scholarly journals Massive Idiosyncratic Exon Skipping Corrects the Nonsense Mutation in Dystrophic Mouse Muscle and Produces Functional Revertant Fibers by Clonal Expansion

2000 ◽  
Vol 148 (5) ◽  
pp. 985-996 ◽  
Author(s):  
Q.L. Lu ◽  
G.E. Morris ◽  
S.D. Wilton ◽  
T. Ly ◽  
O.V. Artem'yeva ◽  
...  
Keyword(s):  
Nonsense Mutation ◽  
Exon Skipping ◽  
Dystrophin Gene ◽  
Expression Vectors ◽  
Mdx Mouse ◽  
Mdx Mice ◽  
Functional Protein ◽  
Nonsense Mutations ◽  
Mouse Muscle ◽  
Dystrophin Expression

Conventionally, nonsense mutations within a gene preclude synthesis of a full-length functional protein. Obviation of such a blockage is seen in the mdx mouse, where despite a nonsense mutation in exon 23 of the dystrophin gene, occasional so-called revertant muscle fibers are seen to contain near-normal levels of its protein product. Here, we show that reversion of dystrophin expression in mdx mice muscle involves unprecedented massive loss of up to 30 exons. We detected several alternatively processed transcripts that could account for some of the revertant dystrophins and could not detect genomic deletion from the region commonly skipped in revertant dystrophin. This, together with exon skipping in two noncontiguous regions, favors aberrant splicing as the mechanism for the restoration of dystrophin, but is hard to reconcile with the clonal idiosyncrasy of revertant dystrophins. Revertant dystrophins retain functional domains and mediate plasmalemmal assembly of the dystrophin-associated glycoprotein complex. Physiological function of revertant fibers is demonstrated by the clonal growth of revertant clusters with age, suggesting that revertant dystrophin could be used as a guide to the construction of dystrophin expression vectors for individual gene therapy. The dystrophin gene in the mdx mouse provides a favored system for study of exon skipping associated with nonsense mutations.

Dystrophin Cytochemistry in Mdx Mouse Muscles Injected with Labeled Normal Myoblasts

1992 ◽  
Vol 1 (1) ◽  
pp. 17-22 ◽  
Author(s):  
Ming Chen ◽  
Hua-Ju Li ◽  
Qiuwen Fang ◽  
Tena G. Goodwin ◽  
J. Ann Florendo ◽  
...  
Keyword(s):  
Mdx Mouse ◽  
Mdx Mice ◽  
Dystrophic Muscle ◽  
Hoechst 33342 ◽  
Mouse Muscle ◽  
Dystrophin Expression ◽  
Host Mouse ◽  
A New Technique ◽  
Phosphate Buffered Saline ◽  
Myoblast Transfer

A new technique enables correlation of dystrophin expression with the location of donor versus host nuclei in the same sections of mdx mouse muscle injected with normal myoblasts. Myoblasts from C57BL/6J mice or from humans were labeled with 0.01% fluoro-gold (FG) in Dulbecco's Modified Eagles Medium (DMEM) for 16 h at 37°C before myoblast transfer. About 3 × 104 myoblasts were injected into the quadriceps muscles of mdx mice immunosuppressed with cyclosporine A (CsA). At 11, 21, or 25 days after myoblast transfer, injected muscles were dissected out and sectioned. These mouse sections were processed for dystrophin and then labeled with a fluorescent nucleus counterstain, 5 μg% Hoechst 33342 in phosphate-buffered saline (PBS), for 10 min at room temperature. Fluoro-gold labeling corresponding with Hoechst 33342 staining indicated survival of normal nuclei in dystrophic muscle. Dystrophin was found in the sarcolemma of myofibers containing FG-labeled nuclei but not of myofibers containing only Hoechst 33342-labeled nuclei. Control muscle samples showed neither FG labeling nor dystrophin. This study demonstrates that the donor human and mouse myoblasts survived and developed in host mouse muscles for at least 25 days after myoblast transfer, and that the localization of their normal nuclei correlates with dystrophin expression in muscle fibers of immunosuppressed mdx host mice.

