.w3.org\/1999\/xhtml\u0026quot;\u0026gt;\u0026lt;strong\u0026gt;Reduction of \u0026lt;em\u0026gt;Sgcg\u0026lt;\/em\u0026gt; transcript and loss of \u0026#x3B3;-sarcoglycan protein expression in \u0026lt;em\u0026gt;Sgcg\u0026lt;\/em\u0026gt; 521\u0026#x394;T muscle.\u0026lt;\/strong\u0026gt; (A) RT-PCR illustrating the presence of \u0026lt;em\u0026gt;Sgcg\u0026lt;\/em\u0026gt; (715\u0026#x2005;bp) and \u0026lt;em\u0026gt;Sgcg\u0026lt;\/em\u0026gt; 521\u0026#x394;T (714\u0026#x2005;bp) transcripts. (B) Sequence chromatograms of the 5T region in the \u0026lt;em\u0026gt;Sgcg\u0026lt;\/em\u0026gt; and \u0026lt;em\u0026gt;Sgcg\u0026lt;\/em\u0026gt; 521\u0026#x394;T transcripts illustrating the deletion of one thymine in exon 6 (red box) of the \u0026lt;em\u0026gt;Sgcg\u0026lt;\/em\u0026gt; 521\u0026#x394;T transcript. (C) \u0026#x3B3;-Sarcoglycan protein (red) was readily detected in WT muscle, but not in 521\u0026#x394;T muscle. Hoechst (blue) marked nuclei. (D) Immunoblot analysis revealed the presence of \u0026#x3B3;-sarcoglycan protein in WT muscle lysates, whereas \u0026#x3B3;-sarcoglycan was not detected in 521\u0026#x394;T muscle lysates. Data are mean\u0026#xB1;s.e.m. *\u0026lt;em\u0026gt;P\u0026lt;\/em\u0026gt;\u0026lt;0.05 (\u0026lt;em\u0026gt;t\u0026lt;\/em\u0026gt;-test). Scale bars: 50\u0026#x2005;\u0026#xB5;m.\u0026lt;\/div\u0026gt;\u0026lt;\/div\u0026gt;\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003E\u003Cspan class=\u0022hw-responsive-img\u0022\u003E\u003Cimg class=\u0022highwire-fragment fragment-image lazyload\u0022 alt=\u0022Fig. 2.\u0022 src=\u0022data:image\/gif;base64,R0lGODlhAQABAIAAAAAAAP\/\/\/yH5BAEAAAAALAAAAAABAAEAAAIBRAA7\u0022 data-src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F2.medium.gif\u0022 width=\u0022440\u0022 height=\u0022299\u0022\/\u003E\u003Cnoscript\u003E\u003Cimg class=\u0022highwire-fragment fragment-image\u0022 alt=\u0022Fig. 2.\u0022 src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F2.medium.gif\u0022 width=\u0022440\u0022 height=\u0022299\u0022\/\u003E\u003C\/noscript\u003E\u003C\/span\u003E\u003C\/a\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cul class=\u0022highwire-figure-links inline\u0022\u003E\u003Cli class=\u0022download-fig first\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F2.large.jpg?download=true\u0022 class=\u0022highwire-figure-link highwire-figure-link-download\u0022 title=\u0022Download Fig. 2.\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload figure\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022new-tab\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F2.large.jpg\u0022 class=\u0022highwire-figure-link highwire-figure-link-newtab\u0022 target=\u0022_blank\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EOpen in new tab\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022download-ppt last\u0022\u003E\u003Ca href=\u0022\/highwire\/powerpoint\/1339099\u0022 class=\u0022highwire-figure-link highwire-figure-link-ppt\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload powerpoint\u003C\/a\u003E\u003C\/li\u003E\u003C\/ul\u003E\u003C\/div\u003E\u003Cdiv class=\u0022fig-caption\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cspan class=\u0022fig-label\u0022\u003EFig. 2.\u003C\/span\u003E \u003Cp id=\u0022p-14\u0022\u003E\u003Cstrong\u003EReduction of \u003Cem\u003ESgcg\u003C\/em\u003E transcript and loss of \u03b3-sarcoglycan protein expression in \u003Cem\u003ESgcg\u003C\/em\u003E 521\u0394T muscle.\u003C\/strong\u003E (A) RT-PCR illustrating the presence of \u003Cem\u003ESgcg\u003C\/em\u003E (715\u2005bp) and \u003Cem\u003ESgcg\u003C\/em\u003E 521\u0394T (714\u2005bp) transcripts. (B) Sequence chromatograms of the 5T region in the \u003Cem\u003ESgcg\u003C\/em\u003E and \u003Cem\u003ESgcg\u003C\/em\u003E 521\u0394T transcripts illustrating the deletion of one thymine in exon 6 (red box) of the \u003Cem\u003ESgcg\u003C\/em\u003E 521\u0394T transcript. (C) \u03b3-Sarcoglycan protein (red) was readily detected in WT muscle, but not in 521\u0394T muscle. Hoechst (blue) marked nuclei. (D) Immunoblot analysis revealed the presence of \u03b3-sarcoglycan protein in WT muscle lysates, whereas \u03b3-sarcoglycan was not detected in 521\u0394T muscle lysates. Data are mean\u00b1s.e.m. *\u003Cem\u003EP\u003C\/em\u003E\u0026lt;0.05 (\u003Cem\u003Et\u003C\/em\u003E-test). Scale bars: 50\u2005\u00b5m.\u003C\/p\u003E\u003Cdiv class=\u0022sb-div caption-clear\u0022\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cspan class=\u0022highwire-journal-article-marker-end\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003Cspan id=\u0022related-urls\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/li\u003E\u003Cli\u003E\u003Cdiv class=\u0022element-fig-frag-data clearfix supplementary-material-caption\u0022\u003E\u003Cdiv class=\u0022highwire-markup\u0022\u003E\u003Cdiv xmlns=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022 id=\u0022content-block-markup\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cdiv class=\u0022fig-expansion \u0022 id=\u0022F3\u0022\u003E\u003Cspan class=\u0022highwire-journal-article-marker-start\u0022\u003E\u003C\/span\u003E\u003Cdiv class=\u0022highwire-figure\u0022\u003E\u003Cdiv class=\u0022fig-inline-img-wrapper\u0022\u003E\u003Cdiv class=\u0022fig-inline-img\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F3.large.jpg?width=800\u0026amp;height=600\u0026amp;carousel=1\u0022 title=\u0022Reduced mass and myofiber area in Sgcg 521\u0026#x394;T mice. (A) Body mass was not significantly different among genotypes. (B) Gluteus\/hamstring (GH) muscle mass normalized to tibia length was reduced in 521\u0026#x394;T mice compared with controls. (C) The total mass from muscle groups normalized to tibia length was significantly reduced in 521\u0026#x394;T mice compared with WT controls (quadriceps, gluteus\/hamstring, triceps, diaphragm, heart, tibialis anterior muscle groups combined). (D) Average myofiber cross-sectional area (CSA) was significantly smaller in 521\u0026#x394;T mice than in WT controls. (E) Percentage of myofibers with \u0026gt;1 internal nuclei was increased in 521\u0026#x394;T muscle, with 4-month-old 521\u0026#x394;T myofibers having the most internal nuclei. (F) Representative images of 521\u0026#x394;T and WT muscle. Anti-dystrophin (green) outlined myofibers. Hoechst (blue) demonstrated nuclei. Data are mean\u0026#xB1;s.e.m. *P\u0022 class=\u0022highwire-fragment fragment-images colorbox-load\u0022 rel=\u0022gallery-fragment-images-363810696\u0022 data-figure-caption=\u0022\u0026lt;div class=\u0026quot;highwire-markup\u0026quot;\u0026gt;\u0026lt;div xmlns=\u0026quot;http:\/\/www.w3.org\/1999\/xhtml\u0026quot;\u0026gt;\u0026lt;strong\u0026gt;Reduced mass and myofiber area in \u0026lt;em\u0026gt;Sgcg\u0026lt;\/em\u0026gt; 521\u0026#x394;T mice.