Quickchange 2 site directed mutagenesis kit

SLCO2A1 mutants A396E, A396T and L563P were generated using QuickChange II site-directed mutagenesis kit (Qiagen) using pDONR221-SLCO2A1 (Addgene, plasmid #131949) as template DNA and primers were obtained from Integrated DNA Technologies (IDT, Leuven, Belgium). Successful mutant generation was confirmed by Sanger sequencing at LGTC (Leiden, the Netherlands). Next, to enable doxycycline-inducible expression of the generated mutants gateway cloning was used to transfer SLCO2A1 mutants from the pDONR vector to a pJTI-R4-DEST-CMV expression vector containing a tet-operon for doxycycline-induction and C-terminal twin-Strep-tag and hemagglutinin (HA)-tag. In brief, 150 ng entry vector pDNOR221-SLCO2A1-mutant (i.e., A396E, A396T or L563P) and 150 ng destination vector pJTI-R4-DEST-CMV were incubated with Gateway LR Clonase II enzyme in TE buffer for 1h at 25°C. The mixture was incubated with Proteinase K solution for 10 min at 37°C prior to transformation into XL-1 blue competent cells. Plasmids were isolated using NucleoBond xtra midiprep kit (Macherey-Nagel, Germany) and sequences were again confirmed by Sanger sequencing. HEK293-JumpIn-parental cells were stably transfected with cDNA of PGT mutants in pJTI-R4 vector using lipofectamine as described previously (Gorostiola González et al., 2023 (link)).