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一种新型的Cre诱导型敲入ARL13B-tRFP融合纤毛报告基因。

A novel Cre-inducible knock-in ARL13B-tRFP fusion cilium reporter.

作者信息

Schmitz Felizitas, Burtscher Ingo, Stauber Michael, Gossler Achim, Lickert Heiko

机构信息

Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Business Campus Garching, Parkring 11, Garching, D-85748, Germany.

Institute of Stem Cell Research, Helmholtz Zentrum München, Ingolstädter Landstraße 1, Neuherberg, D-85764, Germany.

出版信息

Genesis. 2017 Nov;55(11). doi: 10.1002/dvg.23073. Epub 2017 Oct 13.

Abstract

Cilia play a major role in the regulation of numerous signaling pathways and are essential for embryonic development. Mutations in genes affecting ciliary function can cause a variety of diseases in humans summarized as ciliopathies. To facilitate the detection and visualization of cilia in a temporal and spatial manner in mouse tissues, we generated a Cre-inducible cilium-specific reporter mouse line expressing an ARL13B-tRFP fusion protein driven by a CMV enhancer/chicken β actin promotor (pCAG) from the Hprt locus. We detected bright and specific ciliary signals by immunostainings of various mono- and multiciliated tissues and by time-lapse live-cell analysis of cultured embryos and organ explant cultures. Additionally, we monitored cilium assembly and disassembly in embryonic fibroblast cells using live-cell imaging. Thus, the ARL13B-tRFP reporter mouse strain is a valuable tool for the investigation of ciliary structure and function in a tissue-specific manner to understand processes, such as ciliary protein trafficking or cilium-dependent signaling in vitro and in vivo.

摘要

纤毛在众多信号通路的调节中起主要作用,对胚胎发育至关重要。影响纤毛功能的基因突变可导致人类多种疾病,统称为纤毛病。为了便于在小鼠组织中以时空方式检测和可视化纤毛,我们构建了一种Cre诱导的纤毛特异性报告基因小鼠品系,该品系从Hprt位点表达由CMV增强子/鸡β肌动蛋白启动子(pCAG)驱动的ARL13B-tRFP融合蛋白。我们通过对各种单纤毛和多纤毛组织进行免疫染色,以及对培养的胚胎和器官外植体培养物进行延时活细胞分析,检测到明亮且特异的纤毛信号。此外,我们使用活细胞成像监测胚胎成纤维细胞中的纤毛组装和拆卸。因此,ARL13B-tRFP报告基因小鼠品系是一种有价值的工具,可用于以组织特异性方式研究纤毛结构和功能,以了解体外和体内的纤毛蛋白运输或纤毛依赖性信号传导等过程。

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