\u0026lt;\/strong\u0026gt; (A) Body mass was not significantly different among genotypes. (B) Gluteus\/hamstring (GH) muscle mass normalized to tibia length was reduced in 521\u0026#x394;T mice compared with controls. (C) The total mass from muscle groups normalized to tibia length was significantly reduced in 521\u0026#x394;T mice compared with WT controls (quadriceps, gluteus\/hamstring, triceps, diaphragm, heart, tibialis anterior muscle groups combined). (D) Average myofiber cross-sectional area (CSA) was significantly smaller in 521\u0026#x394;T mice than in WT controls. (E) Percentage of myofibers with \u0026gt;1 internal nuclei was increased in 521\u0026#x394;T muscle, with 4-month-old 521\u0026#x394;T myofibers having the most internal nuclei. (F) Representative images of 521\u0026#x394;T and WT muscle. Anti-dystrophin (green) outlined myofibers. Hoechst (blue) demonstrated nuclei. Data are mean\u0026#xB1;s.e.m. *\u0026lt;em\u0026gt;P\u0026lt;\/em\u0026gt;\u0026lt;0.05 (one-way ANOVA with Tukey; \u0026lt;em\u0026gt;n\u0026lt;\/em\u0026gt;\u0026#x2265;5 mice per group). Scale bars: 50\u0026#x2005;\u0026#xB5;m.\u0026lt;\/div\u0026gt;\u0026lt;\/div\u0026gt;\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003E\u003Cspan class=\u0022hw-responsive-img\u0022\u003E\u003Cimg class=\u0022highwire-fragment fragment-image lazyload\u0022 alt=\u0022Fig. 3.\u0022 src=\u0022data:image\/gif;base64,R0lGODlhAQABAIAAAAAAAP\/\/\/yH5BAEAAAAALAAAAAABAAEAAAIBRAA7\u0022 data-src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F3.medium.gif\u0022 width=\u0022440\u0022 height=\u0022286\u0022\/\u003E\u003Cnoscript\u003E\u003Cimg class=\u0022highwire-fragment fragment-image\u0022 alt=\u0022Fig. 3.\u0022 src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F3.medium.gif\u0022 width=\u0022440\u0022 height=\u0022286\u0022\/\u003E\u003C\/noscript\u003E\u003C\/span\u003E\u003C\/a\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cul class=\u0022highwire-figure-links inline\u0022\u003E\u003Cli class=\u0022download-fig first\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F3.large.jpg?download=true\u0022 class=\u0022highwire-figure-link highwire-figure-link-download\u0022 title=\u0022Download Fig. 3.\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload figure\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022new-tab\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F3.large.jpg\u0022 class=\u0022highwire-figure-link highwire-figure-link-newtab\u0022 target=\u0022_blank\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EOpen in new tab\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022download-ppt last\u0022\u003E\u003Ca href=\u0022\/highwire\/powerpoint\/1339097\u0022 class=\u0022highwire-figure-link highwire-figure-link-ppt\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload powerpoint\u003C\/a\u003E\u003C\/li\u003E\u003C\/ul\u003E\u003C\/div\u003E\u003Cdiv class=\u0022fig-caption\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cspan class=\u0022fig-label\u0022\u003EFig. 3.\u003C\/span\u003E \u003Cp id=\u0022p-16\u0022\u003E\u003Cstrong\u003EReduced mass and myofiber area in \u003Cem\u003ESgcg\u003C\/em\u003E 521\u0394T mice.\u003C\/strong\u003E (A) Body mass was not significantly different among genotypes. (B) Gluteus\/hamstring (GH) muscle mass normalized to tibia length was reduced in 521\u0394T mice compared with controls. (C) The total mass from muscle groups normalized to tibia length was significantly reduced in 521\u0394T mice compared with WT controls (quadriceps, gluteus\/hamstring, triceps, diaphragm, heart, tibialis anterior muscle groups combined). (D) Average myofiber cross-sectional area (CSA) was significantly smaller in 521\u0394T mice than in WT controls. (E) Percentage of myofibers with \u0026gt;1 internal nuclei was increased in 521\u0394T muscle, with 4-month-old 521\u0394T myofibers having the most internal nuclei. (F) Representative images of 521\u0394T and WT muscle. Anti-dystrophin (green) outlined myofibers. Hoechst (blue) demonstrated nuclei. Data are mean\u00b1s.e.m. *\u003Cem\u003EP\u003C\/em\u003E\u0026lt;0.05 (one-way ANOVA with Tukey; \u003Cem\u003En\u003C\/em\u003E\u22655 mice per group). Scale bars: 50\u2005\u00b5m.\u003C\/p\u003E\u003Cdiv class=\u0022sb-div caption-clear\u0022\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cspan class=\u0022highwire-journal-article-marker-end\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003Cspan id=\u0022related-urls\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/li\u003E\u003Cli\u003E\u003Cdiv class=\u0022element-fig-frag-data clearfix supplementary-material-caption\u0022\u003E\u003Cdiv class=\u0022highwire-markup\u0022\u003E\u003Cdiv xmlns=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022 id=\u0022content-block-markup\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cdiv class=\u0022fig-expansion \u0022 id=\u0022F4\u0022\u003E\u003Cspan class=\u0022highwire-journal-article-marker-start\u0022\u003E\u003C\/span\u003E\u003Cdiv class=\u0022highwire-figure\u0022\u003E\u003Cdiv class=\u0022fig-inline-img-wrapper\u0022\u003E\u003Cdiv class=\u0022fig-inline-img\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F4.large.jpg?width=800\u0026amp;height=600\u0026amp;carousel=1\u0022 title=\u0022Marked dystrophic histopathology in Sgcg 521\u0026#x394;T mice. Tibialis anterior muscle from 521\u0026#x394;T mice displays hallmark signs of muscle disease including increased immune infiltrate, fibrosis, necrosis, calcification and internal myonuclei visualized through H\u0026amp;E and Masson\u0027s Trichrome staining. Older 521\u0026#x394;T mice (4\u0026#x2005;months) have more severe muscle pathology than younger 521\u0026#x394;T (2\u0026#x2005;months) mice. These features are absent in healthy WT muscle. Scale bars: 100\u0026#x2005;\u0026#xB5;m.