Genetic correction of splice site mutation in purified and enriched myoblasts isolated from mdx5cv mice.
BACKGROUND
Duchenne Muscular Dystrophy (DMD) is an X-linked genetic disorder that results in the production of a dysfunctional form of the protein, dystrophin. The mdx5cv mouse is a model of DMD in which a point mutation in exon 10 of the dystrophin gene creates an artificial splice site. As a result, a 53 base pair deletion of exon 10 occurs with a coincident creation of a frameshift and a premature stop codon. Using primary myoblasts from mdx5cv mice, single-stranded DNA oligonucleotides were designed to correct this DNA mutation.


RESULTS
Single-stranded DNA oligonucleotides that were designed to repair this splice site mutation corrected the mutation in the gene and restored expression of wild-type dystrophin. This repair was validated at the DNA, RNA and protein level. We also report that the frequency of genetic repair of the mdx mutation can be enhanced if RNAi is used to suppress expression of the recombinase inhibitor protein Msh2 in cultures containing myoblasts but not in those heavily enriched in myoblasts.


CONCLUSIONS
Exogenous manipulations, such as RNAi, are certainly feasible and possibly required to increase the successful application of gene repair in some primary or progenitor muscle cells.
Maguire
Katie
K
Department of Biological Sciences, University of Delaware, Newark, DE 19716, USA. katie_maguire78@yahoo.com
Suzuki
Takayuki
T
DiMatteo
Darlise
D
Parekh-Olmedo
Hetal
H
Kmiec
Eric
E
D000818:Animals; D002478:Cells, Cultured; D055785:Gene Knockdown Techniques; D008297:Male; D051379:Mice; D018101:Mice, Inbred mdx; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics; D051718:MutS Homolog 2 Protein / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism*; D034622:RNA Interference; D022821:RNA Splice Sites / Q000235:genetics*; D052416:Targeted Gene Repair / Q000379:methods*
D022821:RNA Splice Sites; C497173:Msh2 protein, mouse; D051718:MutS Homolog 2 Protein