Genetic manipulation and immortalized culture of ex vivo primary human germinal center B cells

Autor: Miriam Di Re, Chun Gong, Rebecca Caeser, Daniel J. Hodson, Jie Gao
Přispěvatelé: Hodson, Daniel J [0000-0001-6225-2033], Apollo - University of Cambridge Repository
Rok vydání: 2021
Předmět:
Zdroj: Nature Protocols. 16:2499-2519
ISSN: 1750-2799
1754-2189
DOI: 10.1038/s41596-021-00506-4
Popis: 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.
Databáze: OpenAIRE
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