Identifying Signalling Pathways Regulated by GPRC5B in β-Cells by CRISPR-Cas9-Mediated Genome Editing
Identifying Signalling Pathways Regulated by GPRC5B in β-Cells by CRISPR-Cas9-Mediated Genome Editing
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DOI：
10.1159/000487159
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发表时间：
2018-01-01
影响因子：
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通讯作者：
Amisten, Stefan
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文献类型：
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作者：
Atanes, Patricio;Ruz-Maldonado, Inmaculada;Amisten, Stefan
背景/目标：CRISPR-Cas9 是一种 RNA 引导的靶向基因组编辑工具，通过提供精确修改 DNA 的能力，彻底改变了基因工程。 GPRC5B 是属于 G 蛋白偶联受体 (GPCR) C 族家族的孤儿受体。在本研究中，我们使用 CRISPR-Cas9 和 Gprc5b 的瞬时过表达分析了 MIN6 β 细胞中 Gprc5b 受体的功能作用。方法：通过细胞分选分析 GFP 质粒递送的效率，确定用于 MIN6 β 细胞的最佳转染试剂。使用 CRISPR-Cas9 技术生成了 Gprc5b 表达被敲低的 MIN6 β 细胞系 (Gprc5b KD)。使用瞬时转染不同浓度的小鼠 Gprc5b 质粒以过表达 Gprc5b 的 Gpcr5b KD MIN6 β 细胞进行 Gprc5b 受体 mRNA 表达、增殖、凋亡、Cignal 45-Pathway Reporter Array 信号传导和蛋白质印迹分析。结果：JetPRIME (R) 是 MIN6 β 细胞转染的最佳候选，提供约 30% 的转染效率。针对 Gprc5b 的 CRISPR-Cas9 技术导致 MIN6 β 细胞中该受体的稳定敲低，其重新表达诱导增殖并增强细胞因子和棕榈酸酯诱导的细胞凋亡。 Cignal 45 Reporter 分析表明细胞凋亡和增殖途径依赖于 Gprc5b 调节，蛋白质印迹证实通过 TGF-β 和 IFNg gamma 激活信号传导。 结论：本研究提供了 CRISPR-Cas9 技术用于下调 MIN6 β 细胞中 Gprc5b 表达的证据。这一策略使我们能够确定将 GPRC5B 受体表达与 b 细胞增殖和凋亡联系起来的信号通路。 (c) 2018 年作者由 S. Karger AG 出版，巴塞尔
Background/Aims: CRISPR-Cas9, a RNA-guided targeted genome editing tool, has revolutionized genetic engineering by offering the ability to precisely modify DNA. GPRC5B is an orphan receptor belonging to the group C family of G protein-coupled receptors (GPCRs). In this study, we analysed the functional roles of the Gprc5b receptor in MIN6 beta-cells using CRISPR-Cas9 and transient over-expression of Gprc5b. Methods: The optimal transfection reagent for use in MIN6 beta-cells was determined by analysing efficiency of GFP plasmid delivery by cell sorting. A MIN6 beta-cell line in which Gprc5b expression was knocked down (Gprc5b KD) was generated using CRISPR-Cas9 technology. Gprc5b receptor mRNA expression, proliferation, apoptosis, Cignal 45-Pathway Reporter Array signalling and western blot assays were carried out using Gpcr5b KD MIN6 beta-cells that had been transiently transfected with different concentrations of mouse Gprc5b plasmid to over-express Gprc5b. Results: JetPRIME (R) was the best candidate for MIN6 beta-cell transfection, providing approximately 30% transfection efficiency. CRISPR-Cas9 technology targeting Gprc5b led to stable knock-down of this receptor in MIN6 beta-cells and its re-expression induced proliferation and potentiated cytokine-and palmitate-induced apoptosis. The Cignal 45 Reporter analysis indicated Gprc5b-dependent regulation of apoptotic and proliferative pathways, and western blotting confirmed activation of signalling via TGF-beta and IFNg gamma Conclusion: This study provides evidence of CRISPR-Cas9 technology being used to down-regulate Gprc5b expression in MIN6 beta-cells. This strategy allowed us to identify signalling pathways linking GPRC5B receptor expression to b-cell proliferation and apoptosis. (c) 2018 The Author(s) Published by S. Karger AG, Basel