Journal: Molecular Brain

Article Title: Deficiency of calretinin in prefrontal cortex causes behavioral deficits relevant to autism spectrum disorder in mice

doi: 10.1186/s13041-025-01233-7

Figure Lengend Snippet: Distribution of enhanced green fluorescent protein (EGFP) and the knockdown efficiency of rAAV-CR shRNA on PFC CR expression in mice. ( A ) Viral genome maps for viruses designed to express CR and EGFP. ( B ) EGFP signals confirmed by immunofluorescence in the PFC around 21 days post injection. ( C ) The boxed area is exhibited in ( B ) at a higher magnification. ( D - E ) Three weeks after the injection of rAAV vectors, CR protein expression in mice with rAAV-CR shRNA intra-PFC injection ( n = 6). ( F ) Representative images depicting CR-positive cells in sections obtained from the rAAV-EGFP- and rAAV-CR shRNA-treated animals. Upper row: Immunohistochemical detection of CR-containing cells in 10 objective lens. Bottom row: regions of PFC sketched by the black boxes in the upper row were visualized using 40 objective lens. Some CR-immunogenic neurons have been identified by arrows. ( G ) Significant expression is detected of CR interneurons in mice exposed with rAAV-CR shRNA injection ( n = 5 each). ** p < 0.01, *** p < 0.001, compared with rAAV-EGFP-exposed animals. Student’s t -test. Data are presented as means ± SEM

Article Snippet: The recombinant adeno-associated virus (rAAV) vectors expressing CR shRNA (rAAV-GAD67-CR-shRNA-CMV-EGFP-WPRE-hGH-ITR, 5.54 × 10 12 vg/ml) were supplied by Shanghai Biotechnology Co., Ltd.

Techniques: Knockdown, shRNA, Expressing, Immunofluorescence, Injection, Immunohistochemical staining

Journal: Molecular Brain

Article Title: Deficiency of calretinin in prefrontal cortex causes behavioral deficits relevant to autism spectrum disorder in mice

doi: 10.1186/s13041-025-01233-7

Figure Lengend Snippet: Deficiency of CR resulted in social impairments in the three-chamber test. ( A ) In three-chamber social interaction test, rAAV-EGFP-injected mice spent obviously more time in the chambers of the unfamiliar animal relative to the novel object. In contrast, rAAV-CR shRNA treatment was associated with these animals having spent equal amounts of time in the chamber containing the novel mouse and the unfamiliar object. ( B ) The rAAV-EGFP-exposed mice spent more time sniffing the novel mouse as compared to the unfamiliar object. Mice with rAAV-CR shRNA injection spent similar time sniffing with the novel mouse and the unfamiliar object. ( C ) The rAAV-CR shRNA group had an evidently lower social preference index than the control animals. n = 10. *** p < 0.001, ### p < 0.001 for novel mouse vs. novel object. ^^^ p < 0.001, compared with rAAV-EGFP-treated group. “ns” indicating no statistical significance. One-way ANOVA with Tukey’s post hoc test. Student’s t -test. Data are means ± SEM. ( D ) In test for social novelty preferences, mice with rAAV-EGFP injection spent more time exploring the compartment containing the stranger mouse compared with familiar controls. In contrast, no considerable difference in the time spent exploring the compartment containing the novel mouse compared with the time spent exploring the familiar animal in rAAV-CR shRNA-operated mice. ( E ) The rAAV-EGFP-injected mice spent significantly more time sniffing and engaging with the novel mouse in comparison with the familiar mouse. In contrast, rAAV-CR shRNA-treated mice spent equal amounts of time sniffing the novel mouse and the familiar animal. ( F ) The rAAV-CR shRNA-administered mice had significantly a lower discrimination index than the control animals. n = 10. &&& p < 0.001, $$$ p < 0.001 for novel mouse vs. familiar mouse. ^^^ p < 0.001, compared with rAAV-EGFP-treated mice. “ns” indicating no statistical significance. One-way ANOVA with Tukey’s post hoc test. Student’s t -test. Data are presented as means ± SEM

Article Snippet: The recombinant adeno-associated virus (rAAV) vectors expressing CR shRNA (rAAV-GAD67-CR-shRNA-CMV-EGFP-WPRE-hGH-ITR, 5.54 × 10 12 vg/ml) were supplied by Shanghai Biotechnology Co., Ltd.

