Institute of Bioengineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
Methods Mol Biol. 2021;2312:15-33. doi: 10.1007/978-1-0716-1441-9_2.
Synthetic receptors control cell behavior in response to environmental stimuli for applications in basic research and cell therapy. However, the integration of synthetic receptors in unexplored contexts is cumbersome, especially for nonspecialist laboratories. Here, I provide a detailed protocol on how to use receptors of the generalized extracellular molecule sensor (GEMS) platform. GEMS is a modular receptor system that can be adapted to sense molecules of choice by using affinity domains that dimerize in response to the target. GEMS consist of an erythropoietin receptor scaffold that has been mutated to no longer bind to erythropoietin. N-terminal fusions with affinity domains, such as single chain variable fragments (scFvs), that bind to two epitopes on the same target activate the receptor. The intracellular receptor domain can be chosen from several signal transduction domains of single-pass transmembrane receptors to activate endogenous signaling pathways. As of now, GEMS have been used for sensing prostate specific antigen (PSA), the synthetic azo dye RR120, caffeine, nicotine, rapamycin, the SunTag peptide, and a de novo designed protein displaying two viral epitopes. The tested intracellular domains were derived from FGFR1, IL-6RB, and VEGFR2, and were used to drive transgene expression from reporter plasmids responsive to the endogenous transcription factors STAT3, NFAT, NF-κB, and a synthetic transcription factor activated by the MAPK pathway. In this protocol, I focus on transient transfections of HEK293T cells and include several general notes about cell handling. While the described methods are optimized for experiments with GEMS, most of the described techniques are general procedures to set up synthetic biology experiments in mammalian cell culture. I outline how to generate stable cell lines and share tips on how to adapt GEMS for new ligands. The main objective of this protocol is to make the GEMS technology accessible also to nonspecialist laboratories to facilitate the use of synthetic receptors in new research contexts.
合成受体可控制细胞行为以响应环境刺激，从而应用于基础研究和细胞治疗。然而，在未知的环境中整合合成受体是繁琐的，特别是对于非专业实验室。在此，我提供了一个关于如何使用广义细胞外分子传感器（GEMS）平台的受体的详细方案。GEMS 是一种模块化受体系统，通过使用对靶标响应二聚化的亲和结构域，可适应对选择分子的检测。GEMS 由已突变为不再与促红细胞生成素结合的促红细胞生成素受体支架组成。与亲和结构域（如单链可变片段（scFv））的 N 端融合，这些结构域可结合同一靶标上的两个表位，从而激活受体。细胞内受体结构域可从单跨膜受体的几种信号转导结构域中选择，以激活内源性信号通路。到目前为止，GEMS 已用于检测前列腺特异性抗原（PSA）、合成偶氮染料 RR120、咖啡因、尼古丁、雷帕霉素、SunTag 肽和新设计的展示两个病毒表位的蛋白质。所测试的细胞内结构域来自 FGFR1、IL-6RB 和 VEGFR2，并用于驱动来自报告质粒的转基因表达，该报告质粒对 STAT3、NFAT、NF-κB 和通过 MAPK 途径激活的合成转录因子作出反应。在本方案中，我专注于 HEK293T 细胞的瞬时转染，并包括有关细胞处理的几个一般注意事项。虽然所描述的方法是针对 GEMS 实验进行了优化，但大多数描述的技术都是在哺乳动物细胞培养中建立合成生物学实验的一般程序。我概述了如何生成稳定细胞系，并分享了一些有关如何为新配体改编 GEMS 的技巧。本方案的主要目的是使 GEMS 技术也可供非专业实验室使用，以促进在新的研究环境中使用合成受体。
