Novel EGFP reporter cell and mouse models for sensitive imaging and quantification of exon skipping
Duchenne muscular dystrophy (DMD) is a fatal X-linked disorder caused by nonsense or frameshift mutations in the DMD gene. Among various treatments available for DMD, antisense oligonucleotides (ASOs) mediated exon skipping is a promising therapeutic approach. For successful treatments, however, it...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
Nature Research
2020
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| _version_ | 1797077308017737728 |
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| author | Hara, Y Mizobe, Y Inoue, YU Hashimoto, Y Motohashi, N Masaki, Y Seio, K Takeda, S Nagata, T Wood, MJA Inoue, T Aoki, Y |
| author_facet | Hara, Y Mizobe, Y Inoue, YU Hashimoto, Y Motohashi, N Masaki, Y Seio, K Takeda, S Nagata, T Wood, MJA Inoue, T Aoki, Y |
| author_sort | Hara, Y |
| collection | OXFORD |
| description | Duchenne muscular dystrophy (DMD) is a fatal X-linked disorder caused by nonsense or frameshift mutations in the DMD gene. Among various treatments available for DMD, antisense oligonucleotides (ASOs) mediated exon skipping is a promising therapeutic approach. For successful treatments, however, it is requisite to rigorously optimise oligonucleotide chemistries as well as chemical modifications of ASOs. To achieve this, here, we aim to develop a novel enhanced green fluorescence protein (EGFP)-based reporter assay system that allows us to perform efficient and high-throughput screenings for ASOs. We design a new expression vector with a CAG promoter to detect the EGFP fluorescence only when skipping of mdx-type exon 23 is induced by ASOs. Then, an accurate screening was successfully conducted in C57BL/6 primary myotubes using phosphorodiamidate morpholino oligomer or locked nucleic acids (LNA)/2′-OMe mixmers with different extent of LNA inclusion. We accordingly generated a novel transgenic mouse model with this EGFP expression vector (EGFP-mdx23 Tg). Finally, we confirmed that the EGFP-mdx23 Tg provided a highly sensitive platform to check the effectiveness as well as the biodistribution of ASOs for exon skipping therapy. Thus, the assay system provides a simple yet highly sensitive platform to optimise oligonucleotide chemistries as well as chemical modifications of ASOs. |
| first_indexed | 2024-03-07T00:16:06Z |
| format | Journal article |
| id | oxford-uuid:7ae2702d-8c80-47a4-8ef4-22385eb5ee8e |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T00:16:06Z |
| publishDate | 2020 |
| publisher | Nature Research |
| record_format | dspace |
| spelling | oxford-uuid:7ae2702d-8c80-47a4-8ef4-22385eb5ee8e2022-03-26T20:47:06ZNovel EGFP reporter cell and mouse models for sensitive imaging and quantification of exon skippingJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:7ae2702d-8c80-47a4-8ef4-22385eb5ee8eEnglishSymplectic ElementsNature Research 2020Hara, YMizobe, YInoue, YUHashimoto, YMotohashi, NMasaki, YSeio, KTakeda, SNagata, TWood, MJAInoue, TAoki, YDuchenne muscular dystrophy (DMD) is a fatal X-linked disorder caused by nonsense or frameshift mutations in the DMD gene. Among various treatments available for DMD, antisense oligonucleotides (ASOs) mediated exon skipping is a promising therapeutic approach. For successful treatments, however, it is requisite to rigorously optimise oligonucleotide chemistries as well as chemical modifications of ASOs. To achieve this, here, we aim to develop a novel enhanced green fluorescence protein (EGFP)-based reporter assay system that allows us to perform efficient and high-throughput screenings for ASOs. We design a new expression vector with a CAG promoter to detect the EGFP fluorescence only when skipping of mdx-type exon 23 is induced by ASOs. Then, an accurate screening was successfully conducted in C57BL/6 primary myotubes using phosphorodiamidate morpholino oligomer or locked nucleic acids (LNA)/2′-OMe mixmers with different extent of LNA inclusion. We accordingly generated a novel transgenic mouse model with this EGFP expression vector (EGFP-mdx23 Tg). Finally, we confirmed that the EGFP-mdx23 Tg provided a highly sensitive platform to check the effectiveness as well as the biodistribution of ASOs for exon skipping therapy. Thus, the assay system provides a simple yet highly sensitive platform to optimise oligonucleotide chemistries as well as chemical modifications of ASOs. |
| spellingShingle | Hara, Y Mizobe, Y Inoue, YU Hashimoto, Y Motohashi, N Masaki, Y Seio, K Takeda, S Nagata, T Wood, MJA Inoue, T Aoki, Y Novel EGFP reporter cell and mouse models for sensitive imaging and quantification of exon skipping |
| title | Novel EGFP reporter cell and mouse models for sensitive imaging and quantification of exon skipping |
| title_full | Novel EGFP reporter cell and mouse models for sensitive imaging and quantification of exon skipping |
| title_fullStr | Novel EGFP reporter cell and mouse models for sensitive imaging and quantification of exon skipping |
| title_full_unstemmed | Novel EGFP reporter cell and mouse models for sensitive imaging and quantification of exon skipping |
| title_short | Novel EGFP reporter cell and mouse models for sensitive imaging and quantification of exon skipping |
| title_sort | novel egfp reporter cell and mouse models for sensitive imaging and quantification of exon skipping |
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