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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

Detection of dystrophin with exon specific antibodies. Cultures containing dystrophic myoblasts were seeded in 8 well chambers and were targeted with a 72 hour recovery time after the addition of the either the nonspecific ODN (A) or mdx47NT (B). These cells were then allowed to differentiate for 96 hours at which point they were fixed and incubated with antibody specific for a rabbit polyclonal antibody that recognizes dystrophin and a secondary Alexa fluor labeled antibody (red). The cell nuclei were stained with DAPI, which is shown in blue.
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Figure 4: Detection of dystrophin with exon specific antibodies. Cultures containing dystrophic myoblasts were seeded in 8 well chambers and were targeted with a 72 hour recovery time after the addition of the either the nonspecific ODN (A) or mdx47NT (B). These cells were then allowed to differentiate for 96 hours at which point they were fixed and incubated with antibody specific for a rabbit polyclonal antibody that recognizes dystrophin and a secondary Alexa fluor labeled antibody (red). The cell nuclei were stained with DAPI, which is shown in blue.

Mentions: The donor splice site in the DMD gene in mdx5cv mice causes a frameshift which results in a translation of truncated dystrophin protein (10 of the 79 exons). As shown above, correction of the mutation at the DNA level resulted in the restoration of intact dystrophin message. To determine whether the corrected mRNA is translated into normal dystrophin that contains exon 11, cells were seeded and once again transfected with mdx47NT or mdx47T under conditions that provided the highest level of gene correction. The entire assay was performed in microscope chambers so that the samples could be probed directly with an antibody specific for the dystrophin protein. Figure 4 reveals the presence of dystrophin after a 72 hour recovery and 96 hour differentiation period, indicating dystrophin is now being produced within myotubes. Within myotubes that stain positive for dystrophin, it is evident that the protein is distributed throughout (see Figure 4B) These data are in agreement with the western blot analyses performed previously by Bertoni et al. [25]. Altogether, the data from DNA, mRNA and protein analyses show that single stranded oligonucleotides without conjugated chemical moieties at their termini (i.e. Cy3) correct the dystrophin gene in cultures containing an enriched population of myoblasts isolated from dystrophic mice.


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)

Detection of dystrophin with exon specific antibodies. Cultures containing dystrophic myoblasts were seeded in 8 well chambers and were targeted with a 72 hour recovery time after the addition of the either the nonspecific ODN (A) or mdx47NT (B). These cells were then allowed to differentiate for 96 hours at which point they were fixed and incubated with antibody specific for a rabbit polyclonal antibody that recognizes dystrophin and a secondary Alexa fluor labeled antibody (red). The cell nuclei were stained with DAPI, which is shown in blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Detection of dystrophin with exon specific antibodies. Cultures containing dystrophic myoblasts were seeded in 8 well chambers and were targeted with a 72 hour recovery time after the addition of the either the nonspecific ODN (A) or mdx47NT (B). These cells were then allowed to differentiate for 96 hours at which point they were fixed and incubated with antibody specific for a rabbit polyclonal antibody that recognizes dystrophin and a secondary Alexa fluor labeled antibody (red). The cell nuclei were stained with DAPI, which is shown in blue.
Mentions: The donor splice site in the DMD gene in mdx5cv mice causes a frameshift which results in a translation of truncated dystrophin protein (10 of the 79 exons). As shown above, correction of the mutation at the DNA level resulted in the restoration of intact dystrophin message. To determine whether the corrected mRNA is translated into normal dystrophin that contains exon 11, cells were seeded and once again transfected with mdx47NT or mdx47T under conditions that provided the highest level of gene correction. The entire assay was performed in microscope chambers so that the samples could be probed directly with an antibody specific for the dystrophin protein. Figure 4 reveals the presence of dystrophin after a 72 hour recovery and 96 hour differentiation period, indicating dystrophin is now being produced within myotubes. Within myotubes that stain positive for dystrophin, it is evident that the protein is distributed throughout (see Figure 4B) These data are in agreement with the western blot analyses performed previously by Bertoni et al. [25]. Altogether, the data from DNA, mRNA and protein analyses show that single stranded oligonucleotides without conjugated chemical moieties at their termini (i.e. Cy3) correct the dystrophin gene in cultures containing an enriched population of myoblasts isolated from dystrophic mice.

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