SNARE Function Analyzed in Synaptobrevin/VAMP Knockout Mice Susanne Schoch, Ferenc Deák, Andreas Königstorfer, Marina Mozhayeva, Yildirim Sara, Thomas C. Sádhof, and Ege T. Kavalali

Generation, maintenance, and morphological analysis of synaptobrevin 2 knockout mice. Genomic -clones containing the murine synaptobrevin 2 gene were isolated using standard techniques, and used to construct a targeting vector (Fig. 1) in principle as described previously (45,46). Two targeting vectors were originally constructed. In both vectors, a neomycin resistance cassette flanked by loxP sites was inserted into a Not1 site at the 5' end of the gene 104 bp upstream of the initiator ATG, and point mutations (V38R and V42R) were introduced into the coding region with the original goal of generating mice in which the synaptobrevin 2 gene is inactivated by the neomycin resistance cassette, but can be reactivated by excision of the cassette which is flanked by loxP sites. However, excision of the neomycin cassette in did not reactivate expression of synaptobrevin 2 but even with only the loxP site the targeted locus was a null mutant, making the mutnat mice useful only as null mutants for synaptobrevin 2. The vectors were electroporated into embryonic stem cells (E14 cells, gift of Dr. K. von Figura, Göttingen), colonies were selected with G418 and gancyclovir (1,2), and double-resistant colonies were analysed for homologous recombination by Southern blotting with an outside probe. Clones containing homologously recombined genes were expanded, confirmed by PCR, and used to generate mice by blastocyst injection. Multiple independent lines were generated that exhibited no phenotypic differences as far analysed. Routine genotyping was performed by PCR using (wild type reaction: SSC 9985 [5'-GCC CAC GCC GCA GTA CCC GGA TG-3'] vs. SSC 9988 ]5'-GCG AGA AGG CCA CCC GAT GGG AG-3'] = 500 bp product; mutant reaction: T1909 [5'-CAC CCT CAT GAT GTC CAC CCG-3'] vs. T1912 [5'-CAG CAG ACC CAG GCC CAG GT3'] or T1910 [5'-CAC CCT CAT GAT GTC CAC CAC-3'] vs. T1911 [5'-CAG CAG ACC CAG GCC CAG CG-3'] = 550 bp product. Mutant mice were maintained as heterozygotes by continuous backcrossing with Black 6 mice. For morphological analyses, brains from knockout and control embryos harvested at embryonic day 18.5 were immersion-fixed in 4% paraformaldehyde overnight under agitation, embedded in paraffin, and Nissl-stained sections were analysed by microscopy. All analyses were performed on littermates derived from heterozygote matings to ensure use of precisely matched controls. Control cultures used were homozygous wild type or heterozygous mutant since in preliminary experiments, no significant difference between hetereozygous and wild type synaptic properties were detected using the analyses described in the current study.

Hippocampal cultures. Neurons from embryonic day 18 hippocampus were dissociated by trypsin (5 mg/ml for 10 min at 37° C), triturated with a siliconized Pasteur pipette, and plated on to 12 mm coverslips coated with poly-L-lysine (~3 coverslips/ hippocampus). Neurons were cultured at 37 °C in a humidified incubator with 95%-air, 5%-CO₂in Minimal Essential Media containing 5 g/l glucose, 0.1 g/l transferrin, 0.25 g/l insulin, 0.3 g/l glutamine, 5-10% heat inactivated FCS, 2% B-27 supplement and 2-4 M cytosine arabinoside, and used at 10-20 days in vitro.

REFERENCES

1. T.W. Rosahl et al., Nature 375, 488 (1995).

2. R. Fernandez-Chacon et al., Nature 410, 41 (2001).

Supplemental Figure 1. Synaptobrevin/VAMP 2 knockout mice. (A) Generation of synaptobrevin 2 knockout mice. Using genomic clones containing the synaptobrevin 2 gene (WT), a targeting vector was constructed in which the neomycin resistance gene flanked by loxP sites was inserted into the 5' end of the gene, and two copies of the TK gene were attached to the short arm (Targeting Vector). Homologous recombination inserts the neomycin resistance cassette into the 5' end of the gene with deletion of the TK gene (KO). Positions of noncoding and coding exon sequences are indicated by white and gray boxes, respectively, and selected restriction enzyme cleavage sites by letters (B = BamH I; E = EcoR I; H = Hind III; N = Not I). (B) Appearance of newborn wild type and mutant mice. Mice from a single litter were photographed immediately after birth. (C to E) Immunoblot analysis of wild type and knockout mice. Proteins from total forebrains (panel C), selected brain regions (panel D), and forebrain and heart (panel E) were analysed with antibodies to the indicated proteins (Syb 2 = synaptobrevin 2; Syp = synaptophysin; Syt 1 = synaptotagmin). Tissues were obtained from wild type (+/+), heterozygous (+/-) and homozygous mutant mice (-/-). All signals were visualized by enhanced chemiluminescence.

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Supplemental Figure 2. Normal brain architecture without neurodegeneration in synaptobrevin 2 knockout mice. Pictures show Nissl-stained coronal sections from wild type (WT) and synaptobrevin knockout brains (KO). Abbreviations used: CP, caudatum and putamen; Ctx, cerebral cortex; ec, external capsule; Hc, hippocampus; Hth, hypothalamus; LHA, lateral hypothalamic area; LP, lateral posterior mucleus of the thalamus; LSr, lateral septal nucleus, rostral part; LV, lateral ventricle; PF, parafascicular nuclei of the thalamus; PH, posterior hypothalamic nucleus; PO, posterior complex of the thalamus; St, Striatum; Th, thalamus; V3, third ventricle; VPL and VPM, ventral posterolateral and posterolmedial nucleus of the thalamus.

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