103: Characterisation of CHRNA3 Cre-ERT2 mouse line

Topic: Basic Science
Subtopic: Pain category (ICD-11), Chronic primary musculoskeletal pain, , , , , , Developing methodology/technology, Transgenic models, , ,

Background and Aims: CHRNA3 is a molecular marker of a population of silent nociceptors that innervate deep tissue such as joint and muscle [1], as well as sympathetic neurons. Using CRISPR-Cas9 gene editing, we have generated a knock-in CHRNA3 Cre ERT2 mouse with the aim to study silent nociceptors in the context of musculoskeletal and cancer pain. In this study we have used Cre dependent viruses and reporter lines to characterise the level of Cre-ERT2 recombination in sensory and sympathetic neurons in perinatal, adolescent and adult mice following tamoxifen administration.

Methods: CHRNA3-CreERT2 mice crossed with floxed tdTomato and CHRNA3-eGFP reporters received 5×75 mg/kg tamoxifen. A separate CHRNA3-CreERT2 cohort received 5 µl intraplantar injections of one of three AAV9 vectors at P4-P6, followed by tamoxifen in week 5. Lumbar DRG (L3–L5) and sympathetic ganglia were collected 2 weeks (reporter cohort) or 3 months (viral cohort) later. Frozen sections were processed for immunohistochemistry and labelled with neuronal markers, anti-CGRP, and anti-GFP/anti-tdTomato antibodies to enhance reporter detection. Slides were imaged by confocal microscopy or wide-field slide scanning. Reporter-positive neurons were semi-quantified using Cellpose2/ImageJ or QuPath.

Results: Tamoxifen induced tdTomato expression in ~0.18±0.02% of L4 and ~10.4±3% of L6 neurons (n=5) in adult CHRNA3-Cre-ERT2×tdTomato mice, with similar levels at P28. In CHRNA3-eGFP mice, 6.5% of L4 neurons expressed eGFP. In triple-transgenics, 2.2% (n=1) of L4 and 11.7±4.4% (n=2) of L6 eGFP neurons were tdTomato-positive. In the stellate ganglion, ~80% of eGFP neurons co-expressed tdTomato. Prenatal tamoxifen was ineffective, yielding two Cre-ERT2-positive pups from four litters, with no DRG and sparse sympathetic expression.
After AAVs, tamoxifen induced sparse recombination in CGRP-positive DRG neurons: ~0.7±0.5% (n=3) for AAV9-CAG-Flex-GCaMP6s, ~0.5±0.4% (n=2) for AAV9-SYN-Flex-PSAM4-GlyR-IRES-eGFP, and ~0.4±0.1% (n=3) for AAV-PHP.S-shortCAG-dlox-ChrimsonR-tdTomato.

Conclusions: Following tamoxifen administration, we find that Cre-ERT2 recombination can be achieved in a low proportion of lumbar sensory neurons, whilst high levels can be achieved in sympathetic neurons. In contrast, a higher proportion of lumbar sensory neurons expressed eGFP in the CHRNA3-eGFP mouse. Given this discrepancy, the expression of CHRNA3, which controls Cre-ERT2 expression, might be insufficient to mediate recombination in sensory neurons of mice aged >p28.

References: [1] Prato V, Taberner FJ, Hockley JRF, Callejo G, Arcourt A, Tazir B, Hammer L, Schad P, Heppenstall PA, St. John Smith E, Lechner SG. “Genetic identification of mechanoinsensitive ‘silent’ nociceptors”. Cell reports 2017;21(11):3102-3115.

Presenting Author: Łukasz Pietras
Submitting Author: George Goodwin

Senior Author: George Goodwin

IASP 2026 Global Year theme of Neuropathic Pain: No

• Łukasz Pietras - Polish Centre for Technology Development, Łukasiewicz-PORT
• Manuela De Las Casas Felgueroso - Universidad Miguel Hernández-CSIC
• Franziska Denk - King's College London
• Mateusz W. Kucharczyk - Polish Centre for Technology Development, Łukasiewicz-PORT