Figure 1.

Mutation of the Syt IV gene by homologous recombination. (A) Partial structure of the wild-type Syt IV gene (Top), the targeting construct (Middle), and the mutated Syt IV gene after homologous recombination (Bottom). In the targeting construct, a neomycin resistance (neo^R) gene was inserted into the exonic sequence, and the genomic fragment was flanked by herpes simplex virus thymidine kinase (tk) genes. Expected sizes of wild type and disrupted Syt IV gene after digestion with NcoI and hybridization with a 3′ flanking probe (solid bar) are indicated. Targeted clones were verified by PCR (data not shown). Relevant restriction enzymes are SacI (S), BamHI (B), and NcoI (N). (B) Southern blot analysis of offspring from a mating of Syt IV heterozygotes. Tail DNAs were digested with NcoI and hybridized with the 3′ flanking probe. Syt IV genotypes are indicated above each lane. (C) Northern blot analysis. Total RNA (10 μg) from the brains of two mice of each genotype were hybridized with Syt IV (Top), GAPDH (Middle), and Syt I (Bottom) cDNA probes. Relative positions of 18S and 28S ribosomal RNAs are indicated in Top.