Mdx4cv Mice Research Articles

DMD mdx3Cv and DMD mdx4Cv dystrophin mutations in mice: rapid polymerase chain reaction genotyping

We devised non-radioactive PCR assays for the DMD mdx3Cv and DMD mdx4Cv mouse dystrophin point mutations, in which mutant and wild type reactions electrophoresed separately diagnose whether the DNA carries the mutant, wild type, or both alleles. This simple and reliable assay facilitates the use of these mutant mouse models, which have an extended inflammatory phase (DMD mdx3Cv), less reversion to wild type (DMD mdx4Cv), and reduced expression of dystrophin mRNAs arising from internal promoter usage than the DMD mdx mouse. The PCR assays described facilitate the use of the DMD mdx3Cv and DMD mdx4Cv mutant mouse models, when maintaining the mutations as heterozygotes, backcrossing into different inbred genetic backgrounds, or when crossing targeted mutations into these dystrophic backgrounds.

Mini- and full-length dystrophin gene transfer induces the recovery of nitric oxide synthase at the sarcolemma of mdx4cv skeletal muscle fibers.

In normal skeletal muscle fibers, dystrophin accumulates at the cytoplasmic face of the sarcolemma where it associates with dystrophin-associated proteins (DAPs). Several studies have recently shown that the neuronal isoform of nitric oxide synthase (nNOS) is also located at the sarcolemma, and that this membrane localization is mediated through interactions of nNOS with one of the DAPs, namely alpha 1-syntrophin. Since the lack of dystrophin in muscle fibers from Duchenne muscular dystrophy patients and mdx mice is accompanied by an absence of sarcolemmal nNOS, we examined in the present study, whether dystrophin gene replacement would lead to the restoration of nNOS at its appropriate subcellular location. To this end, tibialis anterior muscles from mdx4cv mice were directly injected with plasmid DNA encoding either full-length (pRSV-dys) or mini-(pRSV-dyB; lacking exons 17-48) dystrophin. For these experiments, we chose to study 10-week-old mdx4cv mice since at this developmental stage, muscles from these mice have already undergone several cycles of degeneration-regeneration. Immunofluorescence experiments performed on serial cross-sections revealed that approximately 50% of the dystrophin-positive fibers also exhibited significant levels of nNOS at their sarcolemma 2 weeks following gene transfer with pRSV-dys. Similar results were obtained with pRSV-dyB indicating that exons 17-48 of the dystrophin gene are not essential for the correct localization of nNOS in skeletal muscle fibers. Taken together with the recent demonstration that dystrophin gene transfer leads to significant physiological benefits our results suggest that dystrophin gene therapy using full-length or truncated dystrophin, also induces a rapid recovery of biochemical functions.

Mini-dystrophin gene transfer in mdx4cv diaphragm muscle fibers increases sarcolemmal stability.

To date, all dystrophin gene transfer studies have been performed on mdx hindlimb skeletal muscles which in comparison to the severe deficits seen in muscles from patients afflicted with Duchenne muscular dystrophy (DMD), exhibit only modest morphological and functional changes. Since the mdx diaphragm muscle presents the same pathophysiological alterations characteristic of DMD muscles, we therefore injected recombinant plasmid DNA encoding the dystrophin mini-gene (pRSVdy-B) into diaphragm muscles of 10-week-old mdx4cv mice and examined the physiological consequences of dystrophin expression in a muscle that has undergone a phase of massive degeneration and regeneration. Immunoperoxidase and immunofluorescence experiments revealed that 1 and 3 weeks following gene transfer, approximately 17% of the fibers in a bundle of diaphragm muscle expressed dystrophin at the sarcolemma. Most importantly, this level of dystrophin expression was sufficient to protect all fibers present within these diaphragm muscle bundles from the damaging effects of repetitive lengthening contractions. In addition, dystrophin expression partially restored the ability of transduced mdx4cv muscle bundles to generate isometric tetanic tension following lengthening contractions. These results show that mini-dystrophin expression leads to rapid and significant functional improvements in diaphragm muscles of mdx4cv mice. Although these data provide encouraging results for future therapeutic strategies aimed at curing DMD, additional work will none the less be necessary to determine the full impact of dystrophin gene replacement. In this context, it is clear from the data presented here that the diaphragm muscle of the mdx mouse is an invaluable model system to address this critical issue.

The frequency of revertants in mdx mouse genetic models for Duchenne muscular dystrophy.

The mdx mouse has been used for the development of cellular and gene therapies for Duchenne muscular dystrophy. The relatively frequent occurrence of dystrophin-positive muscle cells called revertants has hampered these efforts by interfering with data interpretation. The mdx4cv and mdx5cv dystrophin mouse mutants have approximately 10-fold fewer revertants than the mdx mutant at both 2 and 6 mo. The mdx3cv dystrophin mouse mutant may be a useful model for some types of human dystrophin deficiencies in which the levels of dystrophin are low but not completely absent.