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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

Mutation in dystrophin gene of mdx5cv mice and correcting ODNs. Mdx5cv mice have a single point mutation (A to T) in the dystrophin gene, which is both bolded and underlined in the gene sequence above. This single base mutation will create a donor splice site within exon 10 and cause a 53 base portion of this exon to be spliced out of the mature transcript as depicted by the dashed line in the diagram. A frameshift results in a stop codon in exon 11 (*) and will ultimately result in the production of a truncated dystrophin protein. Because the natural donor site is still present in the transcript there is a possibility that some full-length message, containing exon 10 is made and this is shown as the solid line. The ODNs, mdx47T and mdx47NT, shown above, were designed to correct the substitution mutation and the target base is underlined.
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Figure 2: Mutation in dystrophin gene of mdx5cv mice and correcting ODNs. Mdx5cv mice have a single point mutation (A to T) in the dystrophin gene, which is both bolded and underlined in the gene sequence above. This single base mutation will create a donor splice site within exon 10 and cause a 53 base portion of this exon to be spliced out of the mature transcript as depicted by the dashed line in the diagram. A frameshift results in a stop codon in exon 11 (*) and will ultimately result in the production of a truncated dystrophin protein. Because the natural donor site is still present in the transcript there is a possibility that some full-length message, containing exon 10 is made and this is shown as the solid line. The ODNs, mdx47T and mdx47NT, shown above, were designed to correct the substitution mutation and the target base is underlined.

Mentions: The mdx5cv mouse has a point mutation in exon 10, which creates a splice donor site that results in a 53 base deletion within the exon (see diagram in Figure 2). This deletion results in a reading frameshift wherein a premature stop codon is created 95 bases downstream of the mutation site. If the ODN directs correction of the base, then the correct splice pattern is recreated and the 53 deleted bases remain in the message; in effect, restoring the reading frame. To evaluate the restoration of the 53 base pair insertion, a reverse primer was designed to bind to the deleted portion of exon 10. Thus, amplification can only occur if the targeted splice site corrected and the full-length mature message is produced at detectable levels. To determine the level of expression of dystrophin mRNA where the deleted region had been restored, mRNA was reverse-transcribed using a primer specific for exon 12, which is present in all of the transcripts, corrected or not.


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)

Mutation in dystrophin gene of mdx5cv mice and correcting ODNs. Mdx5cv mice have a single point mutation (A to T) in the dystrophin gene, which is both bolded and underlined in the gene sequence above. This single base mutation will create a donor splice site within exon 10 and cause a 53 base portion of this exon to be spliced out of the mature transcript as depicted by the dashed line in the diagram. A frameshift results in a stop codon in exon 11 (*) and will ultimately result in the production of a truncated dystrophin protein. Because the natural donor site is still present in the transcript there is a possibility that some full-length message, containing exon 10 is made and this is shown as the solid line. The ODNs, mdx47T and mdx47NT, shown above, were designed to correct the substitution mutation and the target base is underlined.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Mutation in dystrophin gene of mdx5cv mice and correcting ODNs. Mdx5cv mice have a single point mutation (A to T) in the dystrophin gene, which is both bolded and underlined in the gene sequence above. This single base mutation will create a donor splice site within exon 10 and cause a 53 base portion of this exon to be spliced out of the mature transcript as depicted by the dashed line in the diagram. A frameshift results in a stop codon in exon 11 (*) and will ultimately result in the production of a truncated dystrophin protein. Because the natural donor site is still present in the transcript there is a possibility that some full-length message, containing exon 10 is made and this is shown as the solid line. The ODNs, mdx47T and mdx47NT, shown above, were designed to correct the substitution mutation and the target base is underlined.
Mentions: The mdx5cv mouse has a point mutation in exon 10, which creates a splice donor site that results in a 53 base deletion within the exon (see diagram in Figure 2). This deletion results in a reading frameshift wherein a premature stop codon is created 95 bases downstream of the mutation site. If the ODN directs correction of the base, then the correct splice pattern is recreated and the 53 deleted bases remain in the message; in effect, restoring the reading frame. To evaluate the restoration of the 53 base pair insertion, a reverse primer was designed to bind to the deleted portion of exon 10. Thus, amplification can only occur if the targeted splice site corrected and the full-length mature message is produced at detectable levels. To determine the level of expression of dystrophin mRNA where the deleted region had been restored, mRNA was reverse-transcribed using a primer specific for exon 12, which is present in all of the transcripts, corrected or not.

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