Targeted mutagenesis of the mouse Avp gene. (a) Targeting strategy. Specific mutations and restriction sites were inserted into exon 1 [A(–1)T; ScaI] or exon 2 [C67X; NheI] by homologous recombination. An additional XbaI site (X*) is created after Cre excision of the loxP-Neo-loxP cassette from the A(–1)T targeted allele. Introduced restriction sites were used to detect mutant and WT Avp genes and reverse-transcribed Avp mRNA. White boxes, Avp gene exons; gray boxes, Oxt gene exons. X, XbaI; H, HindIII; E, EcoRI; A, AccI. (b) Southern blot analysis. XbaI- and ScaI-digested genomic DNA was hybridized with a 1,214-bp probe (HindIII-EcoRI DNA fragment), labeling a 2,375-bp XbaI-digested DNA for the normal allele and 1,507-bp (XbaI-ScaI) and 404-bp (ScaI-XbaI*) fragments for the A(–1)T mutant allele. Digestion with EcoRI and NheI and hybridization with a 912-bp probe (XbaI-AccI DNA fragment) labeled a 4,578-bp EcoRI-digested DNA for the normal allele and 978-bp (EcoRI-NheI) and 3,600-bp (NheI-EcoRI) DNAs for the C67X mutant allele. (c) RT-PCR analysis for the detection of WT and mutant Avp transcripts in the hypothalamus. A 366-bp cDNA spanning the A(–1)T mutation was amplified using forward and reverse primers located within exon 1 and exon 2, respectively. Restriction digestion with ScaI generated a 366-bp band from the normal allele and 265- and 101-bp fragments from the mutant allele. DNA spanning the C67X mutation (267 bp) was amplified by the use of forward (exon 2) and reverse (exon 3) primers. NheI digestion gave rise to 163- and 104-bp fragments derived from the mutant allele.