Mouse Biology Unit, European Molecular Biology Laboratory Monterotondo, Italy.
Front Mol Neurosci. 2013 May 21;6:11. doi: 10.3389/fnmol.2013.00011. eCollection 2013.
Chloride is the most abundant physiological anion and participates in a variety of cellular processes including trans-epithelial transport, cell volume regulation, and regulation of electrical excitability. The development of tools to monitor intracellular chloride concentration ([Cli]) is therefore important for the evaluation of cellular function in normal and pathological conditions. Recently, several Cl-sensitive genetically encoded probes have been described which allow for non-invasive monitoring of [Cli]. Here we describe two mouse lines expressing a CFP-YFP-based Cl probe called Cl-Sensor. First, we generated transgenic mice expressing Cl-Sensor under the control of the mouse Thy1 mini promoter. Cl-Sensor exhibited good expression from postnatal day two (P2) in neurons of the hippocampus and cortex, and its level increased strongly during development. Using simultaneous whole-cell monitoring of ionic currents and Cl-dependent fluorescence, we determined that the apparent EC 50 for Cli was 46 mM, indicating that this line is appropriate for measuring neuronal [Cli] in postnatal mice. We also describe a transgenic mouse reporter line for Cre-dependent conditional expression of Cl-Sensor, which was targeted to the Rosa26 locus and by incorporating a strong exogenous promoter induced robust expression upon Cre-mediated recombination. We demonstrate high levels of tissue-specific expression in two different Cre-driver lines targeting cells of the myeloid lineage and peripheral sensory neurons. Using these mice the apparent EC 50 for Cli was estimated to be 61 and 54 mM in macrophages and DRG, respectively. Our data suggest that these mouse lines will be useful models for ratiometric monitoring of Cli in specific cell types in vivo.
氯离子是最丰富的生理阴离子，参与多种细胞过程，包括跨上皮转运、细胞体积调节和电兴奋性调节。因此，开发监测细胞内氯离子浓度([Cli])的工具对于评估正常和病理条件下的细胞功能非常重要。最近，已经描述了几种可用于非侵入性监测[Cli]的氯离子敏感的遗传编码探针。本文描述了两种表达称为 Cl-Sensor 的 CFP-YFP 基氯离子探针的小鼠品系。首先，我们生成了在 Thy1 微启动子控制下表达 Cl-Sensor 的转基因小鼠。Cl-Sensor 在海马和皮层神经元中从出生后第 2 天（P2）开始表达良好，其水平在发育过程中强烈增加。通过同时监测离子电流和氯离子依赖性荧光，我们确定 Cli 的表观 EC50 为 46 mM，表明该品系适合测量新生小鼠的神经元[Cli]。我们还描述了一种用于 Cre 依赖性条件表达 Cl-Sensor 的转基因小鼠报告基因系，该基因系靶向 Rosa26 基因座，并通过整合强的外源启动子，在 Cre 介导的重组后诱导强烈表达。我们在两种靶向髓系和周围感觉神经元的不同 Cre 驱动系小鼠中证明了高水平的组织特异性表达。使用这些小鼠，估计巨噬细胞和 DRG 中的 Cli 的表观 EC50 分别为 61 和 54 mM。我们的数据表明，这些小鼠品系将成为体内特定细胞类型的 Cli 比率监测的有用模型。
