Dual Fluorescence Splicing Reporter Minigene Identifies an Antisense Oligonucleotide to Skip Exon v8 of the <i>CD44</i> Gene
Splicing reporter minigenes are used in cell-based in vitro splicing studies. Exon skippable antisense oligonucleotide (ASO) has been identified using minigene splicing assays, but these assays include a time- and cost-consuming step of reverse transcription PCR amplification. To make in vitro splic...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2020-11-01
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| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/21/23/9136 |
| Summary: | Splicing reporter minigenes are used in cell-based in vitro splicing studies. Exon skippable antisense oligonucleotide (ASO) has been identified using minigene splicing assays, but these assays include a time- and cost-consuming step of reverse transcription PCR amplification. To make in vitro splicing assay easier, a ready-made minigene (FMv2) amenable to quantitative splicing analysis by fluorescence microscopy was constructed. FMv2 was designed to encode two fluorescence proteins namely, mCherry, a transfection marker and split eGFP, a marker of splicing reaction. The split eGFP was intervened by an artificial intron containing a multicloning site sequence. Expectedly, FMv2 transfected HeLa cells produced not only red mCherry but also green eGFP signals. Transfection of FMv2<i>CD44</i>v8, a modified clone of FMv2 carrying an insertion of <i>CD44</i> exon v8 in the multicloning site, that was applied to screen exon v8 skippable ASO, produced only red signals. Among seven different ASOs tested against exon v8, ASO#14 produced the highest index of green signal positive cells. Hence, ASO#14 was the most efficient exon v8 skippable ASO. Notably, the well containing ASO#14 was clearly identified among the 96 wells containing randomly added ASOs, enabling high throughput screening. A ready-made FMv2 is expected to contribute to identify exon skippable ASOs. |
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| ISSN: | 1661-6596 1422-0067 |