9 research outputs found
Generation of a Homozygous Transgenic Rat Strain Stably Expressing a Calcium Sensor Protein for Direct Examination of Calcium Signaling
In drug discovery, prediction of selectivity and toxicity
require the evaluation of cellular calcium homeostasis. The rat
is a preferred laboratory animal for pharmacology and
toxicology studies, while currently no calcium indicator
protein expressing rat model is available. We established a
transgenic rat strain stably expressing the GCaMP2
fluorescent calcium sensor by a transposon-based methodology.
Zygotes were co-injected with mRNA of transposase and a CAG-
GCaMP2 expressing construct, and animals with one
transgene copy were pre-selected by measuring fluorescence in
blood cells. A homozygous rat strain was generated with high
sensor protein expression in the heart, kidney, liver, and
blood cells. No pathological alterations were found in these
animals, and fluorescence measurements in cardiac tissue slices
and primary cultures demonstrated the applicability of this
system for studying calcium signaling. We show here that the
GCaMP2 expressing rat cardiomyocytes allow the
prediction of cardiotoxic drug side-effects, and provide
evidence for the role of Na+/Ca2+ exchanger and its beneficial
pharmacological modulation in cardiac reperfusion. Our data
indicate that drug-induced alterations and pathological
processes can be followed by using this rat model, suggesting
that transgenic rats expressing a calcium-sensitive protein
provide a valuable system for pharmacological and toxicological
studies
Characterization of a new genetically-encoded Calcium sensor in hippocampal neurons using fluorescence imaging
In vivo functional imaging of the olfactory bulb at single-cell resolution
Functional properties of neuronal circuits can be best studied in vivo in the living mammalian brain. The use of optical methods, like two-photon calcium imaging, permits analyses of network function at single-cell resolution. This chapter provides a step-by-step description of this technique. Using mouse olfactory bulb as a model system, we compare the performance of genetically encoded calcium sensor TN-XXL and smallmolecule calcium indicators; describe how to choose the right calcium indicator and how to load it into the cells of interest; discuss the use of cell type-specific markers and, finally, illustrate the application of this technique for high-resolution in vivo imaging of sensory-driven neuronal activit
Biocompatibility of a genetically encoded calcium indicator in a transgenic mouse model
Engineering efforts of genetically encoded calcium indicators predominantly focused on enhancing fluorescence changes, but how indicator expression affects the physiology of host organisms is often overlooked. Here, we demonstrate biocompatibility and widespread functional expression of the genetically encoded calcium indicator TN-XXL in a transgenic mouse model. To validate the model and characterize potential effects of indicator expression we assessed both indicator function and a variety of host parameters, such as anatomy, physiology, behaviour and gene expression profiles in these mice. We also demonstrate the usefulness of primary cells and organ explants prepared from these mice for imaging applications. Although we find mild signatures of indicator expression that may be further reduced in future sensor generations, the 'green' indicator mice generated provide a well-characterized resource of primary cells and tissues for in vitro and in vivo calcium imaging applications