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Antisense oligonucleotides (ASOs) are commonly used to induce transient changes in gene expression and do not result in mutagenic effects at the DNA level. In this chapter, we describe a novel gene silencing approach using splice-switching oligonucleotides (SSOs) capable of hybridizing at a splice site to induce exon skipping. This strategy is based on the elimination of an exon whose nucleotide length is not a multiple of 3. In this way, exon skipping induces a frameshift in the reading frame and the appearance of a premature termination codon (PTC) on the alternative transcript. After detection of the PTC, alternative transcripts are degraded by the NMD pathway. Here, we present the steps involved in the SSO design, cell treatment, and assessment of gene silencing. We provide evidence for efficient knockdown of Enhancer of Zeste Homolog 2 (EZH2) expression in lymphoid cells.

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Knockdown of Gene Expression Following Activation of the NMD Pathway Using Splice-Switching Oligonucleotides

  • 实验方案

  • 发表日期：

    2025年

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  • Pierre Hirt,
  • Anne Marchalot,
  • Catherine Horiot,
  • Jérôme Moreaux,
  • Laurent Delpy

摘要

Antisense oligonucleotides (ASOs) are commonly used to induce transient changes in gene expression and do not result in mutagenic effects at the DNA level. In this chapter, we describe a novel gene silencing approach using splice-switching oligonucleotides (SSOs) capable of hybridizing at a splice site to induce exon skipping. This strategy is based on the elimination of an exon whose nucleotide length is not a multiple of 3. In this way, exon skipping induces a frameshift in the reading frame and the appearance of a premature termination codon (PTC) on the alternative transcript. After detection of the PTC, alternative transcripts are degraded by the NMD pathway. Here, we present the steps involved in the SSO design, cell treatment, and assessment of gene silencing. We provide evidence for efficient knockdown of Enhancer of Zeste Homolog 2 (EZH2) expression in lymphoid cells.