BAC transgenic mice to study the expression of P2X2 and P2Y1 receptors

Marcus Grohmann,Ronald Jabs,Janka Günther,Stefan M Singheiser,Christian Lohr,Florian Wildner,Daniela Hirnet,Michaela Schumacher,Tanja Nußbaum,Heike Franke,Peter Illes,Günther Schmalzing,Ralf Hausmann,Zoltan Gerevich

doi:10.1007/s11302-021-09792-9

Abstract

Extracellular purines are important signaling molecules involved in numerous physiological and pathological processes via the activation of P2 receptors. Information about the spatial and temporal P2 receptor (P2R) expression and its regulation remains crucial for the understanding of the role of P2Rs in health and disease. To identify cells carrying P2X2Rs in situ, we have generated BAC transgenic mice that express the P2X2R subunits as fluorescent fusion protein (P2X2-TagRFP). In addition, we generated a BAC P2Y1R TagRFP reporter mouse expressing a TagRFP reporter for the P2RY1 gene expression. We demonstrate expression of the P2X2R in a subset of DRG neurons, the brain stem, the hippocampus, as well as on Purkinje neurons of the cerebellum. However, the weak fluorescence intensity in our P2X2R-TagRFP mouse precluded tracking of living cells. Our P2Y1R reporter mice confirmed the widespread expression of the P2RY1 gene in the CNS and indicate for the first time P2RY1 gene expression in mouse Purkinje cells, which so far has only been described in rats and humans. Our P2R transgenic models have advanced the understanding of purinergic transmission, but BAC transgenic models appeared not always to be straightforward and permanent reliable. We noticed a loss of fluorescence intensity, which depended on the number of progeny generations. These problems are discussed and may help to provide more successful animal models, even if in future more versatile and adaptable nuclease-mediated genome-editing techniques will be the methods of choice.

Highlights

The functions of the purine nucleotide ATP as a potent extracellular neurotransmitter via the activation of the P2 receptor (P2R) family is well-established, and virtually every tissue expresses P2Rs, indicating that ATP and related purines act as a common transmitter in almost all tissues and organs [1]
P2X2R subunits have been found in the central (CNS) and peripheral (PNS) nervous systems [1], with high expression levels in the cerebral cortex, basal ganglia, diencephalon, mesencephalon, cerebellum, medulla oblongata, olfactory bulb, and dorsal horn of the spinal cord [6] and the inner ear [5]
To facilitate the morphological identification and functional characterization of cells expressing the P2X2R, we generated C57BL/6 J-Tg(RP23-333M22P2X2-StrepHisTagRFP) Bacterial artificial chromosome (BAC)-transgenic mice expressing the P2X2R protein fused to TagRFP under control of the regulatory elements of the P2RX2 gene

Summary

Introduction

The functions of the purine nucleotide ATP as a potent extracellular neurotransmitter via the activation of the P2 receptor (P2R) family is well-established, and virtually every tissue expresses P2Rs, indicating that ATP and related purines act as a common transmitter in almost all tissues and organs [1]. Despite its widespread CNS expression, P2X2 knockout mice do not exhibit a strong CNS phenotype in terms of general excitability and sensory and motor function [4, 7]. The involvement of P2X2R subunit-containing receptors in sensory transmission has been confirmed in acute and chronic pain models performed with P2X2R subunit knockout mice [7]; for review, see [8]

Methods

Results

Conclusion