Experimental strategy. (A) Step 1: To introduce targets for RMCE, we transfected the insert of plasmid pL1HYGL2 into MELc, and identified clones with single integrated copies by Southern blots. Two such clones were obtained; the insertion sites were termed Random Locus 1 and 2 (RL1 and RL2). These cells were hygromycin (Hyg) resistant but G418 sensitive because the Neo coding sequence (the socket, see text) lacks a promoter. Step 2: RMCE at RL1 and 2 were performed by cotransfecting a CRE expression plasmid, and plasmid pL1PL2 or its derivatives. Exchange of the PGK-HYG gene by the DNA fragment flanked by the L1 and L2 site in plasmid pL1PL2 and derivatives results in insertion of cassette X and in the reconstitution of the MCNeo gene. Therefore, MELc having undergone RMCE can be selected for in G418. The cells also loose Hyg resistance. Initial experiments to demonstrate the feasibility of RMCE were performed at the RL1 and RL2 loci using plasmid pL1PL2 (cassette X = nothing). Insertions with pL1PL2 derivatives (cassette X = cassettes 0, 2, 3 or 234 [see B]) were only performed at RL1. Step 3: After step 2, the reconstituted MCNeo gene is flanked by FLP Recognition Target (F) sites. It can therefore be excised by transient expression of an FLP expression plasmid. Because FLP-mediated excisions are rare events, a GFP plasmid was cotransfected and used to enrich in cells having been transfected. At the end of step 3 the only remaining plasmid sequences are therefore short FRT and L1 sequences flanking cassette X. (B) Cassettes 0, 2, 3, and 234 are represented by the “cassette X” box in (B) HS 2, 3, and 4 were generated from the LAR-β^s construct of Forrester et al20 (see Materials and Methods). The β-globin promoter is represented by the “β-G” box in the diagram. The LacZ fragment contains an SV40-derived intron, the E coli LacZ sequence, and an SV40 derived poly-adenylation signal. All cassettes were inserted with the promoter of the β-globin gene facing away from the MC promoter (see Figs 4C and 5C).