Figure 1.

Analysis of lentiviral vector episome formation under conditions not conducive to homologous recombination. Shuttle vector analysis (see Materials and Methods) was employed on Hirt-extracted episomes from lentiviral shuttle vector-transduced (a) Ercc1-defective Chinese hamster ovary (E1KO7-5) cells, (b) liver cells, and (c) cycle-arrested Chinese hamster ovary (AA8 cells). Panels a–c represent episome formation as a percentage of total episomes; each experiment was performed in triplicate. The term “mutant” refers to circular vector genomes generated by autointegration, as determined by restriction products that do not fit the expected band sizes corresponding to 2-long terminal repeat (LTR) and 1-LTR circular episomes. (d) Episome formation was examined by Southern-blot analysis on DNA extracted 2 days after transduction with an integrating, polypurine tract+ vector (vTK945) from Ercc1-deficient (E1K07-5) (lane 1), Ercc1-wild-type (ATStg) (lane 2), cycling AA8 (lane 3), arrested AA8 (lane 4), human breast cancer BRCA1-deficient (SUM149) cells (lane 5), and human breast cancer (ME16C2) cells containing wild-type BRCA1 (lane 6). DNA was digested with EcoNI and PflMI and probed with the probe complementary to the 3′ region of vTK945, as shown in Figure 3b.