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The amount of proteins present in cells of living organisms depends on various factors, including the rate of transcription of coding sequences, the efficiency with which pre-mRNA transcripts are processed and exported from the nucleus to the cytoplasm, the translatability of a transcript, and the stability of the mRNA. Research has demonstrated that addition of intronsIntrons to coding sequences can significantly increase eukaryotic in vivo expression levelsExpression levels. We present here a protocol for inserting intronsIntrons into coding sequences of interest to optimize expression. This protocol consists of the following steps: (1) codon-optimizing the coding sequence of interest, (2) defining positions for intronIntrons insertion, (3) selecting intronsIntrons to be introduced, (4) in silico insertion of intronsIntrons into coding sequences followed by sequence optimization, (5) finalizing the sequence design by addition of flanking restriction sites, and (6) ordering the sequences from a gene synthesis provider.

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Intronization of Coding Sequences for Optimization of Gene Expression

  • 实验方案

  • 发表日期：

    2025年

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  • Tom Schreiber,
  • Sylvestre Marillonnet

摘要

The amount of proteins present in cells of living organisms depends on various factors, including the rate of transcription of coding sequences, the efficiency with which pre-mRNA transcripts are processed and exported from the nucleus to the cytoplasm, the translatability of a transcript, and the stability of the mRNA. Research has demonstrated that addition of intronsIntrons to coding sequences can significantly increase eukaryotic in vivo expression levelsExpression levels. We present here a protocol for inserting intronsIntrons into coding sequences of interest to optimize expression. This protocol consists of the following steps: (1) codon-optimizing the coding sequence of interest, (2) defining positions for intronIntrons insertion, (3) selecting intronsIntrons to be introduced, (4) in silico insertion of intronsIntrons into coding sequences followed by sequence optimization, (5) finalizing the sequence design by addition of flanking restriction sites, and (6) ordering the sequences from a gene synthesis provider.