Global/temporal gene expression in diaphragm and hindlimb muscles of dystrophin-deficient (mdx) mice

2002 ◽  
Vol 283 (3) ◽  
pp. C773-C784 ◽  
Author(s):  
Karl Rouger ◽  
Martine Le Cunff ◽  
Marja Steenman ◽  
Marie-Claude Potier ◽  
Nathalie Gibelin ◽  
...  
Keyword(s):  
Dystrophin Gene ◽  
Diaphragm Muscle ◽  
Mdx Mouse ◽  
Mdx Mice ◽  
First Year ◽  
Temporal Gene Expression ◽  
Cell Functions ◽  
Hindlimb Muscles ◽  
Genes Encoding ◽  
Dystrophin Protein

The mdx mouse is a model for human Duchenne muscular dystrophy (DMD), an X-linked degenerative disease of skeletal muscle tissue characterized by the absence of the dystrophin protein. The mdx mice display a much milder phenotype than DMD patients. After the first week of life when all mdx muscles evolve like muscles of young DMD patients, mdx hindlimb muscles substantially compensate for the lack of dystrophin, whereas mdx diaphragm muscle becomes progressively affected by the disease. We used cDNA microarrays to compare the expression profile of 1,082 genes, previously selected by a subtractive method, in control and mdx hindlimb and diaphragm muscles at 12 time points over the first year of the mouse life. We determined that 1) the dystrophin gene defect induced marked expression remodeling of 112 genes encoding proteins implicated in diverse muscle cell functions and 2) two-thirds of the observed transcriptomal anomalies differed between adult mdx hindlimb and diaphragm muscles. Our results showed that neither mdx diaphram muscle nor mdx hindlimb muscles evolve entirely like the human DMD muscles. This finding should be taken under consideration for the interpretation of future experiments using mdx mice as a model for therapeutic assays.

scholarly journals An exon skipping-associated nonsense mutation in the dystrophin gene uncovers a complex interplay between multiple antagonistic splicing elements

2006 ◽  
Vol 15 (6) ◽  
pp. 999-1013 ◽  
Author(s):  
A. Disset ◽  
C.F. Bourgeois ◽  
N. Benmalek ◽  
M. Claustres ◽  
J. Stevenin ◽  
...  
Keyword(s):  
Nonsense Mutation ◽  
Exon Skipping ◽  
Dystrophin Gene ◽  
Complex Interplay

scholarly journals Nanopolymers improve delivery of exon skipping oligonucleotides and concomitant dystrophin expression in skeletal muscle of mdx mice

2008 ◽  
Vol 8 (1) ◽  
pp. 35 ◽  
Author(s):  
Jason H Williams ◽  
Rebecca C Schray ◽  
Shashank R Sirsi ◽  
Gordon J Lutz
Keyword(s):  
Skeletal Muscle ◽  
Exon Skipping ◽  
Mdx Mice ◽  
Dystrophin Expression

scholarly journals DMD carrier model with mosaic dystrophin expression in the heart reveals complex vulnerability to myocardial injury

2020 ◽  
Vol 29 (6) ◽  
pp. 944-954
Author(s):  
Tatyana A Meyers ◽  
Jackie A Heitzman ◽  
DeWayne Townsend
Keyword(s):  
Myocardial Injury ◽  
Exon Skipping ◽  
Mdx Mice ◽  
Parental Origin ◽  
Wild Type ◽  
Dystrophin Expression ◽  
High Doses ◽  
Therapy Strategies ◽  
Cellular Dysfunction ◽  
Shed Light

