PCR-restriction-based strategies allow genotyping without sequencing of several allelic variants of the mdx mouse that carry point mutations

Duchenne muscular dystrophy (DMD) is due to the absence of dystrophin. It is characterized by progressive muscle wasting and premature death. The exploration of the pathogenic mechanisms and the evaluation of therapeutic options rely mostly on dystrophic mice. The mdx mouse originates from a spontaneous mutation that arose in a colony of C57BL/10 mice in the 1980s. Since then, it has become the most commonly used animal model of DMD. In the 1990s, several allelic variants of the mdx mouse, namely the mdx2Cv, mdx3Cv, mdx4Cv, and mdx5Cv, have been recovered from chemical mutagenesis in the C57BL/6J background. They have advantages because of the genetic background facilitating the construction of multiple mutants, the location of the mutations on different exons and introns and the association with different levels of “revertant fibres”. These strains all carry point mutations. Conventional genotyping involves PCR amplification, leading to amplicons of equal sizes, followed by allele identification by sequencing. Sequence analysis revealed that the mdx3Cv and mdx5Cv alleles bear restriction sites for AluI, and HphI, respectively, which are absent from the wild type allele. The mdx4Cv mutation disrupts a restriction site for BsmAI. Based on these findings, we designed new strategies for genotyping these dystrophic strains using PCR amplification followed by enzymatic restriction. Compared to strategies proposed by others, our procedure is time and cost effective: It is achieved within a day using standard molecular biology techniques, does not make use of extended primers requiring complicated PCR protocols, and eliminates extra cost for sequencing.