Characterizing the effect of mutations within exon 7 of the murine survival motor neuron gene to model spinal muscular atrophy in the mouse

Spinal muscular atrophy (SMA) is caused by loss of function mutations or deletions in the survival motor neuron gene, SMN1. Humans have two copies of SMN, telemetric SMN1 and centromeric SMN2. The most notable difference between SMN1 and SMN2 is a C-T nucleotide transition within exon 7. The "T" nucleotide in SMN2 transforms a cis exon splicing enhancer to a splicing suppressor. As a result, exon 7 is excluded from 80-90% of total transcripts and the delta 7 truncated protein (Delta7SMN) is non-functional. Fully functional protein (FLSMN) is expressed from the remaining 10-20% full length transcripts. The reduction of FLSMN leads loss of alpha-motor neurons and skeletal muscle atrophy. Animal models are critical to understand the pathogenesis of SMA and the involvement by which SMN causes motor neuron and skeetal muscle specific defects. Mice have only one copy of Smn which does not alternatively splice. In order to model SMA in the mouse, two mutations were introduced into murine Smn exon 7 splice enhancers to induce its alternative splicing. The result is two new mutant alleles, SmnC-T-Neo and Smn2B-Neo. The C-T mutation mimics human SMN2 and the 2B mutation targets a second splice enhancer centrally located in exon 7. Both alleles contain a loxP flanked neo selectable marker within intron 7 which further reduces expression of Smn. The outcome is a lethal phenotype early in embryogenesis. Removal of the neo marker with cre generates SmnC-T and Smn2B alleles and increases Smn expression. SmnC-T/- mice express the lowest levels of protein in a model without motor neuron defects, identifying a new therapeutic threshold for Smn protein expression. The SmnC-T-Neo and Smn2B-Neo alleles can be used to determine the spatial and temporal requirement of Smn expression for rescue of disease phenotype, information which is critical for development of effective therapies for spinal muscular atrophy.