\u0022 class=\u0022highwire-fragment fragment-images colorbox-load\u0022 rel=\u0022gallery-fragment-images-363810696\u0022 data-figure-caption=\u0022\u0026lt;div class=\u0026quot;highwire-markup\u0026quot;\u0026gt;\u0026lt;div xmlns=\u0026quot;http:\/\/www.w3.org\/1999\/xhtml\u0026quot;\u0026gt;\u0026lt;strong\u0026gt;Marked dystrophic histopathology in \u0026lt;em\u0026gt;Sgcg\u0026lt;\/em\u0026gt; 521\u0026#x394;T mice.\u0026lt;\/strong\u0026gt; Tibialis anterior muscle from 521\u0026#x394;T mice displays hallmark signs of muscle disease including increased immune infiltrate, fibrosis, necrosis, calcification and internal myonuclei visualized through H\u0026amp;E and Masson\u0027s Trichrome staining. Older 521\u0026#x394;T mice (4\u0026#x2005;months) have more severe muscle pathology than younger 521\u0026#x394;T (2\u0026#x2005;months) mice. These features are absent in healthy WT muscle. Scale bars: 100\u0026#x2005;\u0026#xB5;m.\u0026lt;\/div\u0026gt;\u0026lt;\/div\u0026gt;\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003E\u003Cspan class=\u0022hw-responsive-img\u0022\u003E\u003Cimg class=\u0022highwire-fragment fragment-image lazyload\u0022 alt=\u0022Fig. 4.\u0022 src=\u0022data:image\/gif;base64,R0lGODlhAQABAIAAAAAAAP\/\/\/yH5BAEAAAAALAAAAAABAAEAAAIBRAA7\u0022 data-src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F4.medium.gif\u0022 width=\u0022440\u0022 height=\u0022290\u0022\/\u003E\u003Cnoscript\u003E\u003Cimg class=\u0022highwire-fragment fragment-image\u0022 alt=\u0022Fig. 4.\u0022 src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F4.medium.gif\u0022 width=\u0022440\u0022 height=\u0022290\u0022\/\u003E\u003C\/noscript\u003E\u003C\/span\u003E\u003C\/a\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cul class=\u0022highwire-figure-links inline\u0022\u003E\u003Cli class=\u0022download-fig first\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F4.large.jpg?download=true\u0022 class=\u0022highwire-figure-link highwire-figure-link-download\u0022 title=\u0022Download Fig. 4.\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload figure\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022new-tab\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F4.large.jpg\u0022 class=\u0022highwire-figure-link highwire-figure-link-newtab\u0022 target=\u0022_blank\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EOpen in new tab\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022download-ppt last\u0022\u003E\u003Ca href=\u0022\/highwire\/powerpoint\/1339094\u0022 class=\u0022highwire-figure-link highwire-figure-link-ppt\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload powerpoint\u003C\/a\u003E\u003C\/li\u003E\u003C\/ul\u003E\u003C\/div\u003E\u003Cdiv class=\u0022fig-caption\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cspan class=\u0022fig-label\u0022\u003EFig. 4.\u003C\/span\u003E \u003Cp id=\u0022p-18\u0022\u003E\u003Cstrong\u003EMarked dystrophic histopathology in \u003Cem\u003ESgcg\u003C\/em\u003E 521\u0394T mice.\u003C\/strong\u003E Tibialis anterior muscle from 521\u0394T mice displays hallmark signs of muscle disease including increased immune infiltrate, fibrosis, necrosis, calcification and internal myonuclei visualized through H\u0026amp;E and Masson\u0027s Trichrome staining. Older 521\u0394T mice (4\u2005months) have more severe muscle pathology than younger 521\u0394T (2\u2005months) mice. These features are absent in healthy WT muscle. Scale bars: 100\u2005\u00b5m.\u003C\/p\u003E\u003Cdiv class=\u0022sb-div caption-clear\u0022\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cspan class=\u0022highwire-journal-article-marker-end\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003Cspan id=\u0022related-urls\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/li\u003E\u003Cli\u003E\u003Cdiv class=\u0022element-fig-frag-data clearfix supplementary-material-caption\u0022\u003E\u003Cdiv class=\u0022highwire-markup\u0022\u003E\u003Cdiv xmlns=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022 id=\u0022content-block-markup\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cdiv class=\u0022fig-expansion \u0022 id=\u0022F5\u0022\u003E\u003Cspan class=\u0022highwire-journal-article-marker-start\u0022\u003E\u003C\/span\u003E\u003Cdiv class=\u0022highwire-figure\u0022\u003E\u003Cdiv class=\u0022fig-inline-img-wrapper\u0022\u003E\u003Cdiv class=\u0022fig-inline-img\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F5.large.jpg?width=800\u0026amp;height=600\u0026amp;carousel=1\u0022 title=\u0022Increased immune infiltrate and fiber type switching in Sgcg 521\u0026#x394;T muscle. (A) Anti-F4\/80 immunofluorescence staining (green) of gastrocnemius muscle from 4-month-old mice revealed an increase in the average number of F4\/80+ macrophages per field in 521\u0026#x394;T muscle compared with WT controls. Hoechst (blue) labels nuclei. Arrows indicate F4\/80+ macrophages. (B) Anti-Ly6 immunofluorescence staining (green) of gastrocnemius muscle from 4-month-old mice revealed an increase in the average number of Ly6+ monocytes\/neutrophils in 521\u0026#x394;T muscle per field. Hoechst (blue) labels nuclei. Arrows indicate Ly6+ monocytes\/neutrophils. (C) Representative images and quantification of the fiber type composition of the gastrocnemius muscle from 4-month-old mice, evaluated through immunostaining against the different myosin isoforms. 521\u0026#x394;T muscle had a significant reduction in the percentage of Type 2B myofibers corresponding with a trend towards an increased number of mixed myofibers (P=0.07). Type 2B fibers (red), Type 2A (green), Type 1 (blue), mixed (composed of Type 2B\/X, 2X, 2A\/X). Data are mean\u0026#xB1;s.e.m. *P\u0022 class=\u0022highwire-fragment fragment-images colorbox-load\u0022 rel=\u0022gallery-fragment-images-363810696\u0022 data-figure-caption=\u0022\u0026lt;div class=\u0026quot;highwire-markup\u0026quot;\u0026gt;\u0026lt;div xmlns=\u0026quot;http:\/\/www.w3.org\/1999\/xhtml\u0026quot;\u0026gt;\u0026lt;strong\u0026gt;Increased immune infiltrate and fiber type switching in \u0026lt;em\u0026gt;Sgcg\u0026lt;\/em\u0026gt; 521\u0026#x394;T muscle.