FIG. 1.

Targeted insertion into the third intron of the Vmat2 gene. (A) The Vmat2 gene in the 129 wild-type mouse genome and mutant sequences after targeted insertion of the vector, with prominent restriction endonuclease sites shown (B, BamHI; H, HindIII; K, KpnI; S, SacI; X, XbaI; and Xh, XhoI). Neomycin resistance sequences (neo) and sequences recognized by the Vmat2 3′ and 5′ hybridization probes are indicated. Homologous sequences in the mouse genome and of the targeting vector and the neomycin resistance (neo) and the herpes simplex virus thymidine kinase (HSV tk) sequences are indicated. (B) Southern blot analysis of hybridization of radiolabeled Vmat2 5′ and 3′ hybridization probes with genomic DNA extracted from the tail tips of wild-type (lane 1), heterozygous (lane 3), and homozygous (lanes 2 and 4) mice digested with the restriction endonucleases HindIII and XbaI (H+X), BamHI (B), KpnI (K), or Xhol (Xh). (C) Sequence of the PCR product generated with a forward primer specific for the 5′ flanking genomic sequences (SF1) and a reverse primer specific for the 5′ end of the transgene (SR1) (see also PCR primer in panel A). (D) Vmat2 RNA expression in the major monoaminergic cell body groups in the brain of homozygous KA1 mice. In situ hybridization was carried out using end-labeled radioactive oligonucleotides complementary to the first and second exon of the Vmat2 gene (see also panel A). The representative dark-field photomicrographs show the expression of Vmat2 mRNA in the substantia nigra (SN) and ventral tegmental (VTA), the dorsal raphe nucleus (RAPHE), and in the locus coruleus (LC) in a wild-type mouse. No signal was detected in the homozygous KA1 mutant.