CRISPR/Cas9 介导的 GFP 报告基因敲入 K562 和 Raji 细胞系用于跟踪免疫细胞杀伤试验。
CRISPR/Cas9-Mediated GFP Reporter Knock-in in K562 and Raji Cell Lines for Tracking Immune Cell Killing Assay.
机构信息
Siriraj Center for Regenerative Medicine, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
出版信息
Methods Mol Biol. 2021;2211:213-229. doi: 10.1007/978-1-0716-0943-9_15.
Cell-mediated cytotoxicity plays an important role in several fundamental immunological processes and is crucial for biological evaluation in in vitro studies. In order to determine the immunological activities of the cells, an assay should be safe, reproducible, and cost-effective. Here, we present a simple and cost-effective approach for evaluation of natural killer (NK) cell-mediated cytotoxicity by generating a CRISPR/Cas9-mediated GFP reporter knock-in in the target cell line, K562, and the non-target cell line, Raji, using a plasmid-based transfection method. The GFP target cells facilitate tracking of the immune cell killing assay, which avoids the need for multiple cell labeling with fluorescent dyes. Our approach is also applicable to the genome editing of other target cell types for functional analysis of effector cells.
细胞介导的细胞毒性在几个基本的免疫学过程中起着重要作用，对于体外研究中的生物学评估至关重要。为了确定细胞的免疫学活性，测定方法应该是安全、可重复且具有成本效益的。在这里，我们提出了一种简单且具有成本效益的方法，通过使用基于质粒的转染方法，在靶细胞系 K562 和非靶细胞系 Raji 中生成 CRISPR/Cas9 介导的 GFP 报告基因敲入，来评估自然杀伤 (NK) 细胞介导的细胞毒性。GFP 靶细胞有助于免疫细胞杀伤测定的跟踪，避免了使用荧光染料对多个细胞进行多次标记的需要。我们的方法也适用于其他靶细胞类型的基因组编辑，用于效应细胞的功能分析。
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