Fig. 1
Conditional forebrain-specific Stau2 knockdown rat model. a Schematic diagram of rat transgenes used to achieve tamoxifen (Tx)-inducible, forebrain-specific Stau2 knockdown. Upon Tx application (+Tx), the inducible form of Cre recombinase (CreERT2), produced under the control of the CaMKIIα promoter (PCaMKIIα) deletes a loxP-flanked “ORF-STOP cassette” (consisting of an ORF and a polyA site (pA)). This allows simultaneous expression of the synthetic miRNA targeting Stau2 (miR(Stau2)) (included within an intronic sequence) and EGFP, driven by the CAG promoter (PCAG). b Immunohistochemical analysis of CreERT2-inducible EGFP production in the hippocampus of double transgenic CaMKIIα-CreERT2 x CAG-STOP-miR(Stau2) animals 6–8 weeks after vehicle (-Tx) or Tx (+Tx; Stau2KD) injection. Arrows indicate the hilus (white) or the granular cell layer (black) of the dentate gyrus (DG). c Quantification of miR(Stau2) expression by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in hippocampal areas of Stau2KD animals (n = 4 animals). Bars represent mean + SEM. d Representative western blot analysis of Stau2 protein levels in hippocampal areas of Tx-injected Stau2KD and CaMKIIα-CreERT2 rats (n = 3 animals/group). Arrowheads indicate the different isoforms of Stau2 protein [7]. e Dual immunofluorescence of EGFP (Alexa488; green), indicating miR(Stau2) expression, and Stau2 (Cy5; red) in hippocampal areas CA1, CA3, and the hilus of the DG (DG-Hi) of Stau2KD animals; arrowheads indicate Stau2-positive immunostaining in EGFP-negative neurons. Scale bars: 50 μm. f Quantification of mRNA levels by qRT-PCR for the indicated genes in control and ubiquitously germline Cre-recombined CAG-STOP-miR(Stau2) rats (Stau2-silenced). Each dot represents the corresponding mRNA level for one animal. Figure shows single values, mean ± SEM. Stau2: Staufen2, RhoA: Ras homolog family member A, Cplx1: Complexin 1, Rgs4: Regulator of small G protein signaling, Ppp2r1b: Protein phosphatase 2 scaffold subunit A, isoform α, Calm3: Calmodulin 3, Arntl: Aryl hydrocarbon receptor nuclear translocator like. PPIA (Peptidylprolyl isomerase A) was used as housekeeping gene (F genotype*genes (6,41) = 47.21; p < 0.0001). Stars represent p values between genotypes obtained by Bonferroni post hoc analysis following two-way ANOVA of repeated measures, ** p < 0.01, ***p < 0.001