Stable integration into HeLa-FlpIn T-REx cells

For stable integration of the construct into the HeLa-FlpIn cell line 2 μg of the FRT site-containing plasmid together with 2 μg pOGG4 Flp-recombinase Expression Vector (Thermo Fisher Scientific, #V600520) were resuspended in 200 μL Opti-MEM (Gibco, #31985-062). 12 μL X-tremeGENE 9 DNA Transfection Reagent (Sigma, #6365787001) were added and the mix incubated at room temperature for 15–30 min. During this incubation period, 3.5 × 10⁶ HeLa-FlpIn cells were seeded in a 10 cm dish, in 10 mL antibiotics-free media and the transfection mix was applied. 24 hr after transfection, the media was changed to DMEM containing antibiotics and 400 μg/mL Hygromycin B (Invitrogen, #10687010) to select positive cells. The day after, the media was changed again and only 150 μg/mL Hygromycin B were added. After about two days, the media was changed again to eliminate dead cells and the remaining cells were grown until the appearence of colonies. Cells were then genotyped, further expanded and frozen in multiple vials.