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. 2017 Feb 1;158(5):945–961. doi: 10.1097/j.pain.0000000000000861

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Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the International Association for the Study of Pain

This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

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Figure 1. — Construction of Grpr-Cre using BAC cloning. (A) Schematic overview of the generation of Grpr-Cre mice. The Cre sequence together with an ampicillin-resistance cassette flanked by 2 frt sequences was inserted through homologous recombination to replace exon 1 in the Grpr locus. Once inserted, the ampicillin-resistance gene was removed by the addition of arabinose, leaving only the Cre sequence under the influence of the Grpr promoter. (B) A pulsed-field gel electrophoresis image showing BAC-wt (180 bp and 8734 bp), BAC-Cre (112 kb, 70 kb, and 8734 bp), and arabinose-treated Cre (181 kb and 8734 bp). (C) Electrophoresis gel image from the single-cell analysis showing a positive and a negative cell for Grpr, ladder 100 bp. iCre (codon-improved Cre), frt (frt site), Amp (ampicillin-resistance gene). (D, E) Induction of action potentials in a tdTomato;Grpr-Cre cell when 300 nM gastrin-releasing peptide (GRP) was applied to the recording chamber. (F) Tonic spike profile exhibited by the recorded tdTomato;Grpr-Cre cell (D) when 30 pA current was applied. (G, H) A subthreshold depolarization response from a tdTomato;Grpr-Cre cell when 300 nM GRP was applied to the bath in the recording chamber. (I) A gap firing spike profile was exhibited by the recorded tdTomato;Grpr-Cre cell (G) when 30 pA of current was applied. Scale bars, 30 μm.