Generation of mouse lines with mutations in the Slc12a2 gene. (a) Restriction maps of the endogenous Slc12a2 locus, the Slc12a2 targeting constructs, and Slc12a2 locus after homologous recombination with the targeting plasmids. Upon homologous recombination, the Slc12a2^Δ1065-1137 construct replaces a 217-bp region of the Slc12a2 gene, which includes exons 24 and 25, with the selectable marker gene neomycin (neo). Relevant restriction sites are abbreviated as follows: B, BamHI; P, PvuI; R, EcoRI; X, XhoI. The Slc12a2^–/– construct is designed, upon homologous recombination, to replace a 344-bp region of the gene encompassing exons 9, 10, and 11 with the neo gene. Relevant restriction sites are abbreviated as follows: B, BamHI; H, HindIII; N, NotI; X, XbaI. The probes used to detect homologous recombination events by Southern blot analysis are indicated. (b) Model of the Na⁺-K⁺-2Cl^– cotransporter with deleted regions indicated. Circles represent amino acid residues. The amino acids encoded by the regions of the gene replaced with neo by Slc12a2^Δ1065-1137 and Slc12a2^Δ506-621 are indicated by filled circles and shaded circles, respectively. (c) Southern blot analyses of offspring generated by the intercross of mice heterozygous for each of the Slc12a2 mutant alleles. An 800-bp BamHI genomic fragment detects the introduction of an XbaI site after targeted integration of the Slc12a2^Δ1065-1137 plasmid (left panel). For Slc12a2 ^Δ506-621 (right panel), a 1.8-kb genomic fragment that hybridizes upstream of the targeted region was used as a probe to detect the change in HindIII restriction fragment length that occurs with proper integration of the targeting construct. (d) Northern analysis of NKCC1 expression in salivary glands of wild-type, Slc12a2^Δ1065-1137 (left panel), and Slc12a2^–/– (right panel) heterozygous and homozygous animals. Abundant NKCC1 RNA is observed in mice homozygous and heterozygous for either mutation. A slightly smaller NKCC1 transcript resulting from the deletion of exons 23 and 24 is seen in RNA prepared from Slc12a2^Δ1065-1137 mice, whereas a novel transcript is present in the samples from the Slc12a2^–/– mice. Analysis with an actin-specific probe indicates equal RNA sample loading. (e) Analysis of the NKCC1 mRNA remaining in the Slc12a2^Δ1065-1137 animals by RT-PCR. RT-PCR of RNA from wild-type animals yields a fragment of approximately 1100 bp, whereas a fragment of approximately 900 bp was amplified from the heterozygous and homozygous mutant cDNAs, consistent with loss of 195-bp of coding sequence in this mutant line. (f) Analysis of the novel RNA transcript present in Slc12a2^–/– mice. To determine the origin of the novel transcript seen in d, the Northern blot was analyzed with a probe specific for the neomycin gene. A band identical in size to that observed using the NKCC1-specific probe is observed in RNA obtained from Slc12a2^–/– mice.