Select and insert strategy for cell line generation. (A) Diagram showing the select and insert strategy used to generate L4 complementing cell lines. LoxP parent cell lines are first isolated by selection for Zeocin resistance and characterised for their inducible expression of GFP. Cre-mediated recombination is then used to replace the GFP gene from LoxP parent cells with an L4 cassette from a promoterless shuttle plasmid. (B) To generate LoxP parent cells, 293TETOFF were transfected with pLoxPGFP and Zeocin-resistant cells were sorted by FACS into two populations based on GFP fluorescence intensity. Left: unsorted cell population; centre: cells sorted for low to medium GFP expression; right: cells sorted for high GFP expression.