Flp in cho cells

Plasmids integrated into CHO Flp-In™ cells (Invitrogen, R758-07) were constructed using restriction cloning on commercial and custom vectors and constructs. The mNF and mPF plasmids integrated into the genomic FRT site with the aid of Flp-recombinase expressed from the pOG44 vector (Invitrogen, V600520). The addition of T2A::PuroR to both plasmids resulted in mNF-PuroR and mPF-PuroR constructs. The molecular cloning extensively used overlap PCR extension to fuse DNA pieces together. For a detailed description of plasmid construction, see Supplementary Methods and Supplementary Table 3 for cloning primers.

HEK293T cells were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle Medium (DMEM, GIBCO) with 10% fetal bovine serum (FBS, Sigma Aldrich), 1x Penicillin/Streptomycin (GIBCO), 1 mM Sodium Pyruvate (GIBCO), 2 mM L-Glutamine (Thermo Fisher Scientific) at 37°C/5% CO₂. The HeLa-derived TZM-bl reporter cell line was sourced from the NIH AIDS Reagent Program and maintained in DMEM containing 10% fetal bovine serum, 1 mM Sodium Pyruvate, 2 mM L-Glutamine (Thermo Fisher Scientific), 50 μg/ml Gentamicin (Sigma-Aldrich), and 25 mM HEPES (Biochrom) at 37°C/5% CO₂. HEK293EBNA1-6E (293-6E) cells were obtained from the National Research Council Canada (NRC) and maintained in Freestyle 293 Expression Medium (Thermo Fisher Scientific) containing 0.2% Penicillin/Streptomycin at 37°C /5% CO₂ with shaking at 90-120 rpm. The sex of these cell lines is unknown. CHO Flp-In™ cells (Invitrogen) were a kind gift from the lab of John Moore (Cornell University) and maintained in Ham’s F-12 Medium supplemented with 10% heat-inactivated FBS (Sigma-Aldrich), 200 U/ml Penicillin/Streptomycin, 2 mM L-Glutamine, 20 mM HEPES, 0.1 mM non-essential amino acids, 1 mM Sodium Pyruvate (GIBCO), and further supplemented with 100 μg/ml Zeocin (Invitrogen).

CHO FlpIn cells were purchased from Invitrogen. DMEM and FBS were purchased from ThermoTrace. Hygromycin B was purchased from Roche Applied Science. PBS and versene were made in-house. Primers used for the generation of mutant receptors were purchased from Bioneer Pacific. The AlphaScreen Surefire pERK1/pERK2 reagents were kindly provided by TGR Biosciences. AlphaScreen streptavidin donor beads, anti-IgG (protein A) acceptor beads and [³H]NMS (specific activity 80 Ci mmol^–1) were purchased from PerkinElmer Life and Analytical Sciences. Xanomeline and analogs were obtained from Karuna Therapeutics. Pilocarpine hydrochloride was purchased from ICN Biomedicals. All other chemicals, including acetylcholine, were from Sigma-Aldrich.