\u0026lt;\/strong\u0026gt; (A) Anti-F4\/80 immunofluorescence staining (green) of gastrocnemius muscle from 4-month-old mice revealed an increase in the average number of F4\/80+ macrophages per field in 521\u0026#x394;T muscle compared with WT controls. Hoechst (blue) labels nuclei. Arrows indicate F4\/80+ macrophages. (B) Anti-Ly6 immunofluorescence staining (green) of gastrocnemius muscle from 4-month-old mice revealed an increase in the average number of Ly6+ monocytes\/neutrophils in 521\u0026#x394;T muscle per field. Hoechst (blue) labels nuclei. Arrows indicate Ly6+ monocytes\/neutrophils. (C) Representative images and quantification of the fiber type composition of the gastrocnemius muscle from 4-month-old mice, evaluated through immunostaining against the different myosin isoforms. 521\u0026#x394;T muscle had a significant reduction in the percentage of Type 2B myofibers corresponding with a trend towards an increased number of mixed myofibers (\u0026lt;em\u0026gt;P\u0026lt;\/em\u0026gt;=0.07). Type 2B fibers (red), Type 2A (green), Type 1 (blue), mixed (composed of Type 2B\/X, 2X, 2A\/X). Data are mean\u0026#xB1;s.e.m. *\u0026lt;em\u0026gt;P\u0026lt;\/em\u0026gt;\u0026lt;0.05 (\u0026lt;em\u0026gt;t\u0026lt;\/em\u0026gt;-test in A,B; two-way ANOVA with Bonferroni\u0027s correction in C; \u0026lt;em\u0026gt;n\u0026lt;\/em\u0026gt;=3 mice per group). Scale bars: 50\u0026#x2005;\u0026#xB5;m (A,B); 1 mm (C).\u0026lt;\/div\u0026gt;\u0026lt;\/div\u0026gt;\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003E\u003Cspan class=\u0022hw-responsive-img\u0022\u003E\u003Cimg class=\u0022highwire-fragment fragment-image lazyload\u0022 alt=\u0022Fig. 5.\u0022 src=\u0022data:image\/gif;base64,R0lGODlhAQABAIAAAAAAAP\/\/\/yH5BAEAAAAALAAAAAABAAEAAAIBRAA7\u0022 data-src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F5.medium.gif\u0022 width=\u0022440\u0022 height=\u0022405\u0022\/\u003E\u003Cnoscript\u003E\u003Cimg class=\u0022highwire-fragment fragment-image\u0022 alt=\u0022Fig. 5.\u0022 src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F5.medium.gif\u0022 width=\u0022440\u0022 height=\u0022405\u0022\/\u003E\u003C\/noscript\u003E\u003C\/span\u003E\u003C\/a\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cul class=\u0022highwire-figure-links inline\u0022\u003E\u003Cli class=\u0022download-fig first\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F5.large.jpg?download=true\u0022 class=\u0022highwire-figure-link highwire-figure-link-download\u0022 title=\u0022Download Fig. 5.\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload figure\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022new-tab\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F5.large.jpg\u0022 class=\u0022highwire-figure-link highwire-figure-link-newtab\u0022 target=\u0022_blank\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EOpen in new tab\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022download-ppt last\u0022\u003E\u003Ca href=\u0022\/highwire\/powerpoint\/1339100\u0022 class=\u0022highwire-figure-link highwire-figure-link-ppt\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload powerpoint\u003C\/a\u003E\u003C\/li\u003E\u003C\/ul\u003E\u003C\/div\u003E\u003Cdiv class=\u0022fig-caption\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cspan class=\u0022fig-label\u0022\u003EFig. 5.\u003C\/span\u003E \u003Cp id=\u0022p-19\u0022\u003E\u003Cstrong\u003EIncreased immune infiltrate and fiber type switching in \u003Cem\u003ESgcg\u003C\/em\u003E 521\u0394T muscle.\u003C\/strong\u003E (A) Anti-F4\/80 immunofluorescence staining (green) of gastrocnemius muscle from 4-month-old mice revealed an increase in the average number of F4\/80+ macrophages per field in 521\u0394T muscle compared with WT controls. Hoechst (blue) labels nuclei. Arrows indicate F4\/80+ macrophages. (B) Anti-Ly6 immunofluorescence staining (green) of gastrocnemius muscle from 4-month-old mice revealed an increase in the average number of Ly6+ monocytes\/neutrophils in 521\u0394T muscle per field. Hoechst (blue) labels nuclei. Arrows indicate Ly6+ monocytes\/neutrophils. (C) Representative images and quantification of the fiber type composition of the gastrocnemius muscle from 4-month-old mice, evaluated through immunostaining against the different myosin isoforms. 521\u0394T muscle had a significant reduction in the percentage of Type 2B myofibers corresponding with a trend towards an increased number of mixed myofibers (\u003Cem\u003EP\u003C\/em\u003E=0.07). Type 2B fibers (red), Type 2A (green), Type 1 (blue), mixed (composed of Type 2B\/X, 2X, 2A\/X). Data are mean\u00b1s.e.m. *\u003Cem\u003EP\u003C\/em\u003E\u0026lt;0.05 (\u003Cem\u003Et\u003C\/em\u003E-test in A,B; two-way ANOVA with Bonferroni\u0027s correction in C; \u003Cem\u003En\u003C\/em\u003E=3 mice per group). Scale bars: 50\u2005\u00b5m (A,B); 1 mm (C).\u003C\/p\u003E\u003Cdiv class=\u0022sb-div caption-clear\u0022\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cspan class=\u0022highwire-journal-article-marker-end\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003Cspan id=\u0022related-urls\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/li\u003E\u003Cli\u003E\u003Cdiv class=\u0022element-fig-frag-data clearfix supplementary-material-caption\u0022\u003E\u003Cdiv class=\u0022highwire-markup\u0022\u003E\u003Cdiv xmlns=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022 id=\u0022content-block-markup\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cdiv class=\u0022fig-expansion \u0022 id=\u0022F6\u0022\u003E\u003Cspan class=\u0022highwire-journal-article-marker-start\u0022\u003E\u003C\/span\u003E\u003Cdiv class=\u0022highwire-figure\u0022\u003E\u003Cdiv class=\u0022fig-inline-img-wrapper\u0022\u003E\u003Cdiv class=\u0022fig-inline-img\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F6.large.jpg?width=800\u0026amp;height=600\u0026amp;carousel=1\u0022 title=\u0022Increased sarcolemmal membrane leak and fibrosis in Sgcg 521\u0026#x394;T mice. (A) Mice were injected with Evans Blue dye. Gross imaging shows increased fibrosis (white; arrow) and increased dye