Generation of IC2C Flp-In T-REx 293 cell lines and large scale expression

Flp-In^TM T-REx^TM293 cells (Thermo Fisher, Cat#{"type":"entrez-nucleotide","attrs":{"text":"R78007","term_id":"853117","term_text":"R78007"}}R78007) harboring IC2C wt or ∆ICN with C-terminal SNAP-FLAG-strepII tagwhich were generated using the FLP/FRT system (Thermo Fisher). Briefly, pcDNA5-FRT-TO construct and pOG44, which expresses Flipase, were co-transfected using Lipofectamine 2000 (Thermo Fisher) into Flp-In^TM T-REx^TM293 cells. After recovery from transfection, cells were grown in DMEM containing 10% FBS, 1% Penicillin-Streptomycin, and 100 µg/mL Hygromycin B to select cells in which pcDNA5 construct was inserted into FRT locus. The expression of tagged IC2C was verified by western blotting with antibodies against StrepII tag (Novus Biologicals). These cell lines were grown in culture vessels (Corning, Fisher Scientific) to 80% confluence, and then tagged IC2C expression was induced with 1 µg/ml doxycycline for two days. Cells were harvested in PBS by tapping the culture vessels, and spun down at 1200 rpm for 5 min in Sorvall Legend XTR. Cell pellets were washed with PBS, snap frozen in liquid nitrogen, and stored at −80 °C.