Recovery of induced mutations for X chromosome-linked muscular dystrophy in mice.

Abstract

We have used elevated levels of plasma creatine phosphokinase activity to identify muscular dystrophy phenotypes in mice and to screen the progeny of chemical mutagen-treated male mice for X chromosome-linked muscular dystrophy mutations. We were not successful in identifying heterozygous carriers of these induced muscular dystrophy mutations in >8000 progeny. However, we were highly successful in identifying three additional alleles of the characterized mdx locus. These alleles of mdx were recovered from various mutagen-treated males and they occur on an X chromosome that carries flanking markers that allow us to follow the mutations in genetic crosses and in the development of corresponding mutant stocks. These alleles have been designated as mdx2Cv, MdX3Cv, and mdx4Cv. Preliminary data show that mice with mdx2CP and mdx3Cv mutatious have muscular dystrophic phenotypes that do not grossly differ from the characterized mdx mutation. These additional mdx mutations expand the value of mouse models of X chromosome-linked muscular dystrophy and potentially derfie additional sites of mutation that impair dystrophin expression.A recessive X chromosome-linked muscular dystrophy mutation (mdx) has been identified in the laboratory mouse that is isogenic on the homozygous inbred mutant strain C57BL/lOSn.mdx (1). Mutant mice have pathological features that indicate that it is a true myopathy. The defect is expressed in all skeletal muscles of mdx/Y males and mdx/mdx females with extensive limb muscle degeneration, inflammation, and regeneration (2) as well as myocardial lesions (3). However, the affected mutant males and females do not show overt signs of wasting or impaired motor function, which is common to the human Duchenne-Becker muscular dystrophy mutation (4). They are also capable of breeding successfully so that the mdx mutation can be directly propagated. Heterozygous mutant carrier females +/mdx do not show physiological symptoms such as elevated levels of muscle enzyme in circulating blood or histopathology associated with the myopathic condition (1). The mdx mutation has been mapped between Hprt and Pgk-J on the mouse X chromosome (5, 6) and within the mouse Dmd locus that encodes dystrophin (7).The chemical N-ethylnitrosourea (ENU) has been identified as a powerful mutagen in mice. Male mice treated with one intraperitoneal dose of ENU transmit germ-line mutations to progeny at a frequency as high as 1 mutation per 700 loci tested (8)(9)(10)(11). In this report, we describe the results of our use of ENU to produce muscular dystrophy mutations in mice. In one series we screened a group of female progeny of ENU-treated males in an attempt to identify heterozygous carriers of muscular dystrophy mutations. No muscular dystrophy mutations were recovered from those progeny.However, in a second series, we screened for muscular dystrophy mutations when mutagen-treated males carrying variant alleles of Hprt and Pgk-J were mated with C57BL/ lOSn.mdx females. The resulting female progeny of these matings were ...

The Frequency of Revertants in mdx Mouse Genetic Models for Duchenne Muscular Dystrophy

“…The pathologic findings were more extensive with increasing age in all the mutants. As previously reported (8), the pathologic findings of the skeletal and cardiac muscles in the four new rndx mutants were similar to those in the original rndx mice.…”

“…The quadriceps muscles of the mutants were analyzed by immunofluorescent staining for dystrophin. As previously reported (8), the vast majority of the skeletal myofibers on the cross-sections from the mdx2, mdx5, and mdx4 mutants lacked dystrophin immunoreactivity. Unexpected results in the mdx3 mutant will be presented below.…”

“…New mutant alleles of rndx were recovered from female progeny of ethylnitrosourea-treated male mice that are designated mdx2", mdx3'', and mdx4" (8). More recently, we have established a fourth allele, mdx5"".…”

AON-Mediated Exon Skipping for Duchenne Muscular Dystrophy

“…Mdx 2Cv has a mutation in a splice site in exon 43 (causing alternative splicing, resulting in out-of-frame transcripts), mdx 3Cv a mutation in intron 65 (inducing a new splice site, resulting in out-of-frame transcripts), mdx 4Cv a mutation in exon 53 (premature stop codon) and mdx 5Cv a mutation in exon 10 (frame-shift by introduction of a new splice site). All these mice have a phenotype comparable to the mdx mouse (Chapman et al, 1989). In addition, several mouse models have been generated that only affect 1 or a few of the different dystrophin isoforms.…”

From Basic Research to Clinical Trials: Preclinical Trial Evaluation in Mouse Models

“…This resulted in the formation of 4 new mouse models with specific mutations in the dystrophin gene and relevant increases in circulating CK levels in the blood. These mouse models have been described previously (Chapman et al 1989;Cox et al 1993;Im et al 1996). Briefly, the mdx 2cv mouse has a mutation in intro 42; the mdx 3cv mouse has a mutant splice acceptor site in nintron 65; the mdx 4cv mouse has a "C" to "T" substitution in exon 53; and mdx 5cv has an "A" to "T" transition in exon 10.…”