Development of a TRPA1 Reporter Mouse Model

Abstract

Transient Receptor Potential Ankyrin 1 (TRPA1) is an ion channel which is activated by environmental irritants (e.g. formaldehyde and acrolein) and pungent stimuli such as mustard oil, garlic and cinnamaldehyde. TRPA1 is known to be expressed in a subset of nociceptive sensory afferent and thus, playing an important role in defensive reflexes. However, TRPA1 has also been implicated in fibroblast, epithelial cell and smooth muscle cell function, thus it is not entirely clear the extent to which TRPA1 function is restricted to sensory neurobiology.In the current study, we used the Flp‐FRT (flipase‐flipase recognition target) system to generate a TRPA1 reporter mouse model. We generated a TRPA1‐Flp knockin stain which incorporated Flpo after the last endogenous TRPA1 exon. Expression of Flpo was linked to the expression of endogenous TRPA1 via a 2A sequence. The TRPA1‐Flp was crossed with a tdTomato reporter strain carrying frt‐flanked STOP condon (RC::FLTG) to generate a mouse model (Flp::RC) that expresses red fluorescent protein tdTomato in TRPA1 expressing cells. Vagal sensory ganglia were collected from those animals and used for characterization. First, the vagal ganglia were cryosectioned and immunostained to determine nociceptive subtypes using anti‐TRPV1 antibody. Second, primary neurons were isolated from the vagal ganglia and used to monitor calcium influx changes after stimulation with allyl isothiocyanate (AITC), the TRPA1 agonist.tdTomato was expressed in a subset of vagal ganglion neurons, but not in other cell types within the ganglion. The majority of the red fluorescent cells were TRPV1 positive cells and approximately 30% of TRPV1 positive cells co‐expressed TRPA1. In our functional studies, a total of 58 dissociated vagal neurons were assayed and 17 cells expressed tdTomato. Among the tdTomato expressing cells, 11 cells responded to 100 μM AITC. Additionally, 9 cells responded to AITC from 41 of non‐tdtomato expressing cells. Overall, our data suggests that the transgenic mouse model generated using Flp‐FRT system successfully labeled TRPA1 cells. Our preliminary findings suggest that the TRPA1 Flp::RC strain can be utilized in investigations involving TRPA1 ion channel physiology.Support or Funding InformationThis study is funded by National Institutes of Health Common Fund SPARC OT2 (2016–2019): “Functional mapping of peripheral and central circuits for airway protection and breathing”This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.