Poster abstracts

Poster number 13 submitted by Jordan Gladman

SMN2 exon 7 knock-in: A new spinal muscular atrophy mouse model.

Jordan T. Gladman (The Center for Childhood Cancer, Columbus Children’s Research Institute, The Department of Pediatrics, The Ohio State University.), Thomas Bebee (The Center for Childhood Cancer, Columbus Children’s Research Institute, The Department of Pediatrics, The Ohio State University.), Dawn S. Chandler (The Center for Childhood Cancer, Columbus Children’s Research Institute, The Department of Pediatrics, The Ohio State University.)

Abstract:
Proximal spinal muscular atrophy (SMA) is caused by loss of the survival motor neuron-1 (SMN1) gene leaving only the SMN2 gene to produce the essential SMN protein. SMN2 varies from SMN1 at a single nucleotide in exon 7 which leads to ineffective processing of the RNA by the cellular splicing machinery causing skipping of exon 7, and the generation of a non-functional protein. We hypothesize that the introduction of the human SMN2 mutation in the mouse Smn gene will more closely resembles the human condition in which exon 7 skipping occurs. In order to test the mouse Smn gene’s ability to skip exon 7 in the presence of the human SMN2 point mutation, we constructed a series of Smn mini-genes directly amplified from the mouse 129Sv/Ev genome. Using these mini-genes in transfection experiments we find that transcripts containing the native sequences yield mRNA containing all three exons while the mini-gene containing the exon 7 point mutation predominantly yields the exon 7 skipped mRNA in both human and mouse cell lines. Additionally we have shown up to 70% skipping of exon 7 using both mouse and human mini-genes. These experiments support the idea that a point mutation in exon 7 of the mouse gene will affect SMN splicing in the mouse tissues, as in humans. To generate a mouse harboring the SMN C>T mutation in exon 7 we designed a targeting construct containing the C>T point mutation. A single targeted ES cell line that has undergone correct homologous recombination was used to generate a germline population mice carrying the SMN2 exon 7 C>T point mutation in their Smn gene. We believe that the resultant mouse will more accurately recapitulate the SMA splicing phenotype observed in the human condition, and can be used to address potential therapies aimed at correcting SMN2 splicing.

Keywords: Proximal Spinal Muscular Atrophy, Survival Motor Neuron, Mouse Model