Genetic manipulation and immortalized culture of ex vivo primary human germinal center B cells
Genetic manipulation and immortalized culture of ex vivo primary human germinal center B cells
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DOI：
10.1038/s41596-021-00506-4
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发表时间：
2021-04-09
期刊：
影响因子：
14.8
通讯作者：
Hodson, Daniel J.
中科院分区：
文献类型：
--
作者：
Caeser, Rebecca;Gao, Jie;Hodson, Daniel J.
纯化原代非恶性人GC B细胞并在培养物中扩增。基因操作和移植到小鼠中使得能够产生基因定制的人类淋巴瘤的合成模型。下一代测序已经改变了我们对淋巴恶性肿瘤遗传学的认识。然而，有限的实验系统可用于模拟这些遗传变化的功能影响及其对治疗的影响。大多数成熟B细胞恶性肿瘤来自B细胞分化的生发中心（GC）阶段。在这里，我们描述了一个详细的协议的纯化和体外扩增的主要，非恶性的人GC B细胞。我们目前的方法，这些细胞的高效率转导，使组合表达推定的癌基因。我们还描述了CRISPR-Cas9介导的推定肿瘤抑制因子缺失的替代方法。模仿淋巴恶性肿瘤中常见的遗传变化导致体外永生化生长，而移植到免疫缺陷小鼠中产生遗传定制的人类淋巴瘤合成模型。该协议是简单和廉价的，可以在任何实验室与访问标准的细胞培养和动物设施。它可以很容易地扩大规模，使高通量筛选，从而提供了一个多功能的平台，淋巴瘤基因组数据的功能询问。
Primary, nonmalignant, human GC B cells are purified and expanded in culture. Genetic manipulation and engraftment into mice enable the generation of genetically customized, synthetic models of human lymphoma.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.