Figure 4.

Gene targeting at the CFTR locus with HD-23.8-CFTR-PACTk. (a) A single reciprocal crossover in the right and left homology arms results in integration of the PACTk marker to the CFTR gene rendering clones puro^R. PB inverted terminal repeats (PB ITRs) flank the PACTk cassette to permit its footprintless excision in the presence of PB transposase. Sizes of the diagnostic ApaI fragments and the locations of the 5′ probe, 3′ probe and PACTk probe used for Southern analyses are shown. The positions of PCR primers used to amplify the allele without targeted vector integration is shown. The ▵I507 and ▵F508 mutations are ~0.2 kb from the site of PACTk insertion. The position of the adenoviral packaging signal (Ψ) and adenoviral inverted terminal repeat (Ad ITR) are shown for each HDAd. (b) Representative Southern blots of genomic DNA extracted from puro^R clones analyzed with the 5′ external probe, the 3′ external probe and the PACTk probe showing targeted vector integration (clones 20, 64, 74, 96, 4, and 6), aberrant targeting (clones 21 and 57), and random vector integration (clones 1, 3, 11, 22, 43, 50, and 75). CFTR, cystic fibrosis transmembrane conductance regulator; HDAd, helper-dependent adenoviral vector, PB, piggyBac; PCR, polymerase chain reaction.