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

Abstract

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.

The ribosomal protein L22 binds theMDM4pre-mRNA and promotes exon skipping to activate p53 upon nucleolar stress

“… A. A splicing reporter assay based on Fukushima et al, 2020 was used to investigate the sequence requirements needed for L22 to bind MDM4 pre-mRNA and regulate its splicing. To generate the control plasmid FMv2, the following sequences were inserted into the pcDNA3.1/Hygro(+) vector containing the CMV promoter by using the restriction enzymes Nhe I and Xho I: the mCherry coding sequence, the 5′ end of the eGFP gene (5′- eGFP ), the 5′ end of DMD intron 18 (5′ DMD intron 18), a Multiple Cloning Site (MCS), the 3′ end of DMD intron 19 (3′ DMD intron 19), and the 3′ end of the eGFP gene (3′- eGFP ).…”

“…For the assay, cells were grown on 24-well plates (3524, Corning Costar/Geyer), transfected with control (ctrl#1) or L22 (L22#1) siRNAs for 72h, and with control plasmid (FMv2) (Fukushima et al, 2020), MDM4 reporter plasmid (FMv2-MDM4) or different FMv2-MDM4 versions for 48h. Using the ZEISS Celldiscoverer 7 live cell fluorescence microscope with 5x magnification, red fluorescence (mCherry, exposure time 100 ms, intensity 30%) and green fluorescence (eGFP, exposure time 20 ms, intensity 30%) were detected, and five to six images per well were taken.…”

“…For the assay, cells were grown on 24-well plates (3524, Corning Costar/Geyer), transfected with control (ctrl) or L22 (L22) siRNAs for 72 h, and with control plasmid (FMv2) (Fukushima et al, 2020), MDM4 reporter plasmid (FMv2-MDM4) or different FMv2-MDM4 versions for 48 h.…”

Successful skipping of abnormal pseudoexon by antisense oligonucleotides in vitro for a patient with beta-propeller protein-associated neurodegeneration

“…Present study illustrates experimental utility of dual fluorescence splicing reporter minigene system in highthroughput screening therapeutically effective antisense oligonucleotides and will be readily applicable to any patients with splicing abnormalities of various genes 11 .…”

“…The fact that pseudoexon insertion is the cause of BPAN indicates that inducing pseudoexon skipping during splicing would normalize the patient's gene product, and that an antisense oligonucleotide (ASO) that induces pseudoexon skipping could be a treatment for this BPAN case. Therefore, to search for ASOs that induce pseudoexon skipping, we applied our recently developed mini-gene FMv2 11 , a dual fluorescent splicing reporter system www.nature.com/scientificreports/ that encodes two fluorescent proteins, mCherry, a transfection marker designed to facilitate evaluation of exon skipping and split eGFP, a splicing reaction marker, that is constructed from pcDNA3.1/Hygro (+) encoding a CMV promoter, a BGH-polyA signal and a drug resistance gene (V87020, Invitrogen by Thermo-Fisher Scientific, Waltham, MA, USA). The split eGFP is mediated by an artificial intron containing a multicloning site sequence where the genomic fragment to be analyzed for splicing can be cloned into.…”