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

Kornélia Szebényi,László Homolya,Zsuzsanna Bősze,Péter Vajdovich,Ágnes Enyedi,András Füredi,Tamás I Orbán,László Héja,József Tóvári,Enikő Pergel,Balázs Sarkadi,Gergely Szakács,Orsolya Kolacsek,Balázs Bender,Ágota Apáti

doi:10.1038/srep12645

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.

Highlights

The importance of proper calcium homeostasis and signaling from the cellular to the complex organ levels is well appreciated: both in physiological and pathological processes cellular free calcium plays a major role[1]
Several drugs with various mechanisms of action had to be withdrawn from the market because of side effects caused by disruption of the calcium homeostasis, including Clobutinol, a cough suppressant[5], Dofetilide, an antiarrhythmic agent[6], Grepafloxacin and Sparfloxacin, antibacterial agents[7], Terfenadine, an antihistamine[8], or Terodiline, a spasmolytic agent[9]
In the present study we describe the generation and validation of a new GCaMP2 expressing rat strain with a single transgene copy per haploid genome

Summary

Introduction

The importance of proper calcium homeostasis and signaling from the cellular to the complex organ levels is well appreciated: both in physiological and pathological processes cellular free calcium plays a major role[1]. Several drugs with various mechanisms of action had to be withdrawn from the market because of side effects caused by disruption of the calcium homeostasis, including Clobutinol, a cough suppressant[5], Dofetilide, an antiarrhythmic agent[6], Grepafloxacin and Sparfloxacin, antibacterial agents[7], Terfenadine, an antihistamine[8], or Terodiline, a spasmolytic agent[9] All these findings suggest that in the process of drug discovery an early prediction of toxicity requires the direct examination of the drug effects on cellular calcium homeostasis and signaling in different target tissues, especially in the heart. CAG promoter specific GCaMP2 expression in blood cells allowed the development of a non-invasive, combined approach of genetic and phenotypic selection, yielding rat strains with high sensor protein expression, in spite of a mono-allelic transgene incorporation

Methods

Results

Conclusion