uptake (blue streaks; arrowhead) in 521\u0026#x394;T mice compared with WT controls. (B) Increased dye uptake was seen in 2-month-old 521\u0026#x394;T muscle by spectrophotometric analysis compared with WT. This increase trended towards a reduction by 4 months of age in 521\u0026#x394;T muscle (compared with 2-month-old 521\u0026#x394;T muscle; P=0.07). Combined dye values from abdominal, quadriceps, gluteus\/hamstring, triceps, diaphragm, heart and gastroc\/soleus muscles. (C) Serum CK was elevated in 521\u0026#x394;T mice compared with controls, with 4-month-old 521\u0026#x394;T serum CK exceeding that of 2-month-old mutant animals. (D) Fibrosis was measured as HOP content. HOP content was elevated in 521\u0026#x394;T muscle, with more seen in 4-month-old 521\u0026#x394;T muscle than at 2\u0026#x2005;months of age. Combined values from abdominal, gastroc\/soleus, diaphragm and triceps. Data are mean\u0026#xB1;s.e.m. *P\u0022 class=\u0022highwire-fragment fragment-images colorbox-load\u0022 rel=\u0022gallery-fragment-images-363810696\u0022 data-figure-caption=\u0022\u0026lt;div class=\u0026quot;highwire-markup\u0026quot;\u0026gt;\u0026lt;div xmlns=\u0026quot;http:\/\/www.w3.org\/1999\/xhtml\u0026quot;\u0026gt;\u0026lt;strong\u0026gt;Increased sarcolemmal membrane leak and fibrosis in \u0026lt;em\u0026gt;Sgcg\u0026lt;\/em\u0026gt; 521\u0026#x394;T mice.\u0026lt;\/strong\u0026gt; (A) Mice were injected with Evans Blue dye. Gross imaging shows increased fibrosis (white; arrow) and increased dye uptake (blue streaks; arrowhead) in 521\u0026#x394;T mice compared with WT controls. (B) Increased dye uptake was seen in 2-month-old 521\u0026#x394;T muscle by spectrophotometric analysis compared with WT. This increase trended towards a reduction by 4 months of age in 521\u0026#x394;T muscle (compared with 2-month-old 521\u0026#x394;T muscle; \u0026lt;em\u0026gt;P\u0026lt;\/em\u0026gt;=0.07). Combined dye values from abdominal, quadriceps, gluteus\/hamstring, triceps, diaphragm, heart and gastroc\/soleus muscles. (C) Serum CK was elevated in 521\u0026#x394;T mice compared with controls, with 4-month-old 521\u0026#x394;T serum CK exceeding that of 2-month-old mutant animals. (D) Fibrosis was measured as HOP content. HOP content was elevated in 521\u0026#x394;T muscle, with more seen in 4-month-old 521\u0026#x394;T muscle than at 2\u0026#x2005;months of age. Combined values from abdominal, gastroc\/soleus, diaphragm and triceps. Data are mean\u0026#xB1;s.e.m. *\u0026lt;em\u0026gt;P\u0026lt;\/em\u0026gt;\u0026lt;0.05 (one-way ANOVA with Tukey; \u0026lt;em\u0026gt;n\u0026lt;\/em\u0026gt;\u0026#x2265;4 mice per group). Scale bars: 5 mm.\u0026lt;\/div\u0026gt;\u0026lt;\/div\u0026gt;\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003E\u003Cspan class=\u0022hw-responsive-img\u0022\u003E\u003Cimg class=\u0022highwire-fragment fragment-image lazyload\u0022 alt=\u0022Fig. 6.\u0022 src=\u0022data:image\/gif;base64,R0lGODlhAQABAIAAAAAAAP\/\/\/yH5BAEAAAAALAAAAAABAAEAAAIBRAA7\u0022 data-src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F6.medium.gif\u0022 width=\u0022440\u0022 height=\u0022369\u0022\/\u003E\u003Cnoscript\u003E\u003Cimg class=\u0022highwire-fragment fragment-image\u0022 alt=\u0022Fig. 6.\u0022 src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F6.medium.gif\u0022 width=\u0022440\u0022 height=\u0022369\u0022\/\u003E\u003C\/noscript\u003E\u003C\/span\u003E\u003C\/a\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cul class=\u0022highwire-figure-links inline\u0022\u003E\u003Cli class=\u0022download-fig first\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F6.large.jpg?download=true\u0022 class=\u0022highwire-figure-link highwire-figure-link-download\u0022 title=\u0022Download Fig. 6.\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload figure\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022new-tab\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F6.large.jpg\u0022 class=\u0022highwire-figure-link highwire-figure-link-newtab\u0022 target=\u0022_blank\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EOpen in new tab\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022download-ppt last\u0022\u003E\u003Ca href=\u0022\/highwire\/powerpoint\/1339095\u0022 class=\u0022highwire-figure-link highwire-figure-link-ppt\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload powerpoint\u003C\/a\u003E\u003C\/li\u003E\u003C\/ul\u003E\u003C\/div\u003E\u003Cdiv class=\u0022fig-caption\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cspan class=\u0022fig-label\u0022\u003EFig. 6.\u003C\/span\u003E \u003Cp id=\u0022p-21\u0022\u003E\u003Cstrong\u003EIncreased sarcolemmal membrane leak and fibrosis in \u003Cem\u003ESgcg\u003C\/em\u003E 521\u0394T mice.\u003C\/strong\u003E (A) Mice were injected with Evans Blue dye. Gross imaging shows increased fibrosis (white; arrow) and increased dye uptake (blue streaks; arrowhead) in 521\u0394T mice compared with WT controls. (B) Increased dye uptake was seen in 2-month-old 521\u0394T muscle by spectrophotometric analysis compared with WT. This increase trended towards a reduction by 4 months of age in 521\u0394T muscle (compared with 2-month-old 521\u0394T muscle; \u003Cem\u003EP\u003C\/em\u003E=0.07). Combined dye values from abdominal, quadriceps, gluteus\/hamstring, triceps, diaphragm, heart and gastroc\/soleus muscles. (C) Serum CK was elevated in 521\u0394T mice compared with controls, with 4-month-old 521\u0394T serum CK exceeding that of 2-month-old mutant animals. (D) Fibrosis was measured as HOP content. HOP content was elevated in 521\u0394T muscle, with more seen in 4-month-old 521\u0394T muscle than at 2\u2005months of age. Combined values from abdominal, gastroc\/soleus, diaphragm and triceps. Data are mean\u00b1s.e.m. *\u003Cem\u003EP\u003C\/em\u003E\u0026lt;0.05 (one-way ANOVA with Tukey; \u003Cem\u003En\u003C\/em\u003E\u22654 mice per group). Scale bars: 5 mm.