Journal: Nature Communications

Article Title: Aberrant neural activity in the peritumoral cortex underlies the progression of tumor-associated seizures

doi: 10.1038/s41467-025-66226-5

Figure Lengend Snippet: a Schematic illustration of experimental design for glioma induction and measurements in the glioma mouse model. Deletion of PTEN , Ink4a/Arf , and overexpression of K-Ras V12 were induced by AAV-GFAP-cre injection in the primary motor cortex of triple conditional mouse. Measurements were carried out on 7, 14, and 21 days post injection (DPI). Brain outlines from Allen Mouse Brain Atlas (atlas.brain-map.org). b Representative hematoxylin-eosin (HE) staining and Ki67 immunolabelling of mouse brain with tumor-bearing cortex. Tumor resembles features of human high-grade glioma, including high cell density, mitoses, giant nuclei, and high density of Ki67 expression. White arrows indicate giant nuclei. c Confocal imaging series of tumor-bearing cortex on 7, 14, and 21 DPI (see “Methods”). Neurons indicated by NeuN (magenta), glial reaction indicated by GFAP (grey), and proliferating cells of growing tumor indicated by Ki67 (green). d Progression of tumor size on 7 DPI (green, N = 3 animals), 14 DPI (blue, N = 7 animals), and 21 DPI (magenta, N = 9 animals). (7 vs 14 DPI P = 0.7733; 14 vs 21 DPI P = 0.0067; 7 vs 21 DPI P = 0.0063) e Progressive changes of cell composition during tumor progression. Top row: Representative images from tumor-bearing cortical regions used for intensity analysis. Bottom row: fluorescent intensity analysis showing progressive reduction of neuron density (NeuN, magenta), expanding GFAP reactive rim (grey), and growing Ki67-positive tumor core (green) in time. Thin lines indicate individual animals ( N = 2 animals per timepoint, see “Methods”), thick lines represent composite mean signal. The center of the tumor is indicated with dashed and the distance from tumor core is indicated on x -axis. f Confocal image of 21 DPI tumor-bearing cortex showing distinct borders of tumor core, infiltrated zone, and outer ridge. g Left: Example images of VGlut1/2 stainings (magenta), NeuN (cyan), Ki67 (green), and GFAP (blue) in peritumoral regions. Right: quantification of the densities of VGlut1/2 positive puncta per 25600 µm 2 in the peritumoral regions. 7-DPI ( N = 2, n = 9), 14-DPI ( N = 2, n = 11), 21-DPI ( N = 2, n = 10); peritumoral regions defined by contralateral (dark blue), outer ridge (grey), infiltrated zone (green), tumor core (light blue). DPI Days post injection. Error bars show mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Statistics: d , g , one-way ANOVA with Sidak’s multiple comparisons test. Source data are provided as a Source Data file.

Article Snippet: AAV viral vectors expressing cre -recombinase under the GFAP promoter (Addgene 105550-AAV5) was used for the induction of tumor growth.

Techniques: Over Expression, Injection, Staining, Expressing, Imaging

Journal: bioRxiv

Article Title: Versatile and efficient non-viral integration of large transgenes in human T cells via CRISPR knock-in and engineered integrases

doi: 10.1101/2025.09.10.675267

Figure Lengend Snippet: ( A ) Schematic showing insertion of an attG landing pad array into the last exon of GAPDH . The HDR template contains an array of attG landing pads for the integrases Pa01, Kp03, Bxb1 and PhiC31. The array is flanked by splice sites to create an artificial intron and is followed by a downstream eGFP reporter. Restoration of a functional GAPDH protein is achieved by in-frame integration of the recoded last exon 9 contained within the HDRT. HA-L/R: Homology-arm left/right; GSG: linker consisting of Gly-Ser-Gly; P2A: self-cleavage protein; pA: poly(A) sequence. ( B ) Integrase-mediated site-specific integration of the circular DNA donor vector into the GAPDH locus on DNA, mRNA and protein level. The promoter-less DNA donor with splice acceptor and P2A following the attV array (corresponding to the attG array on the HDRT) leads to RFP expression through the endogenous GAPDH promoter upon successful integration. Not drawn to scale.

