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

Figure 2. In vivo Excision of the Tkneo Gene Results in Altered Gene Expression.

Figure 2

(A) A schematic diagram demonstrating the in vivo excision of the Tkneo gene. Heterozygous neo-containing SNAP-25b deficient females were mated with heterozygous Prm1Cre males. Male offspring carrying both the mutation in the Snap25 gene and the Cre transgene were mated with B6 females. In vivo excision was demonstrated by PCR, and the Cre transgene was crossed out from the confirmed neo-excised SNAP-25b deficient mice. (B) To investigate total SNAP-25 mRNA levels in mouse brain of neo-excised SNAP-25b deficient mutants at PN14-15, the same radioactive semi-quantitative RT-PCR assay as for neo-containing mouse mutants (see Figure 1B) was used. Total SNAP-25 mRNA levels (SNAP-25a + SNAP-25b) in neo-excised SNAP-25b deficient mice brain at PN14-15 were 117.3±6.8% of WT (n = 6 mice for each genotype, repeated three times each, mean±S.E.M., *p = 0.0625, Wilcoxon's signed-rank test). (C) SNAP-25a/SNAP-25b mRNA ratio at PN14 were for WT littermates 17.5±1.2% and 82.5±1.2%, respectively (mean±S.E.M., ***p<0.0001 Student's t-test), in heterozygous neo-excised SNAP-25b mutants 68.9±2.1% SNAP-25a and 31.1±2.1% SNAP-25b (***p<0.0001), and in homozygous neo-excised SNAP-25b deficient mice 100% SNAP-25a (n = 5 mice for each genotype, repeated twice). (D) Protein levels in brain at PN14-15. In neo-excised SNAP-25b deficient mice, there was 110.9±2.3% (*p = 0.0312) SNAP-25 protein, 106.4±4.2% (p = 0.3125, n.s) SNAP-23, and 99.4±3.5% (p = 0.8438, n.s.) syntaxin 1 relative to WT expression levels (n = 6 mice for each genotype, run in three replicates, mean±S.E.M., Wilcoxon's signed-rank test). (E) Immunoblot demonstrating temperature dependence and stability of the ternary SDS resistant SNARE complex in neo-excised SNAP-25b deficient (KO) and WT mouse brain cortex. The samples separated in lanes 1–5 (from left to right) are from adult WT brain cortex, and the preparations in lanes 6–10 are from neo-excised SNAP-25b deficient mutants (KO only expressing SNAP-25a). The high molecular weight ∼97 kD complex was identified as SNAP-25 in ternary SNARE complex, while the band migrating as ∼28 kDa is free SNAP-25 protein. KO brain homogenates heated to 80°C showed 44±8% SNAP-25 still present in ternary complex compared to 4°C samples, significantly lower than for SNAP-25 in WT brain (71.2±7.2, *p = 0.01). At 85°C and 90°C, the difference in percentage of SNAP-25 remaining in complex between WT and KO was as following: 59.8±7 and 36.9±3.4 (*p = 0.018), and 48.3±12.3 and 4.5±2.2 (**p = 0.0081), respectively. All experiments were repeated at least three times. Results were analyzed with unpaired Student's t-test and summarized as mean±S.E.M.