Transgenic mouse lines for non-invasive ratiometric monitoring of intracellular chloride

Abstract

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.

Intracellular Chloride Regulation in AVP+ and VIP+ Neurons of the Suprachiasmatic Nucleus

“…Therefore, we performed Cl − imaging in VIP+ and AVP+ neurons of the SCN to look for differences in [Cl − ] i regulation between these two populations of neurons. To measure [Cl − ] i in SCN neurons, we used a newly-developed Cre-inducible mouse line, with a floxed Cl-Sensor allele inserted into the Rosa26 locus 33 . To obtain Cl-Sensor expression in the SCN, we crossed these mice with either VIP-IRES-Cre mice or AVP-IRES-Cre mice to give Cl-Sensor expression in either VIP+ or AVP+ neurons 34 , 35 .…”

Calibration and functional analysis of three genetically encoded Cl−/pH sensors

“…These indicators have opened the way for non-invasive monitoring and ratiometric measurement of [Cl − ] i in different cell types in vitro (Pellegrino et al, 2011; Bertollini et al, 2012; Friedel et al, 2013). Expressed in neurons of transgenic mice, they have allowed imaging of Cl − dynamics in inhibitory circuits of different brain areas, including hippocampus, cerebellum, and deep cerebellar nuclei (Berglund et al, 2008, 2011), as well as in intact hippocampus (Dzhala et al, 2012) and a dorsal root ganglia preparation (Batti et al, 2013). Producing a construct consisting of a glycine receptor (GlyR) with Cl-Sensor incorporated into the long cytoplasmic domain (BioSensor-GlyR) provided a tool for non-invasive monitoring activity of these Cl − -selective receptor-operated channels (Mukhtarov et al, 2008).…”

Correlative intravital imaging of cGMP signals and vasodilation in mice

“…This gene locus is known for transcriptional accessibility in most if not all cell types throughout pre- and postnatal development (Soriano, 1999 ). Although technically more challenging, generation of R26 knock-in mice became a popular approach to express fluorescent proteins including biosensors (Zariwala et al, 2012 ; Abe et al, 2013 ; Batti et al, 2013 ). To increase biosensor expression above the relatively low levels that are usually achieved with the endogenous R26 promoter in adult mice, the strong chicken actin/β-globin (CAG) promoter (Niwa et al, 1991 ) can be integrated into the R26 knock-in transgene.…”