Journal: bioRxiv

Article Title: Elimination of myotonia improves myopathy in a muscleblind knockout model of myotonic dystrophy

doi: 10.1101/2025.09.19.677400

Figure Lengend Snippet: (a) Schematic of CRISPR/Cas9 approach to develop a myotonia-resistant mouse model by excision of the exon 7a sequence from the Clcn1 gene. Scissors represent approximate location of the gRNA sequences in the intronic regions flanking the exon 7a (sequences provided in supplemental figure 1). (b) Ethidium bromide-stained agarose gel of RT-PCR products generated with primers flanking exon 7a from RNA isolated from quadriceps muscle from WT, ClC-1 ΔE7a/ΔE7a, Mbnl1 −/− , Mbnl1 −/− ; ClC-1 ΔE7a/ΔE7a, and ADR (NMD control) mice (P18). Each lane represents an individual mouse, with biological replicates denoted by number. Ladder (1kb Plus ladder, Invitrogen) and no template control (NT CTL) shown. Unique transcript isoforms identified by the distinct molecular weight of the amplicon indicated on the right. –E7a represents the adult, wildtype isoform. (c) Quantitation of exon 7a percent spliced in (PSI) based on relative densitometry of the amplicons shown in panel (b), as well as for samples at P7 and 4-6-months of age, with bars shown mean ± SE and individual biological replicates indicated by dots (n=3-4/genotype/timepoint, other agarose gels shown in Fig. S2a). (d) To determine relative RNA stability, the ratio of relative Clcn1 steady-state mRNA to pre-mRNA was determined with the use of separate RT-qPCR reactions with primer-probe sets directed at constitutively spliced exon-exon boarders and intron-exon boarders, respectively. Steady-state and pre-mRNA experiments were each completed with the same reference Tbp primer-probe set and normalized to the average of P18 WT samples to allow for the determination of the ratio of relative Clcn1 steady-state expression / Clcn1 pre-mRNA (n = 3-4 biological replicates/genotype/timepoint; completed in technical triplicate). Dots represent biological replicates and bars plotted mean ± SE. Statistical comparisons were completed with two-way with Tukey’s multiple comparisons (only significant intra-timepoint comparisons shown for simplicity). Significant comparisons indicated as multiplicity-adjusted p-value: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Additionally, for a Clcn1 non-sense mediated decay (NMD) control mouse model, adr-mto2J mice (ADR) were obtained from Jackson Laboratories (Bar Harbor, MA) and extensively backcrossed to the FVB/N background.

Techniques: CRISPR, Sequencing, Staining, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Generated, Isolation, Control, Molecular Weight, Amplification, Quantitation Assay, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Elimination of myotonia improves myopathy in a muscleblind knockout model of myotonic dystrophy

doi: 10.1101/2025.09.19.677400

Figure Lengend Snippet: (a) Representation of the murine Clcn1 gene with constitutive exons (gray) and common alternative splice elements seen in DM1 annotated in distinct colors. Downward projection represents a schematic of post-splicing Clcn1 mRNA containing all the above annotated alternative splicing features not typically present in the adult, ion-channel coding isoform of the transcript. Approximate forward and reverse primer locations—exon 1 and exon 23, respectively-–used for generating the whole- Clcn1 coding region amplicons for long-read sequencing libraries shown as arrows. Whole- Clcn1 amplicon sequencing libraries were generated for both early (P7) and late (P18) post-natal mice with RNA isolated from quadriceps muscle of WT, ClC-1 ΔE7a/ΔE7a, Mbnl1 −/− , and Mbnl1 −/− ; ClC-1 ΔE7a/ΔE7a genotypes as well as late post-natal ADR samples used as the NMD control (n = 3 mice/genotype/timepoint). To determine transcript isoform utilization for each sample, reads were aligned to Ch. 6 (mm39) and collapsed into isoforms with greater than 100 supporting reads and the most abundant reads were annotated for quantification with the use of FLAIR. (b) Quantification of prominent individual splice elements, as expressed as percent spliced in (PSI), based on whole-length transcript reads. Bars shown mean ± SE with individual biological replicates shown as dots. (c) Assessment of transcript isoform utilization with FLAIR with reads quantified for the adult, protein-coding isoform, and the 15 most abundant alternate isoforms seen in the library, shown as a heat map; rows indicate transcript isoform based on the element or combination of elements that differentiate from the adult, protein-coding isoform and columns indicate the different genotype/timepoint conditions assessed. Color intensity of each box represents the average proportion of that isoform in the pool of reads for each sample (n = 3 mice/genotype/timepoint), as per the scale bar to right (note, the broken scale skewed to lower isoform proportions to visually highlight differences in the lower abundance alternative transcript isoforms). Arrows superimposed on boxes represent statistical comparisons between each genotype/timepoint and the age-matched wild-type samples using DESeq2; number of arrows indicates significance level (p < 0.05 ↑, p < 0.01 ↑↑, p < 0.001 ↑↑↑, or p < 0.0001 ↑↑↑↑) and direction indicates up/down regulated compared to age-matched wild-type samples.

