Fukushima Sachiyo, Farea Manal, Maeta Kazuhiro, Rani Abdul Qawee Mahyoob, Fujioka Kazumichi, Nishio Hisahide, Matsuo Masafumi
Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
Research Center for Locomotion Biology, Kobe Gakuin University, Kobe 651-2180, Japan.
Int J Mol Sci. 2020 Nov 30;21(23):9136. doi: 10.3390/ijms21239136.
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 FMv2v8, a modified clone of FMv2 carrying an insertion of 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.
剪接报告基因微型基因用于基于细胞的体外剪接研究。通过微型基因剪接试验已鉴定出外显子可跳跃反义寡核苷酸（ASO），但这些试验包括逆转录PCR扩增这一耗时且昂贵的步骤。为了使体外剪接试验更简便，构建了一种适用于通过荧光显微镜进行定量剪接分析的即用型微型基因（FMv2）。FMv2被设计用于编码两种荧光蛋白，即作为转染标记的mCherry和作为剪接反应标记的分裂型eGFP。分裂型eGFP被一个包含多克隆位点序列的人工内含子所间隔。不出所料，转染FMv2的HeLa细胞不仅产生了红色的mCherry信号，还产生了绿色的eGFP信号。转染FMv2v8（FMv2的一个修饰克隆，在多克隆位点插入了外显子v8）用于筛选可跳跃外显子v8的ASO，结果只产生了红色信号。在针对外显子v8测试的七种不同ASO中，ASO#14产生的绿色信号阳性细胞指数最高。因此，ASO#14是最有效的可跳跃外显子v8的ASO。值得注意的是，在随机添加ASO的96孔板中能够清晰地识别出含有ASO#14的孔，从而实现高通量筛选。预计这种即用型FMv2将有助于鉴定可跳跃外显子的ASO。