Abstract Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease that causes progressive muscle wasting and cardiomyopathy. This X-linked disease results from mutations of the DMD allele on the X-chromosome resulting in the loss of expression of the protein dystrophin. Dystrophin loss causes cellular dysfunction that drives the loss of healthy skeletal muscle and cardiomyocytes. As gene therapy strategies strive toward dystrophin restoration through micro-dystrophin delivery or exon skipping, preclinical models have shown that incomplete restoration in the heart results in heterogeneous dystrophin expression throughout the myocardium. This outcome prompts the question of how much dystrophin restoration is sufficient to rescue the heart from DMD-related pathology. Female DMD carrier hearts can shed light on this question, due to their mosaic cardiac dystrophin expression resulting from random X-inactivation. In this work, a dystrophinopathy carrier mouse model was derived by breeding male or female dystrophin-null mdx mice with a wild type mate. We report that these carrier hearts are significantly susceptible to injury induced by one or multiple high doses of isoproterenol, despite expressing ~57% dystrophin. Importantly, only carrier mice with dystrophic mothers showed mortality after isoproterenol. These findings indicate that dystrophin restoration in approximately half of the heart still allows for marked vulnerability to injury. Additionally, the discovery of divergent stress-induced mortality based on parental origin in mice with equivalent dystrophin expression underscores the need for better understanding of the epigenetic, developmental, and even environmental factors that may modulate vulnerability in the dystrophic heart.

Nonsense but not missense mutations can decrease the abundance of nuclear mRNA for the mouse major urinary protein, while both types of mutations can facilitate exon skipping

1994 ◽  
Vol 14 (9) ◽  
pp. 6326-6336
Author(s):  
P Belgrader ◽  
L E Maquat
Keyword(s):  
Nonsense Mutation ◽  
Premature Termination ◽  
Exon Skipping ◽  
Mrna Translation ◽  
Rna Metabolism ◽  
Urinary Protein ◽  
Missense Mutations ◽  
Major Urinary Protein ◽  
Nonsense Mutations ◽  
Exon 2

In an effort to understand the mechanisms by which nonsense codons affect RNA metabolism in mammalian cells, nonsense mutations were generated within the gene for the secretory major urinary protein (MUP) of mice. The translation of MUP mRNA normally begins within exon 1 and terminates within exon 6, the penultimate exon. Through the use of Northern (RNA) blot hybridization and assays that couple reverse transcription and PCR, a nonsense mutation within codon 50 of exon 2 or codon 143 of exon 5 was found to reduce the abundance of fully spliced, nuclear MUP mRNA to 10 to 20% of normal without an additional reduction in the abundance of cytoplasmic mRNA. In contrast, a nonsense mutation within codon 172 of exon 5 was found to have no effects on the abundance of MUP mRNA. These findings suggest that a boundary between nonsense mutations that do and do not reduce the abundance of nuclear mRNA exists within the exon preceding the exon that harbors the normal site of translation termination. In this way, the boundary is analogous to the boundary that exists within the penultimate exon of the human gene for the cytosolic enzyme triosephosphate isomerase. Assays for exon skipping, i.e., the removal of an exon as a part of the flanking introns during the process of splicing, reveal that 0.1, 2.0, and 0.1% of MUP mRNA normally lack exon 5, exon 6, and exons 5 plus 6, respectively. Relative to normal, the two nonsense mutations within exon 5 increase the abundance of RNA lacking exon 5 on average 20-fold and increase the abundance of RNA lacking exons 5 plus 6 on average 5-fold. Since only one of these nonsense mutations also reduces the abundance of fully spliced nuclear mRNA to 10 to 20% of normal, the two mechanisms by which a nonsense mutation can alter nuclear RNA metabolism must be distinct. The analysis of missense mutations within codons 143 and 172, some of which retain the nonsense mutation, indicates that the reduction in the abundance of fully spliced nuclear mRNA is dependent upon the premature termination of MUP mRNA translation, whereas skipping is attributable to nonsense mutation-mediated changes in exon 5 structure rather than to the premature termination of translation. The increase in exon 5 skipping by either the nonsense or missense mutations within codon 172 correlates with a decrease in the complementarity of exon 5 to U1 snRNA. This suggests that a 5' splice site may extend as far as 12 nucleotides into the upstream exon, which is, to our knowledge, the largest extension.

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