Journal: International journal of molecular sciences

Article Title: A Dual-Function "TRE-Lox" System for Genetic Deletion or Reversible, Titratable, and Near-Complete Downregulation of Cathepsin D.

doi: 10.3390/ijms24076745

Figure Lengend Snippet: Figure 1. Overview of the design and dual-functionality of the TRE-Lox system. (a) Overall structure of the 5′ end of the murine cathepsin D (CatD) gene (CTSD) and its promoter region (PCTSD, dark gray), indicating the relative position of the two gRNAs (black arrows) used for CRISPR/Cas9- assisted homologous recombination. Note the placement of the TATA box (TATA) very close to the main transcription start site (TSS) (right-angle arrow), the presence of the initiation codon (ATG, dashed white line) within Exon 1 (Ex 1, light gray), and the presence of a splice donor (SD) and splice acceptor (SA) flanking Intron 1 (black line). (b) Structure of the TRE-Lox knock-in (KI) insert, illustrating the relative positions of the two tet-operons (tetO2, green) and one LoxP site (LoxP, light blue) within the 5′ untranslated region (5′UTR) and, within Intron 1, a tetracycline response element (TRE) comprised of seven tetO repeats (tetO7) and the second LoxP site. The relative placement of the puromycin resistance cassette (Puror, purple) flanked by two FRT sites (FRT, dark blue), which is excisable by Flp recombinase, is depicted using a curly bracket. (c) Downregulation of CTSD via the action of rtTRKRAB acting on the TRE-Lox insert. In the presence of Dox (red triangles), rtTRKRAB binds to the tetO repeats within both the 5′UTR and Intron 1, triggering methylation of histones in a radius of 2–3 kb, thereby remodeling the chromatin and silencing the CTSD gene. (d) Genetic deletion of CTSD via the action of Cre recombinase on the TRE-Lox insert. The figure depicts the end result of Cre-mediated recombination of the TRE-Lox KI insert, which causes removal of the initiation codon, the first portion of the coding region of Exon 1 encoding the signal peptide of CatD, and the 5′ end of Intron 1.

Article Snippet: Relevant regions (and their sources) were as follows: the 3′ end of Exon 1 and the 5′ portion of Intron 1 of murine CTSD (from C57Bl6/J mouse tail DNA); tetO2 (from Addgene plasmid #113892 [41]); TRE 3G and, separately, an FRT-flanked puromycin resistance cassette (both from Addgene plasmid #156430 [42]).

Techniques: CRISPR, Homologous Recombination, Knock-In, Methylation

Journal: iScience

Article Title: TAOK2 controls synaptic plasticity and anxiety via ERK and calcium signaling

doi: 10.1016/j.isci.2025.113712

Figure Lengend Snippet: MAPK and calcium signaling is reduced in Taok2 cKO neurons (A–D) Luciferase assay using EGR1p (A), SARE- (B), FOSBp- (C), and CRE- (D) sensor-detected MAPK (A–C) and calcium (B, D)-dependent signaling activity in primary cortical neurons. Neurons were treated with AMPA (1 μM), BDNF (10 ng/mL), or bicuculline (BIC, 1 μM) on day in vitro 12 (DIV12) for 4 h. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; n.s., not significant; Student’s t test (two-sided). (E) Imaging of control and Taok2 cKO primary cortical neurons infected with AVVs expressing the synapsin1-driven GCaMP6f calcium sensor. Images were taken before and after stimulating cells with 25 mM KCl. (F) Quantification of cellular GCaMP6f responses to 25 mM KCl. Intensities from responding cells were normalized to the background and pooled for analysis. Gray arrowheads on the x axis represent the time points at which images were taken before and after stimulation as shown in (E). The line represents the mean, and the shaded area represents the SEM; n = 30 responding cells from two independent cultures. ∗∗∗ p < 0.001 and mixed ANOVA. (G) Western blots indicate reduced phosphorylated Mek1/2 (p-Mek1/2) and Erk1/2 (p-Erk1/2) in primary mouse cortical neurons. Neurons were treated with AMPA (1 μM) on DIV12 for 4 h. (H and I) Quantification of western blot data shown in (G). Data of p-Erk1/2 are relative to total Erk1/2 (H), and data of p-Mek1/2 are relative to total Mek1/2 (I). Values are presented as mean ± SD. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; Wilcoxon rank-sum test (two-sided).

Article Snippet: On DIV6, half the medium was changed and the neurons were infected with AAVs for expression of mRuby2-P2A-GCaMP6f under the control of the human synapsin1 promoter (hSyn1p) (Addgene #50943) at an MOI of 1000.

Techniques: Luciferase, Activity Assay, In Vitro, Imaging, Control, Infection, Expressing, Western Blot