Next-generation sequencing has transformed our knowledge of the genetics of lymphoid malignancies. However, limited experimental systems are available to model the functional effects of these genetic changes and their implications for therapy. The majority of mature B-cell malignancies arise from the germinal center (GC) stage of B-cell differentiation. Here we describe a detailed protocol for the purification and ex vivo expansion of primary, nonmalignant human GC B cells. We present methodology for the high-efficiency transduction of these cells to enable combinatorial expression of putative oncogenes. We also describe alternative approaches for CRISPR–Cas9-mediated deletion of putative tumor suppressors. Mimicking genetic changes commonly found in lymphoid malignancies leads to immortalized growth in vitro, while engraftment into immunodeficient mice generates genetically customized, synthetic models of human lymphoma. The protocol is simple and inexpensive and can be implemented in any laboratory with access to standard cell culture and animal facilities. It can be easily scaled up to enable high-throughput screening and thus provides a versatile platform for the functional interrogation of lymphoma genomic data.

下一代测序改变了我们对淋巴恶性肿瘤遗传学的认识。然而，可用于模拟这些基因变化的功能效应及其对治疗的影响的实验系统有限。大多数成熟 B 细胞恶性肿瘤源自 B 细胞分化的生发中心 (GC) 阶段。在这里，我们描述了原代非恶性人类 GC B 细胞的纯化和离体扩增的详细方案。我们提出了这些细胞的高效转导方法，以实现假定癌基因的组合表达。我们还描述了 CRISPR-Cas9 介导的假定肿瘤抑制基因删除的替代方法。模仿淋巴恶性肿瘤中常见的基因变化会导致体外永生化生长，而植入免疫缺陷小鼠体内会产生基因定制的人类淋巴瘤合成模型。该方案简单且廉价，可以在任何能够使用标准细胞培养和动物设施的实验室中实施。它可以轻松扩展以实现高通量筛选，从而为淋巴瘤基因组数据的功能询问提供一个多功能平台。