Figure 1. Generation of dox-inducible HCT116-TGFBR2 cell lines.
(A) Schematic outline of recombination-mediated cassette exchange (RMCE). Retroviral transduction was performed using the proviral vector S2F-cLM2CG-FRT3 that contains a bidirectional dox-inducible promoter (Ptetbi) allowing concurrent expression of two marker genes (luciferase and mCherry) in HCT116-mCherry clones. Expression cassettes are flanked by mutant (F3) and wildtype Flp-recombinase target sites (F) that allow directed cassette exchange via Flpo-recombinase. Retroviral packaging signal (Ψ+) and long terminal repeats (LTR) are indicated. (B) Characterization of viral integration sites by nrLAM-PCR and sequencing. In the upper part, clone-specific integration sites and affected genomic loci (open reading frame (orf); Aldehyde dehydrogenase family 1 member L1 (ALDH1L1)) are depicted. In the lower part, 5′- and 3′- viral LTR DNA sequences (small letters) as well as the flanking genomic DNA sequences (capital letters) are shown.