gfp tagged adeno associated virus aav vector  (Addgene inc)

Journal: Nature Communications

Article Title: Inhibition of histone methyltransferase Smyd3 rescues NMDAR and cognitive deficits in a tauopathy mouse model

doi: 10.1038/s41467-022-35749-6

Figure Lengend Snippet: a Quantitative PCR analysis showing mRNA levels of Fbxo2 in N2A cells transfected with Fbxo2 shRNA or a scrambled control shRNA ( n = 3 cultures/group, t = 29.3, p < 0.0001, t test). b A representative fluorescent image showing viral-infected PFC from a mouse with the stereotaxic injection of Fbxo2 shRNA AAV (GFP-tagged). Scale bar: 1000 μm. This experiment was repeated 3 times independently with similar results. c Quantitative PCR analysis showing Fbxo2 mRNA levels in PFC of Tau mice infected with Fbxo2 shRNA or scrambled shRNA AAV (n = 5 mice/group, t = 3.7, p = 0.02, t test). d Input–output curves of NMDAR-EPSC in response to a series of stimulation intensities in PFC layer V pyramidal neurons in Tau mice infected with Fbxo2 shRNA or scrambled shRNA AAV ( n = 13–16 cells/3 mice/group, F 1,27(group) = 9.1, p = 0.006, two-way rmANOVA). Inset: representative eEPSC traces at different stimulation intensities. e , f Bar graphs showing investigation time on novel (N) and familiar (F) objects ( e ), and discrimination indexes ( f ) for NOR tests of WT or Tau mice infected with Fbxo2 shRNA or scrambled shRNA AAV ( n = 4–6 mice/group, e , F 3,34(int e raction) = 4.1, p = 0.01; f , F 1,17(treatment) = 13.1, p = 0.002, F 1,17(interaction) = 12.9, p = 0.002, two-way ANOVA). g , h Bar graphs showing investigation time on correct (T1) and incorrect (T2) holes ( g ), and spatial memory indexes ( h ) for BM tests of WT or Tau mice infected with Fbxo2 shRNA or scrambled shRNA AAV ( n = 4–6 mice/group, g , F 3,34(interaction) = 4.9, p = 0.006; h , F 1,17(treatment) = 4.2, p = 0.06, F 1,17(interaction) = 21.8, p = 0.0002, two-way ANOVA). In all figures, * p < 0.05, ** p < 0.01, ***p < 0.001. i A schematic model showing that Smyd3 elevation in AD leads to Fbxo2 upregulation because of increased H3K4me3 occupancy, resulting in more NR1 ubiquitination and diminished NMDAR function in PFC, causing cognitive impairment. Smyd3 inhibition or Fbxo2 knockdown provides an intervention avenue to rescue synaptic and behavioral deficits in Tau AD mice. The diagram was created with Biorender. Data are presented as mean values ± SEM. Detailed statistical data are provided in Source Data files.

Article Snippet: The shRNA was cloned into GFP-tagged adeno-associated virus (AAV) vector (Addgene) under the control of U6 promotor.

Techniques: Real-time Polymerase Chain Reaction, Transfection, shRNA, Control, Infection, Injection, Inhibition, Knockdown