2.1. Vector Construction

Plasmids coding for mouse Taar1, Taar5 or Taar8b, with an HA-tag fused to the N-terminus, cloned into a pcDps expression vector (pHA-mTaar1, pHA-mTaar5 and pHA-mTaar8b) were described previously [16,17].

The pHA-mTaar1 plasmid was employed as a template to amplify the mouse Taar1, Taar5 or Taar8b cDNA sequence, omitting the stop codon, while providing overhangs complementary to the XhoI and BamHI restriction sequences to enable ligation into the pEGFP-N1 (Clontech, Heidelberg, Germany) expression vector using T4 DNA ligase (EL0011, Thermo Scientific, Schwerte, Germany). The resultant plasmid coded for a chimeric protein with full-length mouse Taar1, covalently linked to the EGFP tag by a 12-amino acid long spacer peptide linker (pmTaar1-EGFP). The sequence was confirmed using standard pEGFP-N1 forward and reverse primers at Eurofins Genomics (Ebersberg, Germany).

Similarly, the sequence coding for full-length mTaar1 minus the stop codon was cloned into a modified puc2CL6Ipwo lentiviral vector [18,19] at XhoI and AgeI sites of insertion (5′-end and 3′-end, respectively), to obtain a construct coding for the chimeric protein consisting of full-length mTaar1, covalently linked to EGFP by a 12-amino acid long spacer peptide linker (mTaar1-EGFP in puc2CL6Ipwo). Sequences were confirmed at Eurofins Genomics (Ebersberg, Germany) using oSF031Fwd (5′-CGGCGCGCCAGTCCTCCG) and oSF031Rev (5′-TAGACAAACGCACACCGG) sequencing primers.