Design and Evaluation of a Truncated Protein Induction Strategy by Antisense Oligonucleotide-Mediated Exon Skipping.

Antisense oligonucleotide (ASO)-mediated exon skipping is an effective method for generating truncated proteins by altering mRNA splicing. This technique, enabling in situ truncation of proteins, provides a rapid way to investigate the functions of specific domains encoded by the targeted exon(s). In this methodological chapter, we focus on the mouse transcription factor ARID3A (AT-rich interactive domain 3A), which contains a DNA-binding domain (ARID domain) and a nuclear matrix-binding domain (Rekles Beta domain). We describe the design of an ASO targeting the donor splice site of Arid3a exon 8, which primarily encodes the Rekles beta domain, to generate a truncated protein lacking nuclear matrix-binding capacity while preserving an open reading frame. The ASO is specifically designed to hybridize only with Arid3a pre-mRNA, thus minimizing off-target effects. The effect of the ASO is evaluated first by RT-PCR to measure the proportion of alternative transcripts with skipped exon. Alternative transcripts can be subsequently characterized by Sanger sequencing. RT-qPCR is then performed outside the targeted exon to ensure that the overall transcription of the gene is unaffected and to exclude RNA degradation. Finally, protein analysis techniques, such as cellular imaging using immunofluorescence labeling, are used to assess the production of the truncated protein. This ASO-mediated exon skipping approach provides a reliable method for exploring the functional importance of specific protein domains.