FIG. 5.

Characterization of ES cells produced by the hit-and-run procedure. Verification of PCR-positive clones of the hit-and-run procedure by Southern blot analysis. Aliquots containing 5 μg of genomic DNA from ES cell clones (cultured on PMEFs) were double digested with AatII/HindIII and hybridized with intron 16-specific probe 4 after electrophoretic separation through 2% agarose gels and transfer to a nitrocellulose membrane. A single band at 5.6 kb was obtained from wild-type ES cells (wt) while an additional band at 4.6 kb was obtained from hit-and-run clones for the targeted allele carrying the HCHWA-D mutation. From the hit clone 110, which served as precursor for these positive clones, a third band at 5.3 kb was detected, indicating the absence of an HCHWA-D mutation on the 3′ site of the partial genomic duplication. Numbers at tops of lanes are clone designations.