2.7. Lentivirus preparation and transduction of insulin-secreting β-cells

For stable modulation of ERO-1α expression and expression of site-specific HyPer7 in INS-1E and EndoC cells, lentiviral particles were prepared as previously described [48]. In brief, HEK 293FT cells were transfected with 9.375 μg envelope plasmid (pcDNA3/MDG), 28.125 μg packaging plasmid (pPAX2) and 37.5 μg lentiviral transfer plasmid by Ca²⁺ phosphate precipitation. After 48 h, the virus particles were harvested and purified with ultrafiltration columns (Vivaspin 20 Centrifugal Concentrator PES, 100 kDa, Satorius, Göttingen, Germany). Cells were incubated with the respective lentiviral particles at a dilution of 1:100 for 8 h. Thereafter, the virus-containing medium was replaced with fresh cell culture medium. At 48 h post-transfection, single mCherry-positive INS-1E cells were sorted onto ECM-coated 96-well plates using the FACSAria III Fusion system (BD Biosciences, Heidelberg, Germany) at the Cell Sorting Core Facility of Hannover Medical School. As EndoC cell viability primarily depends on cell-cell contact, it is not possible to establish single-cell-derived cell lines [49]. To generate a specific ERO-1α-KO EndoC cell line, CRISPR/Cas9 edited cells were selected using the selection marker puromycin (10 mg/mL stock; InvivioGen, San Diego, USA). Following clonal expansion (INS-1E) and puromycin selection (EndoC), genomic DNA was isolated, and the genotype of the clones was identified by Sanger sequencing. INS-1E^{ERO−1α−KO} represents a stable, clonally-derived CRSIPR/Cas9 cell line, whereas EndoC^{ERO−1α−KO} denotes a stable non-clonal CRISPR/Cas9 cell line. All other cells were selected for (over)expression of ERO-1α or HyPer7 variants using either puromycin or neomycin (25 mg/mL stock in PBS; Merck).