FIG. 3.

Intrachromosomal recombination substrates integrated at the HPRT locus. (A) The HPRT locus at exons 2 and 3 is shown along with gene-targeted HPRT loci containing intrachromosomal repair substrates. The H-DR-WT substrate contains a direct neo repeat of S2neo and pneo-WT, and the H-DR-8mu substrate contains a direct neo repeat of S2neo and pneo-8mu (Fig. 1). Separating the neo repeat in both substrates is a pgk-puro gene for selection. Repair substrates were cloned into the PvuII (Pv) and XbaI (X) sites of the HPRT locus, as indicated, using XhoI (Xh) and XbaI sites of the substrates, and this deleted exon 2. Distances between the BamHI (B) and HindIII (H3) sites are shown in kilobases. Neo1 and Neo3 indicate the primers used for PCR amplification. Neo1 has no overlap with pneo-8mu, and Neo3 has an overlap of 6 nucleotides. (B) Southern blot analysis of BamHI-HindII-digested genomic DNA indicated that the intrachromosomal substrates were correctly targeted and had integrated as a single copy in the wild-type and Msh2^−/− cell lines. The probe was a 1.1-kb XhoI-HindIII fragment containing the entire neo⁺ gene. Genomic DNA from parental wild-type and Msh2^−/− cells did not hybridize with this probe, as expected (data not shown).