RMCE-mediated generation of stable cell lines
For RMCE, 2 × 106 cells were transfected with 2 μg of TV and 2 μg of Cre expression vector using SG Cell Line 4D-Nucleofector® X Kit L (V4XC-3024; Lonza). Transfected cells were selected for 2 weeks with 500 μg/mL G418 (ant-gn-1; Invitrogen). During selection, the medium was changed every 4 d. After selection, the percentage of cells expressing GFP was measured on a Gallios flow cytometer platform (Beckman Coulter, Brea, CA, USA). Enrichment of GFP-positive cells was performed using a BD FACSAria™ cell sorter equipped with a 488 nm laser. Sorted cells were seeded 1 cell per well in 150 μl medium in 96-well plates, and incubated for 15 da. Cells expressing GFP were screened using a fluorescence microscopy (Axiovert 200; Carl Zeiss).