Promoter-trap CHO-K1 Monitoring Cells

The promoter-trap CHO-K1 monitoring cell line generated in a previous study contained a promoter-less EGFP expression cassette [23] (link). A total of 1.0 × 10⁶ cells of monitoring cell lines were transfected with 5 μg of sgRNA1 sgRNA-Cas9-mCherry and 5 μg of each promoter HDR KI donor plasmid using a NEPA21 electroporator for site-specific targeted integration. After 3 d, transfected cells were seeded at 0.3 × 10⁶ cells/mL in suspension 6-well cell culture plate with 3 mL PowerCHO-2CD medium supplemented with 8 mM L-glutamine and 500 μg/mL G418 disulfate salt solution (G8168, Sigma Aldrich). EGFP expression levels were determined using flow cytometry 6 d after transfection.