We have created a transgenic mouse with a hypomorphic allele of the vesicular monoamine transporter 2 (gene (9, 34, 36), these mice survive into adulthood as homozygotes and do not suffer gross physical defects, offering a unique opportunity to examine more subtle aspects of the behavioral and brain phenotypes resulting from abnormal intracellular handling of monoamine transmitters. MATERIALS AND METHODS Targeting vector construction. The mouse locus was cloned from a partial cassette (kindly supplied by Austin Smith, Centre for Genome Research, Edinburgh, United Kingdom) was cloned into the promoter and the leader exon in pBluescript (Stratagene). A 2.2-kb gene was cloned into the blunt-ended gene. (A) The gene in the 129 wild-type mouse genome and mutant sequences after targeted insertion of the vector, with prominent restriction endonuclease sites shown (B, 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 5 and 3 hybridization probes with genomic DNA extracted from the tail tips of wild-type Cabazitaxel cost (lane 1), heterozygous (lane 3), and homozygous (lanes 2 and 4) mice digested with the restriction endonucleases 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 gene (see also panel A). The representative dark-field photomicrographs show the expression of 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. Gene targeting in ES cells. The targeting vector (30 g) was linearized with null allele. Homozygous mice derived from one of these cell lines (GB1/1) died shortly after birth. In the KA1 cell line, one arm of the construct did not recombine as predicted but inserted Rabbit Polyclonal to HTR1B into the locus (Fig. ?(Fig.1A).1A). The insertion site was confirmed by Southern blot analysis by using 3 and 5 hybridization probes (Fig. ?(Fig.1B)1B) and verified by PCR analysis of the junction between the transgene and the genome (Fig. ?(Fig.11C). Open in a separate window FIG. 3 RT-PCR and Western blot analysis of homozygous KA1 insertional mutants and homozygous GB1/1 knockout mice. (A) Ethidium-stained agarose electrophoresis of RT-PCR products. Total midbrain RNA from homozygous (hom) and wild-type (wt) GB1/1 and KA1 neonates was isolated and reverse transcribed. Expression of mRNA was detected using specific primers for exon 2 and exon 12 of the mouse gene (33). These primers amplified cDNA made from homozygous KA1 mice, but no amplification product was generated with cDNA made from homozygous GB1/1 neonates. The quality of all cDNA preparations was examined by performing control RT-PCRs with primers to hypoxanthine phosphoribosyltransferase (wild-type allele, the KA1 insertion allele, and the GB1/1 knockout allele. The wild-type allele is usually transcriptionally active. The KA1 insertion interferes severely with transcription, and only a small amount Cabazitaxel cost of message is usually generated. In the GB1/1 knockout line, the first and second exon of the gene are deleted, completely abolishing the generation of normal message. (C) Western blot of striatal membrane preparations from homozygous and wild-type KA1 mice probed with a polyclonal antibody against the VMAT2 protein. Both lanes contain comparable amounts of protein, as determined by the Bradford method. Quantitative analysis of the blot using SeeScan indicated a decrease in signal of more than 95% in homozygotes below that of the wild-type signal. The position of the VMAT2-specific band is usually indicated by the arrowhead. The higher molecular weight band is usually nonspecific, being found in extracts from controls and mutants. Generation of chimeric KA1 and GB1/1 mice. Chimeric mice were generated by injection of the selected ES cells into blastocysts of C57BL/6 mice using standard techniques (29). Highly chimeric males were bred with C57BL/6 females, and agouti offspring were tested for germ line transmission by Southern blot analysis of DNA extracted from tail-tip specimens. Homozygous mice were obtained by interbreeding heterozygotes. In all subsequent experiments, the genotype of the mice was confirmed by Southern blot analysis of tail tips. In situ hybridization histochemistry. Brains from adult mice were removed, rapidly frozen, and stored at ?70C. Ten-micrometer sections were cut (cryostat) and processed for in situ hybridization by using a mix of four different 35S-labeled oligonucleotide probes directed against mouse mRNA: 5-GCAGCAGCACCAGATCGCTCAGGGCCAT-3 (exon 1, nucleotides 1 to 24); 5-GGATCAGCTTGCGCGAGTGGCGGCTGTCCCGCAGCC-3 (exon 1, nucleotides 28 to 63); 5-GCCTTGGGTGACTCCCCTCCTGGGAGGCCCCCCCGT GGC-3 (exon 2, nucleotides 272 to 310); and 5-CATTTATGCAGAATCCAGCAAACATGG GAATTGGATAGCC-3 (exon 2, nucleotides 179 to 218) (33). Enkephalin mRNA was detected with 35S-labeled antisense oligonucleotide probes for mouse proenkephalin cDNA (nucleotides 2428 to 2463; GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”U09941″,”term_id”:”497272″,”term_text”:”U09941″U09941), and material P mRNA was detected with 35S-labeled oligonucleotide Cabazitaxel cost probes antisense to mouse beta-preprotachykinin A cDNA (nucleotides 234 to 270; Cabazitaxel cost GenBank accession number D1007723). Cabazitaxel cost Hybridization was carried out in 2 SSC (1 SSC is usually 0.15 M NaCl plus 0.015 M Sodium citrate), 50% deionized formamide, 10% dextran.