Strategy for Flp-directed integration of lentiviral DNA circles. (A) Schematic representation of vectors utilized for lentiviral transduction and generation of Sleeping Beauty vector-tagged cell lines. The lentiviral vector, pLV/FRT-hygro, is a third generation SIN vector containing an ATG-deficient FRT-hygro fusion gene located between the packaging signal (ψ) and the central polypurine tract (cPPT, not shown) flanked downstream by a selectable marker expression cassette. In the lentiviral SIN vector pLV/PGK-puro the PGK-driven Flpx9 expression cassette is located downstream from ψ and cPPT sequence (the latter not shown). The SB transposon docking vector, pSBT/RSV-FGIP, contains the left and inverted repeats of SB (LIR and RIR, represented by white arrows) and an expression cassette containing the RSV promoter driving a fusion gene consisting of an ATG-FRT-tagged eGFP gene, an IRES element, the puromycin-resistance gene, and a polyadenylation signal. (B) Schematic representation of site-directed integration of LV DNA circles. After reverse transcription of the viral RNA, circular forms of the viral genomic DNA are generated by either non-homologous end joining (2-LTR) or homologous recombination (1-LTR). These circles are normally considered dead-end products of reverse transcription but the FRT site will enable DNA circles to become substrates for Flp recombination. In cells harboring an engineered FRT site flanked by a promoter and a start codon, Flp-mediated insertion of the virus-derived (i) 1-LTR circles and (ii) 2-LTR circles will generate a functional hygromycin B resistance expression cassette. To block the normal viral integration machinery, integration-defective lentiviral vectors (IDLVs), carrying an inactive viral integrase protein (harboring the D64A mutation), were utilized as carriers of viral RNA. Puro, puromycin resistance gene; LTR, long terminal repeat; FRT, Flp recombination target site.