FIG. 3.
DNA-PK-independent repair of transposase-induced DSBs in vivo in mouse liver. (A) PCR-based strategy to detect repair of transposase-induced DSBs following transposon excision from donor plasmid constructs. The structure of the pThAAT vector DNA is shown in linear format for simplicity but was delivered to mice as a supercoiled plasmid. Prior to DNA transposition, PCR primers (arrows) are located 2.8 kb apart, which is beyond the PCR capacity. Following SB-mediated transposon excision and DSB repair, the primers are brought close enough together to allow PCR amplification. (B) Transposon excision and DSB repair in mouse liver. Normal C57BL/6 and DNA-PKcs-deficient C57BL/6-scid mice received a donor hAAT vector with (pThAAT) or without (phAAT) transposase binding sites, together with either pCMV-SB or pCMV-mSB as a control. Total liver DNA was isolated at either day 2 or day 30 postinjection and analyzed by PCR for the presence of a 271-bp product of excision and repair. (C) Variety of DSB repair products produced in vivo in the presence (C57BL/6) and absence (C57BL/6-scid) of DNA-PK activity. The total numbers of clones with identical characteristics are shown, as well as the sizes in base pairs of both the resulting footprint and deleted region. Footprints are in lowercase. Dashes, deleted nucleotides; NA, not applicable.