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Genetic correction of splice site mutation in purified and enriched myoblasts isolated from mdx5cv mice.

Maguire K, Suzuki T, DiMatteo D, Parekh-Olmedo H, Kmiec E - BMC Mol. Biol. (2009)

Bottom Line: 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.Using primary myoblasts from mdx5cv mice, single-stranded DNA oligonucleotides were designed to correct this DNA mutation.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, University of Delaware, Newark, DE 19716, USA. katie_maguire78@yahoo.com

ABSTRACT

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.

Conclusion: 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.

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Related in: MedlinePlus

Knockdown of Msh2 by RNAi. Purified myoblasts were co-transfected with various amounts of RNAi specific to knockdown Msh2 and 0.75 μg of correcting oligonucleotide, mdx47NT. Total mRNA was isolated after 6 hours and subjected to RT-PCR.
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Figure 9: Knockdown of Msh2 by RNAi. Purified myoblasts were co-transfected with various amounts of RNAi specific to knockdown Msh2 and 0.75 μg of correcting oligonucleotide, mdx47NT. Total mRNA was isolated after 6 hours and subjected to RT-PCR.

Mentions: Since RNAi-directed Msh2 knockdowns had elevated correction efficiencies in myoblast-enriched cultures, we applied this strategy to cultures of purified myoblasts where active conversion was shown (above) to take place. Purified myoblast cultures were treated simultaneously with various doses of the RNAi used in the experiments presented in Figure 5 and either 0.3 μg, 0.6 μg or 0.9 μg of targeting ODN, mdx47NT. The fold change in the evolution of wild type dystrophin DNA was measured after 24 hours and the results are presented in Figure 8A, B and 8C, respectively. No statistically significant difference in correction activity is observed at any RNAi level, although a clear reduction in Msh2 RNA levels is taking place (Figure 9). These data differ from the results reported in Figure 5 suggesting perhaps that enhancement of gene repair by RNAi may not have been occurring in myoblasts.


Genetic correction of splice site mutation in purified and enriched myoblasts isolated from mdx5cv mice.

Maguire K, Suzuki T, DiMatteo D, Parekh-Olmedo H, Kmiec E - BMC Mol. Biol. (2009)

Knockdown of Msh2 by RNAi. Purified myoblasts were co-transfected with various amounts of RNAi specific to knockdown Msh2 and 0.75 μg of correcting oligonucleotide, mdx47NT. Total mRNA was isolated after 6 hours and subjected to RT-PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654480&req=5

Figure 9: Knockdown of Msh2 by RNAi. Purified myoblasts were co-transfected with various amounts of RNAi specific to knockdown Msh2 and 0.75 μg of correcting oligonucleotide, mdx47NT. Total mRNA was isolated after 6 hours and subjected to RT-PCR.
Mentions: Since RNAi-directed Msh2 knockdowns had elevated correction efficiencies in myoblast-enriched cultures, we applied this strategy to cultures of purified myoblasts where active conversion was shown (above) to take place. Purified myoblast cultures were treated simultaneously with various doses of the RNAi used in the experiments presented in Figure 5 and either 0.3 μg, 0.6 μg or 0.9 μg of targeting ODN, mdx47NT. The fold change in the evolution of wild type dystrophin DNA was measured after 24 hours and the results are presented in Figure 8A, B and 8C, respectively. No statistically significant difference in correction activity is observed at any RNAi level, although a clear reduction in Msh2 RNA levels is taking place (Figure 9). These data differ from the results reported in Figure 5 suggesting perhaps that enhancement of gene repair by RNAi may not have been occurring in myoblasts.

Bottom Line: 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.Using primary myoblasts from mdx5cv mice, single-stranded DNA oligonucleotides were designed to correct this DNA mutation.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, University of Delaware, Newark, DE 19716, USA. katie_maguire78@yahoo.com

ABSTRACT

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus