Skip to main content
. 2008 Nov 28;4(11):e1000278. doi: 10.1371/journal.pgen.1000278

Figure 1. Generation of SNAP-25b Deficient Mouse Mutants.

Figure 1

(A) Schematic diagram demonstrating the targeting construct and the development of a modified Snap25 allele with eliminated SNAP-25b expression. The mouse genomic sequence was derived from the WT Snap25 gene and addition of an additional exon 5a was performed using PCR. A Tkneo selection gene, surrounded by loxP repeats, was inserted at the EcoRI (E) site located downstream of the two tandemly arranged exons 5a and upstream of exon 6. Letters denote restriction sites: E, EcoRI; H, HindIII; P, PstI; S, SacI; X, XbaI. (B) Total SNAP-25 mRNA level in mouse brain at PN14 was investigated by semi-quantitative RT-PCR. Levels of SNAP-25 mRNA, quantified relative to GAPDH mRNA levels, were determined in neo-containing SNAP-25b deficient mutants (KO) and compared to WT littermates. SNAP-25 mRNA in neo-containing SNAP-25b deficient mutant brain was significantly reduced to 55.5±2.7% compared to WT (mean±S.E.M., n = 6 mice for each genotype, each sample repeated three times, *p = 0.0312, Wilcoxon's signed-rank test). (C) A semi-quantitative RT-PCR restriction enzyme assay was used to determine relative levels of SNAP-25a and SNAP-25b isoform mRNAs in WT (+/+), neo-containing heterozygous (+/−) and neo-containing homozygous (−/−) mutant brains at PN14. A PvuI restriction site exclusive for exon 5a and a StyI site only present in exon 5b was used to determine relative levels of SNAP-25a and SNAP-25b. WT mice had 16.7±1.9% SNAP-25a mRNA, neo-containing heterozygous mutants 51.6±1.7% SNAP-25a mRNA, and homozygous SNAP-25b deficient mutants only, the SNAP-25a mRNA isoform (n = 5 mice of each genotype, run in two replicates, mean±S.E.M., ***p<0.0001, Student's t-test). (D) Western blotting was used to analyze synaptic protein levels in brain at PN14-15, standardized against α-tubulin. The level of SNAP-25 protein in neo-containing SNAP-25b deficient mutants was 80.7±3.6% compared to WT (mean±S.E.M., *p = 0.0312, Wilcoxon's signed-rank test), whereas expression of SNAP-23 protein was 98.9±3.5% (p = 1, n.s.) and that of syntaxin 1 protein 107.6±4.9% (p = 0.4375, n.s.), compared to WT animals. The levels of SNAP-25 protein differed significantly in neo-containing SNAP-25b deficient mutants compared to WT littermates, but not the levels of SNAP-23 and syntaxin 1 proteins (n = 6 mice of each genotype, run in three replicates). (E) Representative images of SNAP-25 immunoreactivity in coronal sections at low magnification (i, iii) and in the hippocampus at higher magnification (ii, iv). No obvious differences in immunoreactivity pattern was observed between neo-containing SNAP-25b deficient (iii, iv) and WT (i, ii) mice at PN14 (n = 2 mice of each genotype, and three levels were analyzed from each animal). Identical microscope settings were used for WT and KO (neo-containing SNAP-25b deficient mutants) images. Scale bar = 1 mm for the low magnification and 200 µm for the high magnification figures. (F) Weight curves of homozygous neo-containing SNAP-25b deficient mutants compared to WT littermates between PN5 to PN15. Body weight gain was significantly reduced in young neo-containing SNAP-25b deficient mutants when compared to WT (***p<0.0001, two-way repeated measures ANOVA, n = 7 mice of each genotype). (G) Bone growth is affected in neo-containing SNAP-25b deficient mice. Mean±S.E.M. thickness of the hypertrophic, proliferative, and reserve zones in femur and tibia in WT (black bars) and neo-containing SNAP-25b deficient (white bars) PN14 mice. *p<0.05 (n = 4). Data was analyzed with unpaired Student's t-test.