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Somatic hypermutation (SHM) is a critical process in adaptive immunity, enabling the generation of high-affinity antibodies through targeted mutations in immunoglobulin variable (IgV) regions. Here, we provide a comprehensive workflow combining immunization, molecular biology, and bioinformatics to investigate SHM mechanisms and outcomes. This is a general protocol for studying SHM at the VH186.2 region in C57BL/6 mice. While this method can be applied broadly, this chapter will detail the protocol used to test the effects of exonuclease 1(EXO1) knock-in mutation (Exo1D173A, or Exo1DA) or knock-out (KO) on hypermutation post-immunization by immunizing age-matched mice with NP(33)-CGG on alum. We start by immunizing and sacrificing the animals to obtain spleens for RNA extraction. We then create cDNA libraries and investigate VH186.2 region mutation to analyze SHM.

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The Experimental Procedure of Somatic Hypermutation Analysis in Splenic B Cells During Primary Response

  • 实验方案

  • 发表日期：

    2026年

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  • Seth Greene,
  • Alyssa Fennig-Victor,
  • Yongwei Zhang,
  • Shanzhi Wang

摘要

Somatic hypermutation (SHM) is a critical process in adaptive immunity, enabling the generation of high-affinity antibodies through targeted mutations in immunoglobulin variable (IgV) regions. Here, we provide a comprehensive workflow combining immunization, molecular biology, and bioinformatics to investigate SHM mechanisms and outcomes. This is a general protocol for studying SHM at the VH186.2 region in C57BL/6 mice. While this method can be applied broadly, this chapter will detail the protocol used to test the effects of exonuclease 1(EXO1) knock-in mutation (Exo1D173A, or Exo1DA) or knock-out (KO) on hypermutation post-immunization by immunizing age-matched mice with NP(33)-CGG on alum. We start by immunizing and sacrificing the animals to obtain spleens for RNA extraction. We then create cDNA libraries and investigate VH186.2 region mutation to analyze SHM.