Figure 4

Southern blot and PCR analysis confirm precise insertion of lentiviral derived DNA circles (A) Southern blot analysis of representative hygromycin B-resistant clones containing inserted lentiviral circles. Genomic DNA was digested with XbaI, which cuts within the duplicated FRT sites, generating a ~5400/5630-bp fragment (1-LTR and 2-LTR insertions, respectively) recognized by the indicated hygro probe. Length differences between 1- and 2-LTR insertions could be resolved on a shorter exposure of the membrane (not shown). (B) PCR-based characterization of vector insertions in transduced cells. Genomic DNA from ten (taken from (3B)) and twelve (taken from (3D)) hygromycin B-resistant clones was used for PCR analysis to confirm site-specific insertion into an engineered FRT site (panel I–II and V–VI), integration of circular DNA substrates containing one or two LTRs (panel III and VII), and finally to verify that unspecific insertions of IDLV/FRT-hygro or IDLV/PGK-Flp had not occurred (panel IV and VIII–IX). N and P indicate negative (HEK/FGIP1) and positive (pLV/FRT-hygro or pLV/PGK-Flp) control clones, respectively.