Research Group Medical Biotechnology & Bioengineering, Faculty of Applied Natural Sciences, TH Köln - University of Applied Sciences, Campus Leverkusen, Leverkusen, Germany.
Institute of Technical Chemistry, Leibniz University Hannover, Hannover, Germany.
Methods Mol Biol. 2023;2681:361-371. doi: 10.1007/978-1-0716-3279-6_20.
Suspension cells derived from human embryonic kidney cells (HEK 293) are attractive cell lines for retroviral vector production in gene therapeutic development studies and applications. The low-affinity nerve growth factor receptor (NGFR) is a genetic marker frequently used as a reporter gene in transfer vectors to detect and enrich genetically modified cells. However, the HEK 293 cell line and its derivatives endogenously express the NGFR protein. To eradicate the high background NGFR expression in future retroviral vector packaging cells, we here employed the CRISPR/Cas9 system to generate human suspension 293-F NGFR knockout cells. The expression of a fluorescent protein coupled via a 2A peptide motif to the NGFR targeting Cas9 endonuclease enabled the simultaneous depletion of cells expressing Cas9 and remaining NGFR-positive cells. Thus, a pure population of NGFR-negative 293-F cells lacking persistent Cas9 expression was obtained in a simple and easily applicable procedure.
悬浮细胞来源于人胚肾细胞（HEK 293），是基因治疗开发研究和应用中逆转录病毒载体生产的有吸引力的细胞系。低亲和力神经生长因子受体（NGFR）是一种遗传标记，常用于转移载体中作为报告基因，以检测和富集基因修饰细胞。然而，HEK 293 细胞系及其衍生物内源性表达 NGFR 蛋白。为了消除未来逆转录病毒载体包装细胞中高背景的 NGFR 表达，我们在此使用 CRISPR/Cas9 系统生成了人悬浮 293-F NGFR 敲除细胞。通过 2A 肽基序与 NGFR 靶向 Cas9 内切酶偶联的荧光蛋白的表达，使表达 Cas9 的细胞和剩余的 NGFR 阳性细胞同时耗竭。因此，通过简单且易于应用的程序获得了缺乏持续 Cas9 表达的纯 NGFR 阴性 293-F 细胞群体。
