PCR analysis of muscular dystrophy in mdx mice.

PCR amplification has enabled a variety of studies to be performed on the murine dystrophin transcripts. Figure 7.12 displays a summary of the features of the murine dystrophin mRNA that have been described in this article. The location of the mutation in the original mdx mouse is indicated, as are the different spliced forms of the dystrophin transcript. Also shown are the location of various PCR primer binding sites that were used to deduce the alternative splicing pattern of the gene. It is likely that conventional cloning efforts aimed at identifying the variety of dystrophin spliced forms would have taken years to perform, particularly since several of the isoforms are expressed at levels significantly below the estimated 0.02% of total mRNA that dystrophin represents in skeletal muscle (Hoffman et al., 1987a, b). Amplification of dystrophin mRNA simplifies scanning methods for the identification of DNA sequence variations. Attempts to re-isolate and sequence the 14 kb cDNA to determine the mutation in separate strains of mdx mice are not likely to be time or cost effective. PCR enables these types of questions to be answered in a relatively short period of time, and similar types of analyses can be applied to human DMD tissues. Knowledge of the transcript diversity displayed by the dystrophin gene will enable the role of these separate isoforms to be addressed. Despite considerable effort by a variety of laboratories over the last five years, the precise functional role played by dystrophin remains unclear, and it can only be assumed that the separate isoforms act to modulate the functional role of dystrophin in separate tissues or in response to differing physiological states. PCR amplification of the dystrophin isoforms has enabled the variable regions of the transcript to be subcloned (Bies et al., 1992). These clones have been used to reintroduce the variable regions into full-length mini-gene expression vectors, which are currently being tested for functional activity through the generation of transgenic mdx mice. The transgenic mice can be easily identified through the PCR-ASO assays described in this article, and the reverse transcriptase PCR assays will enable a detailed analysis of the expression pattern of the introduced mini-genes. It is hoped that such analyses will further attempts to determine the feasibility of using gene therapy as a treatment for DMD/BMD.