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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

Enhancement of Dystrophin repair by knockdown of Msh2. A: Myoblasts derived from mdx5cv mice were co-transfected with various amounts of RNAi specific to knockdown Msh2 and 3 μg of correcting oligonucleotide mdx47NT. Total genomic DNA was isolated after 24 hours and 25 ng were subjected to absolute quantification using a FAM labeled probe specific only for wild type dystrophin. The fold change in wild-type dystrophin was determined from three independent experiments assayed in triplicate and significance was deciphered by a student's t-test. Differences were considered significant only when the p < 0.05. B: Knockdown of Msh2 by RNAi. Myoblasts were co-transfected with various amounts of RNAi specific to knockdown Msh2 and 3 μg of correcting oligonucleotide mdx47NT. Total mRNA was isolated after 6 hours and subjected to RT-PCR.
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Figure 5: Enhancement of Dystrophin repair by knockdown of Msh2. A: Myoblasts derived from mdx5cv mice were co-transfected with various amounts of RNAi specific to knockdown Msh2 and 3 μg of correcting oligonucleotide mdx47NT. Total genomic DNA was isolated after 24 hours and 25 ng were subjected to absolute quantification using a FAM labeled probe specific only for wild type dystrophin. The fold change in wild-type dystrophin was determined from three independent experiments assayed in triplicate and significance was deciphered by a student's t-test. Differences were considered significant only when the p < 0.05. B: Knockdown of Msh2 by RNAi. Myoblasts were co-transfected with various amounts of RNAi specific to knockdown Msh2 and 3 μg of correcting oligonucleotide mdx47NT. Total mRNA was isolated after 6 hours and subjected to RT-PCR.

Mentions: We and others have shown that a transient knockdown of the mismatch repair protein Msh2 in repair proficient cells can increase the overall level of gene repair [26,30]. Msh2 is thought to inhibit the repair reaction through its action as an anti-recombinase [31,32] by precluding the binding of the oligonucleotide. Therefore, by knocking down the levels of this protein, we might predict that the oligonucleotide will bind more stably to the target site with higher efficiency and facilitate oligonucleotide binding. In order to determine whether the repair reaction could be stimulated by knocking down Msh2 in primary cells, we treated our cultures enriched in myoblasts with increasing concentrations of RNAi and quantified the amount of wild-type dystrophin DNA appearing after incubation with the targeting oligonucleotide for 24 hours. The correction frequency was enhanced 2 and 3 fold at the 1 nM and 10 nM concentrations of the RNAi (Figure 5). The efficiency decreases at higher concentrations of RNAi presumably due to the elevated levels of RNAi toxicity often observed in treated cells [33]. The enriched cell cultures used above were purified further to obtain myoblasts. This was accomplished by plating the cells on a collagen coated dish in F10 media. Once confluency was reached, the myoblasts were detached and separated from fibroblasts (etc.); this procedure was carried our repeatedly until the population of cells in the pooled culture was predominantly purified myoblast. The steps in this procedure are illustrated in Figure 6. Panels i and ii display the enriched culture of cells (i) and after differentiation into myotubes (ii); the arrows point to fibroblasts. In panels (iii) and (iv), the purified myoblast culture is illustrated before (i) and after (ii) differentiation into myotubes. Panels (v) and (vi) display cells in dark field after staining with Desmin (red). The fact that the vast majority of cells are stained in panel (vi) indicated that differentiation into myotubes has occurred. Thus, the procedure has enabled the isolation of a purified culture of myoblasts that can be targeted with the oligonucleotide.


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)

Enhancement of Dystrophin repair by knockdown of Msh2. A: Myoblasts derived from mdx5cv mice were co-transfected with various amounts of RNAi specific to knockdown Msh2 and 3 μg of correcting oligonucleotide mdx47NT. Total genomic DNA was isolated after 24 hours and 25 ng were subjected to absolute quantification using a FAM labeled probe specific only for wild type dystrophin. The fold change in wild-type dystrophin was determined from three independent experiments assayed in triplicate and significance was deciphered by a student's t-test. Differences were considered significant only when the p < 0.05. B: Knockdown of Msh2 by RNAi. Myoblasts were co-transfected with various amounts of RNAi specific to knockdown Msh2 and 3 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

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

Figure 5: Enhancement of Dystrophin repair by knockdown of Msh2. A: Myoblasts derived from mdx5cv mice were co-transfected with various amounts of RNAi specific to knockdown Msh2 and 3 μg of correcting oligonucleotide mdx47NT. Total genomic DNA was isolated after 24 hours and 25 ng were subjected to absolute quantification using a FAM labeled probe specific only for wild type dystrophin. The fold change in wild-type dystrophin was determined from three independent experiments assayed in triplicate and significance was deciphered by a student's t-test. Differences were considered significant only when the p < 0.05. B: Knockdown of Msh2 by RNAi. Myoblasts were co-transfected with various amounts of RNAi specific to knockdown Msh2 and 3 μg of correcting oligonucleotide mdx47NT. Total mRNA was isolated after 6 hours and subjected to RT-PCR.
Mentions: We and others have shown that a transient knockdown of the mismatch repair protein Msh2 in repair proficient cells can increase the overall level of gene repair [26,30]. Msh2 is thought to inhibit the repair reaction through its action as an anti-recombinase [31,32] by precluding the binding of the oligonucleotide. Therefore, by knocking down the levels of this protein, we might predict that the oligonucleotide will bind more stably to the target site with higher efficiency and facilitate oligonucleotide binding. In order to determine whether the repair reaction could be stimulated by knocking down Msh2 in primary cells, we treated our cultures enriched in myoblasts with increasing concentrations of RNAi and quantified the amount of wild-type dystrophin DNA appearing after incubation with the targeting oligonucleotide for 24 hours. The correction frequency was enhanced 2 and 3 fold at the 1 nM and 10 nM concentrations of the RNAi (Figure 5). The efficiency decreases at higher concentrations of RNAi presumably due to the elevated levels of RNAi toxicity often observed in treated cells [33]. The enriched cell cultures used above were purified further to obtain myoblasts. This was accomplished by plating the cells on a collagen coated dish in F10 media. Once confluency was reached, the myoblasts were detached and separated from fibroblasts (etc.); this procedure was carried our repeatedly until the population of cells in the pooled culture was predominantly purified myoblast. The steps in this procedure are illustrated in Figure 6. Panels i and ii display the enriched culture of cells (i) and after differentiation into myotubes (ii); the arrows point to fibroblasts. In panels (iii) and (iv), the purified myoblast culture is illustrated before (i) and after (ii) differentiation into myotubes. Panels (v) and (vi) display cells in dark field after staining with Desmin (red). The fact that the vast majority of cells are stained in panel (vi) indicated that differentiation into myotubes has occurred. Thus, the procedure has enabled the isolation of a purified culture of myoblasts that can be targeted with the oligonucleotide.

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