Transgenic mouse lines for non-invasive ratiometric monitoring of intracellular chloride.
Transgenic mouse lines for non-invasive ratiometric monitoring of intracellular chloride.
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DOI：
10.3389/fnmol.2013.00011
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发表时间：
2013
影响因子：
4.8
通讯作者：
Heppenstall PA
中科院分区：
文献类型：
--
作者：
Batti L;Mukhtarov M;Audero E;Ivanov A;Paolicelli RC;Zurborg S;Gross C;Bregestovski P;Heppenstall PA
氯离子是最丰富的生理阴离子，参与多种细胞过程，包括跨上皮运输、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，一些对cl敏感的基因编码探针被描述为允许对[Cli]进行非侵入性监测。在这里，我们描述了两种表达基于cfp - yfp的Cl探针的小鼠系，称为Cl- sensor。首先，我们在小鼠Thy1迷你启动子的控制下产生了表达Cl-Sensor的转基因小鼠。Cl-Sensor从出生后第2天（P2）开始在海马和皮质神经元中表现出良好的表达，在发育过程中表达水平显著增加。利用离子电流和cl依赖性荧光同时全细胞监测，我们确定了Cli的表观EC50为46 mM，表明该线适用于产后小鼠神经元[Cli]的测量。我们还描述了一种转基因小鼠报告系，用于cre介导的Cl-Sensor条件表达，该报告系以Rosa26位点为靶点，通过加入一个强外源启动子诱导cre介导的重组的强表达。我们在两种不同的cre驱动系中证明了高水平的组织特异性表达，这些系靶向髓系细胞和外周感觉神经元。在这些小鼠中，巨噬细胞和DRG对Cli的表观EC50分别为61和54 mM。我们的数据表明，这些小鼠系将成为体内特定细胞类型中Cli比率监测的有用模型。
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 EC50 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 EC50 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.
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影响因子：
4.5
作者：
Depry, Charlene;Mehta, Sohum;Zhang, Jin
通讯作者：
Zhang, Jin
影响因子：
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作者：
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Feng, Guoping
影响因子：
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作者：
Clausen, BE;Burkhardt, C;Förster, I
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Förster, I
影响因子：
5.6
作者：
CASTRANOVA, V;BOWMAN, L;MILES, PR
通讯作者：
MILES, PR
影响因子：
16.2
作者：
Glykys J;Dzhala VI;Kuchibhotla KV;Feng G;Kuner T;Augustine G;Bacskai BJ;Staley KJ
通讯作者：
Staley KJ