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Open AccessArticle

Dual Fluorescence Splicing Reporter Minigene Identifies an Antisense Oligonucleotide to Skip Exon v8 of the CD44 Gene

1
Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan
2
Research Center for Locomotion Biology, Kobe Gakuin University, Kobe 651-2180, Japan
3
KNC Department of Nucleic Acid Drug Discovery, Faculty of Rehabilitation, Kobe Gakuin University, Kobe 651-2180, Japan
4
Department of Occupational Therapy, Faculty of Rehabilitation, Kobe Gakuin University, Kobe 651-2180, Japan
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Int. J. Mol. Sci. 2020, 21(23), 9136; https://doi.org/10.3390/ijms21239136
Received: 2 October 2020 / Revised: 25 November 2020 / Accepted: 29 November 2020 / Published: 30 November 2020
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 FMv2CD44v8, a modified clone of FMv2 carrying an insertion of CD44 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. View Full-Text
Keywords: antisense oligonucleotide; minigene; exon skipping; splicing; dual fluorescence; CD44 antisense oligonucleotide; minigene; exon skipping; splicing; dual fluorescence; CD44
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MDPI and ACS Style

Fukushima, S.; Farea, M.; Maeta, K.; Rani, A.Q.M.; Fujioka, K.; Nishio, H.; Matsuo, M. Dual Fluorescence Splicing Reporter Minigene Identifies an Antisense Oligonucleotide to Skip Exon v8 of the CD44 Gene. Int. J. Mol. Sci. 2020, 21, 9136. https://doi.org/10.3390/ijms21239136

AMA Style

Fukushima S, Farea M, Maeta K, Rani AQM, Fujioka K, Nishio H, Matsuo M. Dual Fluorescence Splicing Reporter Minigene Identifies an Antisense Oligonucleotide to Skip Exon v8 of the CD44 Gene. International Journal of Molecular Sciences. 2020; 21(23):9136. https://doi.org/10.3390/ijms21239136

Chicago/Turabian Style

Fukushima, Sachiyo; Farea, Manal; Maeta, Kazuhiro; Rani, Abdul Q.M.; Fujioka, Kazumichi; Nishio, Hisahide; Matsuo, Masafumi. 2020. "Dual Fluorescence Splicing Reporter Minigene Identifies an Antisense Oligonucleotide to Skip Exon v8 of the CD44 Gene" Int. J. Mol. Sci. 21, no. 23: 9136. https://doi.org/10.3390/ijms21239136

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