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改造和优化 GCaMP8f 使其可用于秀丽隐杆线虫。

Adapting and optimizing GCaMP8f for use in Caenorhabditis elegans.

机构信息

Max Planck Research Group Neural Information Flow, Max Planck Institute for Neurobiology of Behavior-caesar, Bonn 53175, Germany.

International Max Planck Research School for Brain and Behavior, Bonn 53175, Germany.

出版信息

Genetics. 2024 Oct 7;228(2). doi: 10.1093/genetics/iyae125.

Abstract

Improved genetically encoded calcium indicators (GECIs) are essential for capturing intracellular dynamics of both muscle and neurons. A novel set of GECIs with ultrafast kinetics and high sensitivity was recently reported by Zhang et al. (2023). While these indicators, called jGCaMP8, were demonstrated to work in Drosophila and mice, data for Caenorhabditis elegans were not reported. Here, we present an optimized construct for C. elegans and use this to generate several strains expressing GCaMP8f (fast variant of the indicator). Utilizing the myo-2 promoter, we compare pharyngeal muscle activity measured with GCaMP7f and GCaMP8f and find that GCaMP8f is brighter upon binding to calcium, shows faster kinetics, and is not disruptive to the intrinsic contraction dynamics of the pharynx. Additionally, we validate its application for detecting neuronal activity in touch receptor neurons which reveals robust calcium transients even at small stimulus amplitudes. As such, we establish GCaMP8f as a potent tool for C. elegans research which is capable of extracting fast calcium dynamics at very low magnifications across multiple cell types.

摘要

新型的超快动力学和高灵敏度的基因编码钙指示剂(GECIs)最近由 Zhang 等人报道。虽然这些被称为 jGCaMP8 的指示剂已被证明可在果蝇和小鼠中发挥作用,但尚未报告其在秀丽隐杆线虫中的数据。在这里,我们为秀丽隐杆线虫提供了一个经过优化的构建体,并利用它生成了几种表达 GCaMP8f(该指示剂的快速变体)的菌株。利用肌球蛋白-2 启动子,我们比较了用 GCaMP7f 和 GCaMP8f 测量的咽肌活性,发现 GCaMP8f 与钙结合时更亮,动力学更快,并且不会破坏咽的内在收缩动力学。此外,我们验证了其在检测触须感觉神经元中的神经元活性的应用,即使在小刺激幅度下也能产生强烈的钙瞬变。因此,我们确立了 GCaMP8f 作为秀丽隐杆线虫研究的有力工具,它能够在多个细胞类型中以非常低的放大倍数提取快速钙动力学。

相似文献

2
Intracellular Ca2+ imaging in C. elegans.秀丽隐杆线虫中的细胞内钙离子成像
Methods Mol Biol. 2006;351:253-64. doi: 10.1385/1-59745-151-7:253.

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