Construction of a tri-chromatic reporter cell line for the rapid and simple screening of splice-switching oligonucleotides targeting DMD exon 51 using high content screening

PloS One
Takenori ShimoSatoshi Obika

Abstract

Splice-switching oligonucleotides (SSOs) that can modulate RNA splicing are used for the treatment of many genetic disorders. To enhance the efficacy of modulating splicing, it is important to optimize SSOs with regard to target sites, GC content, melting temperature (Tm value), chemistries, and lengths. Thus, in vitro assay systems that allow for the rapid and simple screening of SSOs are essential for optimizing SSO design. In this study, we established a novel tri-chromatic reporter cell line for SSO screening. This reporter cell line is designed to express three different fluorescent proteins (blue, green, and red) and was employed for high content screening (HCS, also known as high content analysis; HCA) for the evaluation of SSO-induced exon skipping by analyzing the expression levels of fluorescent proteins. The blue fluorescent protein is stably expressed throughout the cell and is useful for data normalization using cell numbers. Furthermore, both the green and red fluorescent proteins were used for monitoring the splicing patterns of target genes. Indeed, we demonstrated that this novel reporter cell line involving HCS leads to a more rapid and simple approach for the evaluation of exon skipping than widely used metho...Continue Reading

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Citations

Dec 14, 2018·Journal of Personalized Medicine·Yusuke EchigoyaToshifumi Yokota
Dec 4, 2020·International Journal of Molecular Sciences·Sachiyo FukushimaMasafumi Matsuo
Aug 14, 2021·Nature Biotechnology·Nicolas CardozoJeff Nivala

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Methods Mentioned

BETA
fluorescence microscopy
PCR
transfection
antisense oligonucleotides

Software Mentioned

ESE finder
Primer blast
GGRNA
EZR
ImageJ
ToxInsight

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