HEK293 cell transfection and fluorescence-activated cell sorting (FACS)

To assess EGFP expression background, HEK293 cells were seeded in a 48-well plate at 50,000 cells/well. After 24 h, the medium was changed to medium lacking FBS and cells were transfected using 0.25 μg ssEGFP.HBB plasmid per reaction with lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. Cells were incubated for 72 h and proceeded with FACS. For FACS analysis, cells were dissociated with accutase, and centrifuged at 400 g for 4 min and then washed with DPBS. All measurements were obtained with the MACSQuant Analyzer (MiltenyiBiotec, Art. Nr. 160-001-287).

For stable cell line generation, HEK293 cells stably expressing a nuclear localized Cre recombinase (SC004-Puro, GenTarget Inc.) were plated at a density of 2 x 10⁶ on 100 mm PLL-coated (Sigma) dishes. After 24 h, the medium was changed to medium lacking P-S and cells were transfected with Super PiggyBac Transposase (PB210PA-1, System Biosciences) and CAG.LSL.mKate2.ssEGFP.HBB reporter vector (1:2.5) using lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The CAG.LSLmKate2.ssEGFP.HBB vector was synthesized with flanking piggyBac inverted terminal repeats (ITRs) and cloned into a pBR322 vector, to generate a 12,692 base-pair dsDNA construct (Genscript). Medium was exchanged 24 h post transfection. Cells were cultured and split regularly when confluent. Every 1–2 weeks FACS was used to isolate mKate2+ cells until a pooled stable cell line was obtained. Cell sorting was performed on a BD FACSAria Fusion equipped with 5 lasers (355, 405, 488, 561, and 638 nm). The instrument was set up with a 100 μm nozzle size at 32 kHz and 20 psi. All FACS data were analyzed with FlowJo 10.4.2 (BD Life Sciences).