Article Snippet: Selected plasmids created in this study will be made available via Addgene: (1) the plasmids encoding the TRAC -Bxb1.attG or the TRAC -Pa01.attG landing pad HDR-template (Addgene plasmids #247037, #247039), (2) the promoterless integration plasmids carrying the CD19-CAR with a Bxb1 or a Pa01 docking site (Addgene plasmids #247038 and #247040), and (3) the plasmid encoding for the NLS-enhanced Pa01 (ePa01) integrase used for in vitro mRNA transcription (Addgene plasmid #247041).

Techniques: Functional Assay, Sequencing, Plasmid Preparation, Expressing

Journal: Cell

Article Title: Astrocytic insulin signaling couples brain glucose uptake with nutrient availability

doi: 10.1016/j.cell.2016.07.028

Figure Lengend Snippet: (A) Mouse brain overview and high magnification depicting the localization of the area infected by AAV5 hGFAP-Cre (GFP-recombined cells under the control of the promoter of hGFAP) and showing GFAP immunoreactivity (red; Alexa 647) in tdTomato/eGFP mice (see also Fig. S3N and Table 2). (B) IR mRNA expression levels corresponding to the hypothalamic region containing virus-targeted astrocytes from C57BL6J mice (n=6) or IRf/f mice (n=5) crossed with tdTomato/eGFP mice, which were injected with AAV-hGFAP-Cre in the MBH. (C) Number of GFAP-positive cells that (co)localize with pAkt in the hypothalamus of IRf/f mice injected with AAV-hGFAP-GFP (Hyp GFAP-IR WT) or AAV-hGFAP-Cre mice (Hyp-GFAP-IR KO) in the MBH 15 min after vehicle (PBS) or insulin injection peripherally (3 U/kg bw; n= 4 mice/group). (D) Images depicting GFAP-positive cells (red; Alexa 555) and pAkt Ser473 (green; Alexa 488) in the hypothalamus of Hyp GFAP-IR WT mice versus Hyp-GFAP-IR KO mice. (E) Blood glucose in Hyp GFAP-IR WT mice (n=9) or Hyp-GFAP-IR KO mice (n=8) after ip glucose. Food intake over the following 4 h in Hyp GFAP-IR WT (F) or Hyp GFAP-IR KO (G) mice in response to vehicle or ip 2DG (n=9–10/group). (H) 4-h accumulated individual changes in food intake after vehicle or ip 2DG in Hyp GFAP-IR WT mice and Hyp GFAP-IR KO mice (n=9–10/group). (I) Glucose and (J) insulin content in blood, (K) glucose content in the CSF and (L) the ratio of glucose between CSF versus blood 30 min after ip vehicle or glucose in GFAP-IR WT mice versus GFAP-IR KO mice (n=4–8/group). (M) 18FDG accumulation in the brain of GFAP-IR WT mice versus GFAP-IR KO mice assessed by positron emission tomography (PET). (N) 18FDG fold change and (O) relative mRNA expression levels of GLUT-1 observed in the brain of GFAP-IR WT mice or GFAP-IR KO (n=4–8/group). (P) Individual changes of 4-h food intake after fasting and icv injection of vehicle (aCSF) or glucose (1 mg) and (Q) individual changes of 4-h food intake after icv injection of vehicle (aCSF) or 2-DG (1 mg) in mice with IR expression in astrocytes in the MBH (Hyp GFAP-IR WT mice; n=10) or without (Hyp GFAP-IR KO mice; n=10). AAV: adeno-associated virus; aCSF: artificial cerebrospinal fluid; GFAP: glial fibrillary acidic protein; GLUT-1: glucose transporter-1; GTT: glucose tolerance test; hGFAP: human glial fibrillary acidic protein; Hyp: hypothalamic; IR: insulin receptor; MBH: the mediobasal hypothalamus; 2DG: 2-deoxy-D-glucose; 18FDG: [18F] fluorodeoxyglucose. P values= ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05. NS: No significant differences between groups. Data are presented as the mean ± SEM. Scale bars: (A): left panel: 1 mm and right panel: 250 μm; (D): 10 μm.

Article Snippet: We stereotaxically injected AAV-hGFAP-GFP (hypothalamic GFAP-IR WT) or AAV–hGFAP-Cre (hypothalamic GFAP-IR KO) particles bilaterally (2×10 9 viral genome particles per side) into the MBH of IR f/f littermates by using a motorized stereotaxic device (Neurostar, Tubingen, Germany).

Techniques: Infection, Expressing, Injection, Positron Emission Tomography