Generation of Slc12a6 ^flox/flox transgenic mice. A, Schematic representation of mouse genomic DNA encompassing exons 15–20 of the Slc12a6 gene, the targeting construct and selection markers, and the predicted recombination between the 5′ and 3′ arms of the construct and the genomic Slc12a6 DNA. The modified allele contains LoxP sites (▶) in introns 17 and 18. The PGK-neo cassette was also flanked with Frt sequences (×) and resides in intron 17 in the same orientation. Arrows (→) indicate primers used for PCR or to generate probes for Southern blot screening of ES clones and animals. P, PstI; Xb, XbaI; B, BamHI; K, KpnI; S, SalI; X, XhoI. B, C, Southern blot analysis and PCR screening experiments used to identify 5′ and 3′ targeted recombination in positive clones using probes and specific primers (see Materials and Methods). D, A representative Western blot analysis of brain and kidney lysates from Slc12a6 ^flox/flox transgenic animals and WT littermates probed with anti-KCC3 antibody or anti-tubulin antibody (n = 3).