Figure 1.

Homologous recombination by a replacement vector. (A) The exons encoding the two Kv3.1 splice variants Kv3.1a and Kv3.1b are shown at the top. (B) The two genomic DNA clones in EMBL3 used for the construction of the replacement vector are depicted with their relevant restriction sites. (C) The coding region between EcoRI and MscI (35 bp in the S2–S3 linker) was removed and replaced with the neomycin-resistance cassette (PGKneobpA). Homologous recombination between the DNA replacement construct and the targeted locus replaced a functional Kv3.1 allele with the neo cassette-interrupted, nonfunctional Kv3.1 sequence. Homologous recombination was detected by PCR, using primers C and D, or by Southern blot analysis of HindIII-digested genomic DNA hybridized to external probe A or internal probe B. Solid boxes show Kv3.1 coding regions; hatched boxes show the structural gene of the neo cassette; the stippled boxes indicate untranslated regions. (D) Identification of mutant animals. The internal probe B hybridized to a 5.1-kb BamHI fragment derived from the targeted allele and to a 3.5-kb fragment from the 129SvEMS wild-type allele in a Southern blot of genomic tail DNA. The wild-type allele inherited from C57BL/6 contains an additional BamHI site, leading to a 1.0-kb and a 2.5-kb fragment in mice of mixed genetic background.