4.4. Recombinant Retrovirus Production

Phoenix A [98] were seeded (1.5–2 × 10⁶ cells) in 10 cm dishes coated with 10 µg/mL rat tail collagen by incubating at 37 °C for 1–2 h. The next day these cells were transfected with TransIT-LT1 transfection reagent (cat # MIR 2306; Mirus Bio) in serum free medium according to manufacturer’s protocol. Transfected cells were then selected with puromycin at a concentration of 2 µg/mL. Once the cells reached 90% confluence, the medium was changed and left for 24 h at 32 °C for virus production. Viral supernatants were harvested for 3 rounds and spun down for 1 h at 4300 rpm at 4 °C, snap frozen in liquid nitrogen and stored at −80 °C.

The construct with hygromycin selection were transfected into Phoenix E and 72 h post-transfection the supernatant was used to transduce PT67 cells [99], which were selected with 300 µg/mL hygromycin, and the viral supernatant was harvested as described above.