Department of Biology, The City College of New York and The Graduate Center of The City University of New York.
Department of Microbiology, Icahn School of Medicine at Mount Sinai.
J Vis Exp. 2021 Apr 20(170). doi: 10.3791/61551.
Within the germinal centers of lymphoid organs, mature B cells alter their expressed immunoglobulin (Ig) by introducing untemplated mutations into the variable coding exons of the Ig heavy and light chain gene loci. This process of somatic hypermutation (SHM) requires the enzyme activation-induced cytidine deaminase (AID), which converts deoxycytidines (C), into deoxyuridines (U). Processing the AID-generated U:G mismatches into mutations by the base excision and mismatch repair pathways introduces new Ig coding sequences that may produce a higher affinity Ig. Mutations in AID or DNA repair genes can block or significantly alter the types of mutations observed in the Ig loci. We describe a protocol to quantify JH4 intron mutations that uses fluorescence activated cell sorting (FACS), PCR, and Sanger sequencing. Although this assay does not directly measure Ig affinity maturation, it is indicative of mutations in Ig variable coding sequences. Additionally, these methods utilize common molecular biology techniques which analyze mutations in Ig sequences of multiple B cell clones. Thus, this assay is an invaluable tool in the study of SHM and Ig diversification.
在淋巴器官的生发中心，成熟 B 细胞通过在免疫球蛋白（Ig）重链和轻链基因座的可变编码外显子中引入无模板突变来改变其表达的 Ig。这个体细胞超突变（SHM）的过程需要酶激活诱导的胞嘧啶脱氨酶（AID），它将脱氧胞嘧啶（C）转化为脱氧尿嘧啶（U）。通过碱基切除和错配修复途径处理 AID 产生的 U:G 错配，将新的 Ig 编码序列引入到可能产生更高亲和力 Ig 的突变中。AID 或 DNA 修复基因的突变可以阻断或显著改变 Ig 基因座中观察到的突变类型。我们描述了一种使用荧光激活细胞分选（FACS）、PCR 和 Sanger 测序定量 JH4 内含子突变的方案。虽然该测定法不能直接测量 Ig 亲和力成熟，但它表明 Ig 可变编码序列中的突变。此外，这些方法利用常见的分子生物学技术来分析多个 B 细胞克隆的 Ig 序列中的突变。因此，该测定法是研究 SHM 和 Ig 多样化的宝贵工具。
