Flp in cho cell line

CHO cells expressing LI-cadherin mutants were established as described previously^{9 (link)}. The DNA sequence of the LI-cadherin constructs fused with monomeric GFP was cloned into the pcDNA5/FRT vector (Thermo Fisher Scientific). The Flp-In-CHO Cell Line (Thermo Fisher Scientific) was transfected with the plasmid, following the manufacturer’s protocol. Cloning was performed using the limiting dilution-culture method. Cells were observed using an In Cell Analyzer 2000 instrument (Cytiva) with the FITC filter (490/20 excitation, 525/36 emission), and the cells expressing GFP were selected. The cells were cultivated in Ham’s F-12 Nutrient Mixture (ThermoFisher Scientific) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, 1% GlutaMAX-I (ThermoFisher Scientific), 1% penicillin–streptomycin, and 0.5 mg mL⁻¹ hygromycin B at 37 °C and 5.0% CO₂.

The CHO-K1 cell line (Chinese hamster ovary) was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA); the T47D cell line (human breast cancer) was provided by Dr. P. Dallas, Telethon Kids Institute (Subiaco, WA, Australia). Cell lines were maintained in a humidified incubator at 37 °C with 5% CO₂. CHO-K1 and T47D cells were cultured in RPMI 1640 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM Glutamax, 100 U/mL penicillin-streptomycin.
The CHO-K1/SpyC_C-BLA monoclonal stable cell line was made using FlpIn™ technology (Flp-In™-CHO Cell Line, Invitrogen, Carlsbad, CA, USA). To make the FlpIn plasmid for stable transformation, a coding sequence for SpyC_C-BLA fusion protein (Table A2) was synthesized (ATUM) and cloned into the BamHI and XhoI sites of pcDNA5/FRT (Invitrogen). Then the Amp^R gene was excised from the plasmid backbone, using BglII and BspHI restriction enzymes (New England Biolabs (NEB), Ipswich, MA, USA), and replaced with a Kan^R cassette cloned via NcoI and BglII (NEB). Stable cell line CHO-K1/SpyC_C-BLA was cultured in HAM’s F12-K (Kaighn) medium (Gibco), supplemented with 10% heat-inactivated FCS (Rowe Scientific, Minto, NSW, Australia), 2 mM Glutamax, 100 U/mL penicillin-streptomycin, and 800 µg/mL Hygromycin B (Gibco).

The DsRed expression vector including an amber codon at codon position 131 (pcDNA5-DsRed-¹³¹_amber) was prepared by subcloning the DsRed gene from pDsRed-Express-N1 (Clontech) into pcDNA5/FRT/TO Flp-In expression vector (Invitrogen) between BamHI and XhoI sites, and then mutating the codon position 131 by using a QuikChange site-directed mutagenesis kit (Stratagene). CHO cells stably expressing DsRed mRNA-¹³¹_amber (CHO-DsRed-¹³¹_amber) were prepared by transfecting the Flp-In CHO cell line (Invitrogen) with pcDNA5-DsRed-¹³¹_amber according to the standard protocol of the Flp-In system. The CHO-DsRed-¹³¹_amber cells were grown at 37 °C and 5% CO₂ in Ham's F12 medium supplemented with 10% fetal bovine serum (Sigma) and 1% streptomycin/penicillin (Gibco).
CHO-DsRed-¹³¹_amber cells plated in a 35 mm dish (∼70% confluence, in Ham's F12 without antibiotics) were microinjected with 1 × PBS solution including 36 μM DEACM-PPG-tRNA_CUA and, as an injection marker, 10 μM Alexa Fluor 488 C5 maleimide (Molecular Probes). Microinjections were performed using Eppendorf Femtojet microinjection equipment and Femtotip microinjection capillary tips at 100–150 hPa. The cells were irradiated for 30 s by using the Hg–Xe lamp (∼405 nm, 125 mW cm⁻²) and then incubated for 4 h at 37 °C and 5% CO₂. Cellular fluorescence was imaged using a fluorescence microscope (Olympus IX51/IX2-FL-1/MP5Mc/OL-2).

Adherent Flp-In CHO cell line (Invitrogen, RRID:CVCL_U424) was cultured in Ham’s F-12 Nutrient Mix (Gibco) supplemented with 2 mM GlutaMAX (Gibco) and 10% fetal bovine serum (Sigma-Aldrich) at +37°C, 5% CO₂ according to the manufacturer’s recommendations. The serum-free suspension adapted landing pad cell line was cultured in FectoCHO CD Medium (Polyplus), supplemented with 2 mM GlutaMAX at +37°C, 5% CO₂ at 120 rpm orbital shaker with 19 mm throw. ExpiCHO cells (Gibco, RRID:CVCL_5J31) were cultured in ExpiCHO Expression Medium (Gibco) at +37°C, 8% CO₂.

All materials were purchased from Sigma-Aldrich Merck (Gillingham, UK) unless otherwise stated. Vectors pCR8/GW/TOPO and pSecTag/FRT/V5-His-TOPO, and the Gateway (GW) vector conversion system were obtained from Invitrogen (Carlsbad, CA, USA). LR Clonase II and the Flp-In-CHO cell line were also from Invitrogen (Carlsbad, CA, USA). FITC-labelled lectins SNA-I and MAA were from EY Laboratories (San Mateo, CA, USA) and biotinylated SNA-I, RCA-I, and MAA lectins were from Vector Laboratories (Peterborough, UK). Oligonucleotide primer synthesis and DNA sequencing was carried out by Eurofins. Proof-reading Phusion High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA) was used for PCR amplification of cDNA during construction and standard Taq polymerases (Promega, Madison, WI, USA) for screening. PNGase F was from New England Biolabs (Ipswich, MA, USA).