\u003C\/p\u003E\u003Cdiv class=\u0022sb-div caption-clear\u0022\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cspan class=\u0022highwire-journal-article-marker-end\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003Cspan id=\u0022related-urls\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/li\u003E\u003Cli\u003E\u003Cdiv class=\u0022element-fig-frag-data clearfix supplementary-material-caption\u0022\u003E\u003Cdiv class=\u0022highwire-markup\u0022\u003E\u003Cdiv xmlns=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022 id=\u0022content-block-markup\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cdiv class=\u0022fig-expansion \u0022 id=\u0022F7\u0022\u003E\u003Cspan class=\u0022highwire-journal-article-marker-start\u0022\u003E\u003C\/span\u003E\u003Cdiv class=\u0022highwire-figure\u0022\u003E\u003Cdiv class=\u0022fig-inline-img-wrapper\u0022\u003E\u003Cdiv class=\u0022fig-inline-img\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F7.large.jpg?width=800\u0026amp;height=600\u0026amp;carousel=1\u0022 title=\u0022Reduced muscle function in Sgcg 521\u0026#x394;T mice. (A) Average forelimb grip strength was decreased in 521\u0026#x394;T mice. (B) Increased maximum tetanic force was decreased in 521\u0026#x394;T mice at 4\u0026#x2005;months of age, measured through in situ force analysis of the tibialis anterior muscle. (C) Specific force was also decreased in 521\u0026#x394;T muscle compared with WT control (tibialis anterior, 4-month-old mice). (D) Respiratory muscle activity was also impaired. Enhanced pause (Penh) was elevated, consistent with abnormal breathing in 521\u0026#x394;T mice at 4\u0026#x2005;months, compared with WT controls. Data are mean\u0026#xB1;s.e.m. *P\u0022 class=\u0022highwire-fragment fragment-images colorbox-load\u0022 rel=\u0022gallery-fragment-images-363810696\u0022 data-figure-caption=\u0022\u0026lt;div class=\u0026quot;highwire-markup\u0026quot;\u0026gt;\u0026lt;div xmlns=\u0026quot;http:\/\/www.w3.org\/1999\/xhtml\u0026quot;\u0026gt;\u0026lt;strong\u0026gt;Reduced muscle function in \u0026lt;em\u0026gt;Sgcg\u0026lt;\/em\u0026gt; 521\u0026#x394;T mice.\u0026lt;\/strong\u0026gt; (A) Average forelimb grip strength was decreased in 521\u0026#x394;T mice. (B) Increased maximum tetanic force was decreased in 521\u0026#x394;T mice at 4\u0026#x2005;months of age, measured through \u0026lt;em\u0026gt;in situ\u0026lt;\/em\u0026gt; force analysis of the tibialis anterior muscle. (C) Specific force was also decreased in 521\u0026#x394;T muscle compared with WT control (tibialis anterior, 4-month-old mice). (D) Respiratory muscle activity was also impaired. Enhanced pause (Penh) was elevated, consistent with abnormal breathing in 521\u0026#x394;T mice at 4\u0026#x2005;months, compared with WT controls. Data are mean\u0026#xB1;s.e.m. *\u0026lt;em\u0026gt;P\u0026lt;\/em\u0026gt;\u0026lt;0.05 (one-way ANOVA with Tukey in A; \u0026lt;em\u0026gt;t\u0026lt;\/em\u0026gt;-test in B,C,D; \u0026lt;em\u0026gt;n\u0026lt;\/em\u0026gt;\u0026#x2265;5 mice per group).\u0026lt;\/div\u0026gt;\u0026lt;\/div\u0026gt;\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003E\u003Cspan class=\u0022hw-responsive-img\u0022\u003E\u003Cimg class=\u0022highwire-fragment fragment-image lazyload\u0022 alt=\u0022Fig. 7.\u0022 src=\u0022data:image\/gif;base64,R0lGODlhAQABAIAAAAAAAP\/\/\/yH5BAEAAAAALAAAAAABAAEAAAIBRAA7\u0022 data-src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F7.medium.gif\u0022 width=\u0022440\u0022 height=\u0022149\u0022\/\u003E\u003Cnoscript\u003E\u003Cimg class=\u0022highwire-fragment fragment-image\u0022 alt=\u0022Fig. 7.\u0022 src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F7.medium.gif\u0022 width=\u0022440\u0022 height=\u0022149\u0022\/\u003E\u003C\/noscript\u003E\u003C\/span\u003E\u003C\/a\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cul class=\u0022highwire-figure-links inline\u0022\u003E\u003Cli class=\u0022download-fig first\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F7.large.jpg?download=true\u0022 class=\u0022highwire-figure-link highwire-figure-link-download\u0022 title=\u0022Download Fig. 7.\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload figure\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022new-tab\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F7.large.jpg\u0022 class=\u0022highwire-figure-link highwire-figure-link-newtab\u0022 target=\u0022_blank\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EOpen in new tab\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022download-ppt last\u0022\u003E\u003Ca href=\u0022\/highwire\/powerpoint\/1339096\u0022 class=\u0022highwire-figure-link highwire-figure-link-ppt\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload powerpoint\u003C\/a\u003E\u003C\/li\u003E\u003C\/ul\u003E\u003C\/div\u003E\u003Cdiv class=\u0022fig-caption\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cspan class=\u0022fig-label\u0022\u003EFig. 7.\u003C\/span\u003E \u003Cp id=\u0022p-24\u0022\u003E\u003Cstrong\u003EReduced muscle function in \u003Cem\u003ESgcg\u003C\/em\u003E 521\u0394T mice.\u003C\/strong\u003E (A) Average forelimb grip strength was decreased in 521\u0394T mice. (B) Increased maximum tetanic force was decreased in 521\u0394T mice at 4\u2005months of age, measured through \u003Cem\u003Ein situ\u003C\/em\u003E force analysis of the tibialis anterior muscle. (C) Specific force was also decreased in 521\u0394T muscle compared with WT control (tibialis anterior, 4-month-old mice). (D) Respiratory muscle activity was also impaired. Enhanced pause (Penh) was elevated, consistent with abnormal breathing in 521\u0394T mice at 4\u2005months, compared with WT controls. Data are mean\u00b1s.e.m. *\u003Cem\u003EP\u003C\/em\u003E\u0026lt;0.05 (one-way ANOVA with Tukey in A; \u003Cem\u003Et\u003C\/em\u003E-test in B,C,D; \u003Cem\u003En\u003C\/em\u003E\u22655 mice per group).