Generation of a Homozygous Transgenic Rat Strain Stably Expressing a Calcium Sensor Protein for Direct Examination of Calcium Signaling

Abstract

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.

Generation of transgenic marmosets expressing genetically encoded calcium indicators

“…GECI molecules, including GCaMPs, have been used more largely in the field of neuroscience as an effective monitoring system for neural activity-dependent calcium signaling 5 6 15 . Since calcium signaling is involved in a variety of intracellular signaling pathways, GCaMP is also being applied in human pluripotent stem cells and in transgenic rodents engineered to express GCaMP predominantly in smooth muscle cells, cardiomyocytes, and kidney proximal tubules, thus allowing calcium imaging without additional manipulation 65 66 67 68 69 70 . Owing to the limited access of tissues from the founder animals, a more thorough characterization of GCaMP expression, as well as a deeper assessment of the functionality of the transgene in vivo still remains to be performed.…”

Calcium signaling in human pluripotent stem cells

“…In most cases, under proper expression conditions, GECIs have minimum effects on cellular activity, can be used in vivo as well, and therefore are also suitable for long-term studies (see Ref. [18]), although potential calcium buffering and cellular signal perturbations may still occur ( [19]) (as reviewed in Refs. [20,21]).…”

Ct shift: A novel and accurate real-time PCR quantification model for direct comparison of different nucleic acid sequences and its application for transposon quantifications

“…Gene copies are considered as corpuscular units, which uniformly present in individual cells in case of a cell clone or in the germline of an animal, offering a good candidate to prove that a particular qPCR quantification method is reliable. We have established a transgenic rat line expressing fluorescent calcium sensor protein which contains an SB-CAG-GCaMP2 transposon cassette in a homozygous form (Szebenyi et al, 2015a;Szebenyi et al, 2015b). Phenotypic analysis combined with transgene copy number determination of founders and progenies of subsequent generations enabled an effective and rapid selection of animals, resulting in a stable homozygous line in a short time schedule of crossings.…”

“…Human transgenic cell clones (Kolacsek et al, 2011) and the transgenic rat line (Szebenyi et al, 2015b) were established and genomic DNA was isolated as described earlier.…”