Figure 1.
Generation of synaptotagmin-2 KO mice by homologous recombination. A, Strategy for mutating synaptotagmin-2 in the mouse. The diagrams depict the structures of the wild-type synaptotagmin-2 gene, which contains all eight exons for synaptotagmin-2 (top), the targeting vector constructed for the mutation of the synaptotagmin-2 gene (middle), and the mutant gene resulting from homologous recombination (bottom). In the final product, part of exon 2 through exon 7 were replaced by the poliovirus IRES (IR), the cDNA for lacZ with a nuclear localization signal (lacZ) and PGK-Neo (Neo). This last gene was used for positive selection, whereas the MC1 thymidine kinase gene (TK) at the 3′ end of the construct was used for negative selection. The positions of the oligonucleotide primers used to identify the wild-type and mutant alleles are indicated by arrows (P1, P2, and P3). The SalI recognition sites (S) and template for the probe used for Southern blotting are also indicated. B, Western blots indicate the lack of synaptotagmin-2 in mutant mouse brain and spinal cord. Other synaptic proteins that were quantified (Table 1) in homogenate of brain, cerebellum/brainstem, and spinal cord homogenates are also showed in the figure. C, X-gal staining of whole-mount mouse CNS in WT and synaptotagmin-2 KO. Note that dorsal root ganglia attached to the spinal cord also show positive staining. Syt 2, Synaptotagmin-2; Syt 1, synaptotagmin-1; Syb 2, synaptobrevin-2; Syp 1, synaptophysin-1.