\u003C\/p\u003E\u003Cdiv class=\u0022sb-div caption-clear\u0022\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cspan class=\u0022highwire-journal-article-marker-end\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003Cspan id=\u0022related-urls\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/li\u003E\u003Cli class=\u0022last\u0022\u003E\u003Cdiv class=\u0022element-fig-frag-data clearfix supplementary-material-caption\u0022\u003E\u003Cdiv class=\u0022highwire-markup\u0022\u003E\u003Cdiv xmlns=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022 id=\u0022content-block-markup\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cdiv class=\u0022fig-expansion \u0022 id=\u0022F8\u0022\u003E\u003Cspan class=\u0022highwire-journal-article-marker-start\u0022\u003E\u003C\/span\u003E\u003Cdiv class=\u0022highwire-figure\u0022\u003E\u003Cdiv class=\u0022fig-inline-img-wrapper\u0022\u003E\u003Cdiv class=\u0022fig-inline-img\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F8.large.jpg?width=800\u0026amp;height=600\u0026amp;carousel=1\u0022 title=\u0022In vivo exon skipping in Sgcg 521\u0026#x394;T muscle via IM injection of antisense oligonucleotides. (A) Schematic showing the correction of the 521\u0026#x394;T frameshift mutation (red triangle) with a multi-AON exon-skipping strategy targeting exons 4, 5, 6 and 7 (yellow boxes). This strategy corrects the reading frame by generating a transcript encoding Mini-Gamma, comprising exons 2, 3 and 8. The arrows depict the location of the PCR primers, and the expected amplicon size is designated. (B) Design and results from in vivo study 1, in which analysis was conducted 3\u0026#x2005;days after IM injections of AONs into the tibialis anterior (TA) and gastrocnemius\/soleus (Gas) muscle. Muscles were injected with either the four-AON cocktail or vehicle control (PBS; ctrl). Gel electrophoresis of RT-PCR products from treated muscles demonstrated the 521\u0026#x394;T transcript in PBS-treated muscle (black arrow), as well as a dose-dependent generation of the predicted transcript encoding Mini-Gamma in muscle treated with the Mini-Gamma vivo-PMO cocktail at a low and mid dose (red arrow). (C) Similar treatment of two additional mice for 7\u0026#x2005;days demonstrated similar results but suggested the need for higher AON dosing. (D) Design and results from in vivo study 2. RT-PCR results indicate robust generation of the transcript encoding Mini-Gamma after two injections, two weeks apart at both the mid and high doses (red arrow). A concomitant reduction in the unskipped 521\u0026#x394;T was also seen with both these exposures (black arrow). Mini-Gamma expression was evident 4\u0026#x2005;weeks after a single mid- or high-dose injection. However, expression was diminished compared with the two-injection treatment, and there were more intermediate products. (E) Gel extraction and subsequent Sanger sequencing confirmed that this product was in-frame Mini-Gamma.\u0022 class=\u0022highwire-fragment fragment-images colorbox-load\u0022 rel=\u0022gallery-fragment-images-363810696\u0022 data-figure-caption=\u0022\u0026lt;div class=\u0026quot;highwire-markup\u0026quot;\u0026gt;\u0026lt;div xmlns=\u0026quot;http:\/\/www.w3.org\/1999\/xhtml\u0026quot;\u0026gt;\u0026lt;strong\u0026gt;\u0026lt;em\u0026gt;In vivo\u0026lt;\/em\u0026gt; exon skipping in \u0026lt;em\u0026gt;Sgcg\u0026lt;\/em\u0026gt; 521\u0026#x394;T muscle via IM injection of antisense oligonucleotides.\u0026lt;\/strong\u0026gt; (A) Schematic showing the correction of the 521\u0026#x394;T frameshift mutation (red triangle) with a multi-AON exon-skipping strategy targeting exons 4, 5, 6 and 7 (yellow boxes). This strategy corrects the reading frame by generating a transcript encoding Mini-Gamma, comprising exons 2, 3 and 8. The arrows depict the location of the PCR primers, and the expected amplicon size is designated. (B) Design and results from \u0026lt;em\u0026gt;in vivo\u0026lt;\/em\u0026gt; study 1, in which analysis was conducted 3\u0026#x2005;days after IM injections of AONs into the tibialis anterior (TA) and gastrocnemius\/soleus (Gas) muscle. Muscles were injected with either the four-AON cocktail or vehicle control (PBS; ctrl). Gel electrophoresis of RT-PCR products from treated muscles demonstrated the 521\u0026#x394;T transcript in PBS-treated muscle (black arrow), as well as a dose-dependent generation of the predicted transcript encoding Mini-Gamma in muscle treated with the Mini-Gamma vivo-PMO cocktail at a low and mid dose (red arrow). (C) Similar treatment of two additional mice for 7\u0026#x2005;days demonstrated similar results but suggested the need for higher AON dosing. (D) Design and results from \u0026lt;em\u0026gt;in vivo\u0026lt;\/em\u0026gt; study 2. RT-PCR results indicate robust generation of the transcript encoding Mini-Gamma after two injections, two weeks apart at both the mid and high doses (red arrow). A concomitant reduction in the unskipped 521\u0026#x394;T was also seen with both these exposures (black arrow). Mini-Gamma expression was evident 4\u0026#x2005;weeks after a single mid- or high-dose injection. However, expression was diminished compared with the two-injection treatment, and there were more intermediate products. (E) Gel extraction and subsequent Sanger sequencing confirmed that this product was in-frame Mini-Gamma.\u0026lt;\/div\u0026gt;\u0026lt;\/div\u0026gt;\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003E\u003Cspan class=\u0022hw-responsive-img\u0022\u003E\u003Cimg class=\u0022highwire-fragment fragment-image lazyload\u0022 alt=\u0022Fig. 8.\u0022 src=\u0022data:image\/gif;base64,R0lGODlhAQABAIAAAAAAAP\/\/\/yH5BAEAAAAALAAAAAABAAEAAAIBRAA7\u0022 data-src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F8.medium.gif\u0022 width=\u0022440\u0022 height=\u0022285\u0022\/\u003E\u003Cnoscript\u003E\u003Cimg class=\u0022highwire-fragment fragment-image\u0022 alt=\u0022Fig. 8.\u0022 src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F8.medium.gif\u0022 width=\u0022440\u0022 height=\u0022285\u0022\/\u003E\u003C\/noscript\u003E\u003C\/span\u003E\u003C\/a\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cul class=\u0022highwire-figure-links inline\u0022\u003E\u003Cli class=\u0022download-fig first\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F8.large.jpg?download=true\u0022 class=\u0022highwire-figure-link highwire-figure-link-download\u0022 title=\u0022Download Fig. 8.\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload figure\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022new-tab\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/F8.large.jpg\u0022 class=\u0022highwire-figure-link highwire-figure-link-newtab\u0022 target=\u0022_blank\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EOpen in new tab\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022download-ppt last\u0022\u003E\u003Ca href=\u0022\/highwire\/powerpoint\/1339093\u0022 class=\u0022highwire-figure-link highwire-figure-link-ppt\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload powerpoint\u003C\/a\u003E\u003C\/li\u003E\u003C\/ul\u003E\u003C\/div\u003E\u003Cdiv class=\u0022fig-caption\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cspan class=\u0022fig-label\u0022\u003EFig. 8.