Geneticin

HEK 293 cells (wild type or stably expressing rat MOR) were maintained in DMEM media (Sigma-Aldrich) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in the absence or presence of 0.1 mg/ml geneticin (Biochrom AG, Berlin, Germany), respectively, in 5% CO₂ at 37 °C. Depending on their density, cells were passaged 1:3–1:10 every second to third day from P8 to P28^{26 (link)}.

The HEK-293 (human embryonic kidney-293) cell line encoding the simian virus 40 (SV40) large T antigen (heretofore, 293T cells) was used for the production and characterization of lentiviral vectors (both research grade and GMP grade). A master cell bank (MCB) of 293T cells was established at EUFETS GmbH (Idar-Oberstein, Germany). Two randomly picked 293T MCB vials were tested for sterility, endotoxins, mycoplasma and adventitious viruses in full compliance with the GMP requirements of the local regulatory authorities. These safety tests have shown that the MCB was devoid of bovine, porcine and human viruses. HT1080 (human fibrosarcoma cell line) was used for titration of the lentivirus produced with GMP grade materials. 293T cells and HT1080 cells were cultured at 37°C and 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS, HyClone, Fischer Scientific GmbH, Bonn, Germany). The reference K562 cells co-expressing HLA-A02^*01 and pp65 were cultured in RPMI supplemented with 10% FBS, 1% penicillin/streptomycin and 1 mg/mL geneticin (Biochrom AG, Berlin, Germany).

Wild type and MOR-expressing HEK293 cells were maintained in DMEM media (Sigma-Aldrich, Taufkirchen, Germany) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in the absence or presence of 0.1 mg/ml geneticin (Biochrom AG, Berlin, Germany), respectively, in 5% CO₂ at 37 °C. According to their confluence, cells were passaged 1:3–1:10 every second to third day from P8 to P28^{18 (link)}.