Article Snippet: Additionally, for a Clcn1 non-sense mediated decay (NMD) control mouse model, adr-mto2J mice (ADR) were obtained from Jackson Laboratories (Bar Harbor, MA) and extensively backcrossed to the FVB/N background.

Techniques: Alternative Splicing, Sequencing, Amplification, Generated, Isolation, Control

Journal: bioRxiv

Article Title: Elimination of myotonia improves myopathy in a muscleblind knockout model of myotonic dystrophy

doi: 10.1101/2025.09.19.677400

Figure Lengend Snippet: (a) Overlap of genes mis-spliced in muscle of myotonic Mbnl1 KO mice (Mbnl1 −/− ) and Mbnl1 KO mice without myotonia (Mbnl1 −/− ; ClC-1 ΔE7a/ΔE7a ), both compared to WT mice. Most genes (52%, 340 genes) are commonly mis-spliced (FDR > 90%, delta-PSI > 0.1 & < −0.1), while there are 207 and 113 genes uniquely mis-spliced in myotonic and non-myotonic Mbnl1 KO muscle, respectively (note that unique genes might have the same event in both conditions, but miss the threshold set for delta-PSI to be significantly different). Unique genes were analyzed regarding biological pathway (dot plot, top 10 GO terms), which revealed that genes that are only mis-spliced in myotonic Mbnl1KO mice are involved in cytoskeletal and chromatin organization as well as RNA splicing, whereas in non-myotonic Mbnl1 KO mice, unique genes fall mainly in cytoskeletal organization. (b) Splicing events in Mbnl1 −/− and Mbnl1 −/− ;ClC-1 ΔE7a/ΔE7a were each compared to WT mice and the PSI-values were plotted as a scatter plot (each dot represents one ASE with some transcripts having multiple ASE). Events that are not changing between Mbnl1 −/− and WT are displayed in grey, while events with PSI-value above 0.1 and below −0.1 and an FDR above 80% are shown in blue. Pink events are partial rescues defined as having more than a 0.15 PSI shift in Mbnl1 −/− ; ClC-1 ΔE7a/ΔE7a towards control. Events with a complete rescue (changing in Mbnl1 −/− vs WT and non-changing in Mbnl1 −/− ;ClC-1 ΔE7a/ΔE7a vs WT at an FDR of > 95%) in mice without myotonia are shown in red and are labeled. The positive control Clcn1 exon7 skipping is marked in bold. (c) Heatmap of selected genes ranging from complete rescue to unaffected. Rows represent individual splice events (labels right) and columns represent biological replicates, arranged by unsupervised sorting (genotype denoted by colored box below). (d) Tpm3 shows the strongest rescue in mice without myotonia. In Mbnl1 −/− mice, exon 3 and exon 9 are included with a PSI-value of +0.65 and 0.7, respectively. The isoform including exons 3 and 9 is Tpm3-201, in which exon 3 becomes the first exon, while the exon 5 codes for exon 5. In wildtype mice however, exons 1,2, 10, 14 and 15 which belong to isoform Tpm3-205 (Fig. S8).

Article Snippet: Additionally, for a Clcn1 non-sense mediated decay (NMD) control mouse model, adr-mto2J mice (ADR) were obtained from Jackson Laboratories (Bar Harbor, MA) and extensively backcrossed to the FVB/N background.

Techniques: Control, Labeling, Positive Control