\u003C\/span\u003E \u003Cp id=\u0022p-27\u0022\u003E\u003Cstrong\u003E\u003Cem\u003EIn vivo\u003C\/em\u003E exon skipping in \u003Cem\u003ESgcg\u003C\/em\u003E 521\u0394T muscle via IM injection of antisense oligonucleotides.\u003C\/strong\u003E (A) Schematic showing the correction of the 521\u0394T frameshift mutation (red triangle) with a multi-AON exon-skipping strategy targeting exons 4, 5, 6 and 7 (yellow boxes). This strategy corrects the reading frame by generating a transcript encoding Mini-Gamma, comprising exons 2, 3 and 8. The arrows depict the location of the PCR primers, and the expected amplicon size is designated. (B) Design and results from \u003Cem\u003Ein vivo\u003C\/em\u003E study 1, in which analysis was conducted 3\u2005days after IM injections of AONs into the tibialis anterior (TA) and gastrocnemius\/soleus (Gas) muscle. Muscles were injected with either the four-AON cocktail or vehicle control (PBS; ctrl). Gel electrophoresis of RT-PCR products from treated muscles demonstrated the 521\u0394T transcript in PBS-treated muscle (black arrow), as well as a dose-dependent generation of the predicted transcript encoding Mini-Gamma in muscle treated with the Mini-Gamma vivo-PMO cocktail at a low and mid dose (red arrow). (C) Similar treatment of two additional mice for 7\u2005days demonstrated similar results but suggested the need for higher AON dosing. (D) Design and results from \u003Cem\u003Ein vivo\u003C\/em\u003E study 2. RT-PCR results indicate robust generation of the transcript encoding Mini-Gamma after two injections, two weeks apart at both the mid and high doses (red arrow). A concomitant reduction in the unskipped 521\u0394T was also seen with both these exposures (black arrow). Mini-Gamma expression was evident 4\u2005weeks after a single mid- or high-dose injection. However, expression was diminished compared with the two-injection treatment, and there were more intermediate products. (E) Gel extraction and subsequent Sanger sequencing confirmed that this product was in-frame Mini-Gamma.\u003C\/p\u003E\u003Cdiv class=\u0022sb-div caption-clear\u0022\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cspan class=\u0022highwire-journal-article-marker-end\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003Cspan id=\u0022related-urls\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/li\u003E\u003C\/ul\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cdiv id=\u0022fig-data-tables\u0022 class=\u0022group frag-tables\u0022\u003E\u003Cdiv class=\u0022fig-data-title-jump clearfix\u0022\u003E\u003Ch3 id=\u0022fig-frag-data-title\u0022 class=\u0022fig-data-group-title\u0022\u003ETables\u003C\/h3\u003E\u003Cdiv class=\u0022fig-data-jump-links\u0022\u003E\u003Cul class=\u0022fig-data-jump-links-list links inline\u0022\u003E\u003Cli class=\u0022figures first last\u0022\u003E\u003Ca href=\u0022#fig-data-figures\u0022 class=\u0022fig-data-jump-link fig-data-jump-link-figures link-icon\u0022\u003E\u003Ci class=\u0022icon-caret-up\u0022\u003E\u003C\/i\u003E \u003Cspan class=\u0022title\u0022\u003EFigures\u003C\/span\u003E\u003C\/a\u003E\u003C\/li\u003E\u003C\/ul\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cdiv class=\u0022item-list\u0022\u003E\u003Cul id=\u0022fig-frag-fig\u0022 class=\u0022fig-frag-data-list clearfix\u0022\u003E\u003Cli class=\u0022first last\u0022\u003E\u003Cdiv class=\u0022element-fig-frag-data clearfix supplementary-material-caption\u0022\u003E\u003Cdiv class=\u0022highwire-markup\u0022\u003E\u003Cdiv xmlns=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022 id=\u0022content-block-markup\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cdiv class=\u0022table-expansion \u0022 id=\u0022T1\u0022\u003E\u003Cspan class=\u0022highwire-journal-article-marker-start\u0022\u003E\u003C\/span\u003E\u003Cdiv class=\u0022highwire-figure\u0022\u003E\u003Cdiv class=\u0022fig-inline-img-wrapper\u0022\u003E\u003Cdiv class=\u0022fig-inline-img\u0022\u003E\u003Cspan class=\u0022hw-responsive-img\u0022\u003E\u003Cimg class=\u0022highwire-fragment fragment-table lazyload\u0022 alt=\u0022Table\u0026#xA0;1.\u0022 src=\u0022data:image\/gif;base64,R0lGODlhAQABAIAAAAAAAP\/\/\/yH5BAEAAAAALAAAAAABAAEAAAIBRAA7\u0022 data-src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/T1.medium.gif\u0022 width=\u0022420\u0022 height=\u0022130\u0022\/\u003E\u003Cnoscript\u003E\u003Cimg class=\u0022highwire-fragment fragment-table\u0022 alt=\u0022Table\u0026#xA0;1.\u0022 src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/2\/dmm040832\/T1.medium.gif\u0022 width=\u0022420\u0022 height=\u0022130\u0022\/\u003E\u003C\/noscript\u003E\u003C\/span\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cspan class=\u0022highwire-journal-article-marker-end\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003Cspan id=\u0022related-urls\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/li\u003E\u003C\/ul\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/div\u003E \u003C\/div\u003E\n\n \n \u003C\/div\u003E\n\u003Cdiv class=\u0022panel-separator\u0022\u003E\u003C\/div\u003E\u003Cdiv class=\u0022panel-pane pane-earthchem\u0022 \u003E\n \n \n \n \u003Cdiv class=\u0022pane-content\u0022\u003E\n \u003Ca href=\u0022http:\/\/ecp.iedadata.org\/doidata\/10.1242\/dmm.040832\u0022 class=\u0022\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003E\u003Cimg src=\u0022http:\/\/ecp.iedadata.org\/doibanner\/10.1242\/dmm.040832\u0022 alt=\u0022\u0022 \/\u003E\u003C\/a\u003E \u003C\/div\u003E\n\n \n \u003C\/div\u003E\n\u003C\/div\u003E\n \u003C\/div\u003E\n\u003C\/div\u003E\n\u003C\/div\u003E\u003Cscript type=\u0022text\/javascript\u0022 src=\u0022https:\/\/www.stormoverpacific.com\/sites\/default\/files\/js\/js_hZg96SP9gBcOluDp2mGc57d8sP8uJ7g8P_JYsCISOgQ.js\u0022\u003E\u003C\/script\u003E\n\u003C\/body\u003E\u003C\/html\u003E"}Ļ