Generation of TAC4^−/− mice. (A) Comparison of amino acid sequences of mouse/rat (m/r) HK-1, human (h) HK-1, SP, NKA, and NKB. The tachykinin motif FXGLM is indicated in bold type. (B) Targeted mutagenesis of the TAC4 gene. The map shows the WT locus, the construct, and the targeted allele after homologous recombination. Exons are represented as black boxes. Markers NEO and DT-A are shown as gray boxes. DNA probes for Southern hybridization and primers used for PCR typing (arrows) are indicated. Restriction enzymes used for Southern blot analysis are BglI (B) and NcoI (N). (C) Two TAC4^+/+, 2 heterozygote (+/−), and 2 TAC4^−/− mice were typed by PCR analysis for the presence of NEO as well as the WT and/or mutant band of TAC4. (D) Two TAC4^+/+, 3 heterozygote (+/−), and 2 TAC4^−/− mice were typed by Southern blot analysis for the presence of NEO as well as the WT and/or mutant band of TAC4. (E) RT-PCR analysis for TAC4 mRNA expression in bone marrow (BM), spleen, and thymus of a TAC4^−/− and TAC4^+/+ mouse. β-actin was used to confirm the presence of equal amounts of cDNA. (F) Northern blot analysis for TAC4 mRNA in the olfactory epithelium (OE) of a TAC4^−/− and TAC4^+/+ mouse. A total of 15 μg of total RNA was loaded per lane. 70Z/3 RNA was used as a positive control for TAC4 expression. L32 was used to assure equal loading of the gel.