Journal: Scientific Reports

Article Title: Supernova: A Versatile Vector System for Single-Cell Labeling and Gene Function Studies in vivo

doi: 10.1038/srep35747

Figure Lengend Snippet: ( a ) Schematic for Supernova-mediated single-cell knockout (KO) of endogenous gene of interest (GOI) flanked by two loxP sites. ( b ) α 2-Chimaerin protein is expressed ubiquitously in the hippocampal CA1, while it is undetected specifically in SnRFP-labeled neurons (arrows), indicating that Cre-based Supernova-mediated gene knockout is highly specific to the labeled cells. Cre-SnRFP vectors were introduced into α2-chimaerin ( α2-Chn ) flox/flox mouse CA1 by IUE. α 2-chimaerin immunohistochemistry and DAPI-staining were performed. Upper panels show the hippocampus of α2-Chn flox/flox mouse. A set of enlarged example images is shown in the bottom. Note that because extremely high intensity signal of Supernova labeling hinders detection of α 2-chimaerin signal, we partially photobleached SnRFP signal in this experiment. ( c,d ) Quantification of Supernova-dependent α2-Chn knockout efficiency and specificity. ( c ) α 2-Chimaerin expression was detected in almost all of SnRFP-negative CA1 hippocampal cells (97.7% ± 0.1%, n = 3 mice; 785 cells/804 cells, 464 cells/474 cells, 1011 cells/1036 cells), while only in 5.9% ± 3.0% (n = 3 mice; 3 cells/31 cells, 0 cell/18 cells, 3 cells/37 cells) of SnRFP-positive CA1 neurons, indicating labeled cell-specific knockout. All values represent as mean ± SEM; (**): 0.001 < P < 0.01; Welch’s t -test. ( d ) Intensities of α 2-chimaerin signal in RFP + cells ( α2-Chn KO cells) and RFP - cells (control cells) that surround RFP + cells were measured (n = 14 cells, 3 mice for each group). (***) P < 0.001, Welch’s t -test. Scale bars: 500 μm ( b , upper); 100 μm ( b , bottom).

Article Snippet: For GFP immunohistochemistry, anti-GFP rat antibody (1:1000, Nacalai Tesque #04404–84) and Alexa 488 Donkey anti-rat IgG (H+L) (1:1000, Invitrogen) was used ( ). β-gal immunohistochemistry was performed to detect the LacZ expression.

Techniques: Knock-Out, Labeling, Gene Knockout, Immunohistochemistry, Staining, Expressing, Control

Journal: Scientific Reports

Article Title: Supernova: A Versatile Vector System for Single-Cell Labeling and Gene Function Studies in vivo

doi: 10.1038/srep35747

Figure Lengend Snippet: ( a ) Schematic representation of the adeno-associated virus (AAV)-based Supernova vector set, which consists of AAV-TRE-Cre-WPRE (AAV-TRE-Cre) and AAV-EF1 α -DIO-tTA-P2A-RFP-WPRE (AAV-EF1 α -DIO-tTA-RFP) vectors. DIO: Double-floxed Inverted Open reading frame. ( b–g ) AAV-based Supernova RFP (AAV-SnRFP) labeled hippocampal CA1 pyramidal neurons ( b–d ) and L5 pyramidal neurons in the cortex ( e–g ) so sparsely and brightly that detailed morphologies such as dendritic spines and axonal boutons of labeled neurons were clearly visible. AAV injection was performed at P10-P13, and brains were fixed at 30 days post-infection (DPI). AAV-EF1 α -GFP-WPRE (AAV-EF1 α -GFP) was co-injected as control. ( c ) Higher-magnification images of the squares in ( b ). The rectangle in c was further enlarged in ( d) . ( f,g ) Enlarged images of squares in ( e ). Scale bars, 500 μm ( b ), 100 μm ( c,e ), 10 μm ( d ), 25 μm ( f,g ). ( h ) Cre-mediated genomic DNA recombination detected using RNZ reporter mice was specific to AAV-SnRFP-labeled neurons in the hippocampus (upper panels) and cortex (bottom panels). AAV-SnRFP was injected into the hippocampus or cortex of RNZ mice at P10, and brains were fixed at 40DPI. Coronal sections were prepared and stained with an anti-β-gal antibody, which detects LacZ expression, and DAPI. Scale bars, 50 μm. ( i ) α 2-Chn was disrupted specifically in an AAV-SnRFP-labeled neuron in the hippocampal CA1. AAV-SnRFP was injected into the hippocampus of α2-Chn flox/flox mice at P2, and brains were dissected and sectioned at P18. Immunohistochemistry was performed to detect α 2-chimaerin expressing cells. α 2-chimaerin was expressed in most cells (DAPI) but not in an AAV-SnRFP-labeled cell (arrow) in the hippocampal CA1. Scale bar, 50 μm. Note that we partially photobleached AAV-SnRFP signal in these experiments ( h,i ) to avoid high intensity signal of Supernova labeling overwhelms α 2-chimaerin and LacZ signals.

