Characterization of mu opioid receptor cells in a new Oprm1-cre mouse

we derived, validated, and applied a novel MOR-specific Cre mouse line, inserting a T2A cleavable peptide sequence and the Cre coding sequence into the MOR 3’UTR. Importantly, this line shows specificity and fidelity of MOR expression throughout the brain and with respect to function, there were no differences in behavioral responses to morphine when compared to wild type mice, nor are there any alterations in Oprm1 gene expression or receptor density. To assess Cre recombinase activity, MOR-Cre mice were crossed with the floxed GFP-reporters, RosaLSLSun1-sfGFP or RosaLSL-GFP-L10a. The latter allowed for cell type specific RNA sequencing via TRAP (Translating Ribosome Affinity Purification) of striatal MOR+ neurons following opioid withdrawal. RNA was isolated from striatal cells expressing GFP-L10a by TRAP. Briefly, 24 hours after the last chronic morphine (100mg/kg) or saline dose, Oprm1Cre/Cre; RosaLSL-GFP-L10a mice were euthanized via cervical dislocation, and striatum was isolated (both hemispheres, 2 brains pooled per sample), homogenized with lysis buffer, and incubated with magnetic beads that were pre-conjugated to anti-GFP antibodies (clones htz-GFP-19F and htz-GFP-19C8, Memorial Sloan-Kettering Monoclonal Antibody Facility, New York, New York, USA) to affinity purify RNA that was bound by GFP-L10a fusion protein.