5.7. Stable cell line generation

PC3 and HT-29 stable cell lines expressing iNOS under the control of tetracycline were generated by lentiviral transduction using pLIX403-hNOS2. pLIX403-hNOS2 was a gift from Edward Morgan (Addgene plasmid #110800; http://n2t.net/addgene:110800; RRID:Addgene_110800) [25]. Lentiviruses were produced in Lenti-X 293T cells seeded into 10 cm plates at 2 × 10⁶ cells per plate. For each plate of Lenti-X cells, a transfection mix comprising 3 μg of the transfer vector, 2 μg packaging plasmid, 1 μg envelope plasmid and 18 μg PEI was prepared in 1 mL OptiMEM and added dropwise to the cells. Cells were transfected for 16 h, after which the medium was replaced with fresh RPMI/10% FBS. Lentivirus-containing supernatant was collected after 24 h, replaced with fresh medium and collected again a further 24 h later. The supernatants were pooled, centrifuged at 1000×g for 5 min to remove cell debris, filtered through a 0.45 μm syringe filter, aliquoted and stored at −80 °C. PC3 and HT-29 cells (2 × 10⁶) were reverse-transduced by seeding into 10 cm plates in growth medium plus lentivirus-containing supernatant (1:5 ratio of virus:growth medium) and 8 μg/mL polybrene (Sigma #H9268). Cells were incubated with medium containing virus for 16 h, which was replaced with fresh growth medium to allow cells to recover for 24 h. Stable polyclonal cell lines were selected in 5 μg/mL puromycin for 14 days and expression of iNOS was confirmed by immunoblotting and Griess assay following doxycycline treatment.