Article Snippet: For GFP immunohistochemistry, anti-GFP rat antibody (1:1000, Nacalai Tesque #04404–84) and Alexa 488 Donkey anti-rat IgG (H+L) (1:1000, Invitrogen) was used ( ). β-gal immunohistochemistry was performed to detect the LacZ expression.

Techniques: Virus, Plasmid Preparation, Labeling, Injection, Infection, Control, Staining, Expressing, Immunohistochemistry

Journal: Scientific Reports

Article Title: The ATF6β-calreticulin axis promotes neuronal survival under endoplasmic reticulum stress and excitotoxicity

doi: 10.1038/s41598-021-92529-w

Figure Lengend Snippet: Protection of the primary hippocampal neurons by ATF6β. ( A ) Primary hippocampal neurons were treated with Tg (300 nM) or Tm (1 µg/ml) for 24 h, and cell survival/death was evaluated by the LIVE/DEAD viability assay. Representative fluorescent microscopic images from four independent experiments are shown. The graph depicts the percentages of dead cells. n = 4 experiments. Data are shown as mean ± SEM. ***p < 0.001 by a two-way ANOVA followed by the Bonferroni test. Scale bar: 20 μm. ( B ) Apoptosis was evaluated by immunocytochemical staining of cleaved caspase-3 (red), βIII tubulin (green) and DAPI staining (blue). Representative fluorescent microscopic images from four independent experiments are shown. The graph depicts the percentages of cleaved caspase-3-positive cells. n = 4 experiments. Data are shown as mean ± SEM. ***p < 0.001 by a two-way ANOVA followed by the Bonferroni test. Scale bar: 20 μm. ( C ) Apoptosis was evaluated by immunocytochemical staining of cleaved caspase-3 (red) and GFP (green) using primary hippocampal neurons transfected with vector, ATF6β cDNA or ATF6α cDNA together with GFP cDNA, followed by Tm treatment for 24 h. Representative fluorescent microscopic images are shown. Arrows indicate caspase-3-positive and GFP-positive cells. The graph depicts the percentages of cleaved caspase-3-positive cells out of GFP-positive cells. n = 400 cells per condition from two independent experiments. Data are shown as mean ± SEM. **p < 0.01, ***p < 0.001 by a two-way ANOVA followed by the Bonferroni test. Scale bar: 10 μm. Note that transfection of ATF6β cDNA, but not ATF6α cDNA, rescued Atf6b −/− neurons.

Article Snippet: Cultured hippocampal neurons and Neuro 2a cells were fixed in 4% paraformaldehyde for 15 min at room temperature, and permeabilized in 0.3% Trinton-X100 for 10 min. Primary antibodies against GFP (MBL598; Merck; 1:500), Myc (sc-40; Santa Cruz Biotechnology; 1:200), cleaved caspase-3 (Asp175, Cell Signaling Technology, Inc; 1:800) andβIII tubulin (MAB1637; Merck; 1:500) were used.

Techniques: Viability Assay, Staining, Transfection, Plasmid Preparation