Techniques: Injection, shRNA, Control, Comparison

Journal: Molecular Brain

Article Title: Deficiency of calretinin in prefrontal cortex causes behavioral deficits relevant to autism spectrum disorder in mice

doi: 10.1186/s13041-025-01233-7

Figure Lengend Snippet: rAAV-CR shRNA injection caused stereotyped/repetitive behaviors, anxiety and memory impairments in naïve mice. ( A ) In the marble burying test, the number of marbles buried in animal with CR downregulation was increased than that of rAAV-EGFP-operated mice. ( B ) In the grooming test, mice with rAAV-CR shRNA injection spent more time in grooming relative to corresponding controls. ( C - D ) In the open-field test, animal with CR knockdown spent less time in the center area as compared to that of corresponding controls. No dramatical difference for the distance moved between two groups. ( E - F ) In the elevated plus maze test, the total amount of time the mouse with CR knockdown spent in the open arms was less than that of rAAV-EGFP-treated mice. No statistically apparent difference in open-arm entries was found between two groups. ( G - H ) In the novel object recognition test, rAAV-GFP-injected animals spent more time exploring the unfamiliar object as compared to the familiar one. Mice exposed with rAAV-CR injection presented significantly decreased time spent exploring the novel object in comparison with the familiar one. Animals treated with rAAV-CR shRNA exhibited decreased discrimination ratio relative to rAAV-GFP group. n = 10. *** p < 0.001, ** p < 0.01, compared with rAAV-GFP-treated mouse. ### p < 0.001 for novel object vs. familiar object. “ns” indicating no statistical significance. Student’s t -test. Data are represented as means ± SEM

Article Snippet: The recombinant adeno-associated virus (rAAV) vectors expressing CR shRNA (rAAV-GAD67-CR-shRNA-CMV-EGFP-WPRE-hGH-ITR, 5.54 × 10 12 vg/ml) were supplied by Shanghai Biotechnology Co., Ltd.

Techniques: shRNA, Injection, Knockdown, Comparison

Journal: Molecular Brain

Article Title: Deficiency of calretinin in prefrontal cortex causes behavioral deficits relevant to autism spectrum disorder in mice

doi: 10.1186/s13041-025-01233-7

Figure Lengend Snippet: In vitro whole-cell patch recordings showing the impact of CR knockdown on neuronal excitability in the PFC slices. ( A ) Fluorescent (EGFP, upper panel) and infrared differential interference contrast (DIC, bottom panel) image showing EGFP-labeled cell patched in the PFC from rAAV-EGFP and rAAV-CR shRNA mice. ( B ) Representative spiking pattern of EGFP-containing neurons traces in response to 200 pA current injection. ( C - E ) Quantitative comparisons of the resting membrane potential, action potential threshold, and rheobase current. ( F ) Evoked spike rates vs. current magnitudes in the PFC between two groups, suggesting a significant increase in mean firing frequency in rAAV-CR shRNA-treated mice compared to rAAV-EGFP group ( n = 11 cells for rAAV-GFP from 5 animals and 11 cells for rAAV-CR shRNA 5 animals). *** p < 0.001, ** p < 0.01, “ns” indicating no statistical significance; repeated measures ANOVA. Data are means ± SEM

Article Snippet: The recombinant adeno-associated virus (rAAV) vectors expressing CR shRNA (rAAV-GAD67-CR-shRNA-CMV-EGFP-WPRE-hGH-ITR, 5.54 × 10 12 vg/ml) were supplied by Shanghai Biotechnology Co., Ltd.

Techniques: In Vitro, Knockdown, Labeling, shRNA, Injection, Membrane

Journal: Molecular Therapy. Nucleic Acids

Article Title: Ligand-modified rAAV6 vectors with nanoblades allow high-level gene knockin in HSPCs without compromising cell survival

doi: 10.1016/j.omtn.2025.102495

Figure Lengend Snippet: Bio-conjugation of rAAV6 with mannose allowed high-level nanoblade-mediated knockin into CD34 + cells and reduced cell death (A and B) K562 cells were transduced at an MOI of 50,000 (vg/cell) with the unmodified rAAV2 and rAAV6 vectors (Ctrl) or with rAAV2 and rAAV6 bio-conjugated with mannose sugars at their lysine residues (K3E5 or K3E6) or tyrosine residues (Y3E5) in the presence of nanoblades. At day 3 and 6 post incubation with rAAV vectors and nanoblades, the % of knockin was determined by % of (A) GFP-expressing K562 cells and their survival using DAPI staining (B) analyzed by FACS; mean (SD); n = 3; three biological repeats; multiple Student’s t test, Tukey’s multiple comparisons test; ∗∗∗∗ p < 0.001. (C and D) CD34 + cells were pre-stimulated with cytokines overnight and were then transduced at an MOI of 50,000 (vg/cell) with the unmodified rAAV2 and rAAV6 vectors (Ctrl) or with rAAV2 and rAAV6 bio-conjugated with mannose sugars at their lysine residues (K3E5 or K3E6) or tyrosine residues (Y3E5; see also Table S1 ) in the presence of nanoblades. At day 3 and 6 post transduction, the % of GFP-positive CD34 + cells (% knockin; C) and their survival (D) were determined using FACS analysis; mean (SD); n = 3; three biological repeats for which three independent CD34 + cell donors were used; multiple Student’s t test, Tukey’s multiple comparisons test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (E) Representative FACS analysis for the data in (C) showing gene knockin by analysis of GFP expression in the CD34 + cells at day 6.

Article Snippet: rAAV vectors were loaded at a dose of 10 10 vg on a nitrocellulose paper soaked briefly in PBS prior to assembling the dot-blot manifold (Bio-Rad).

Techniques: Conjugation Assay, Knock-In, Incubation, Expressing, Staining, Transduction