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).