tre3g egfp  (Addgene inc)

Journal: bioRxiv

Article Title: The PST repeat region of MDC1 is a tunable multivalent chromatin tethering domain

doi: 10.1101/2025.01.10.632395

Figure Lengend Snippet: (A) Quantification of clonogenic survival assays using the PARP inhibitor olaparib of U2OS cells expressing HaloTagged MDC1 or MDC ΔPST from its endogenous locus. (N = 3 biological replicates plated in triplicate, Mean ± S.D., Two-way ANOVA) (B) Images of fixed interphase U2OS cells expressing HaloTagged MDC1 or MDC ΔPST from its endogenous locus untreated or treated with zeocin to induce DNA breaks. Cells were stained with JFX650 HaloTag-ligand and Hoechst, and immuno-labeled with antibodies targeting RAD51 and γH2AX. (C) Images of fixed interphase U2OS cells expressing Halo-MDC1 from its endogenous locus, MDC1 knock-out cells (ΔMDC1), and U2OS ΔMDC1 stably expressing HaloTagged MDC1 variants untreated or treated with zeocin to induce DNA breaks. Cells were stained with JFX650 HaloTag-ligand and Hoechst, and immuno-labeled with an antibody targeting RAD51. (D) Quantification of the number of RAD51 foci of the experiment in (C). An individual replicate is plotted on the left (red line = median, >100 cells per condition) and 3 biological replicates are plotted on the right (Median ± S.D., Two-way ANOVA). (E) Images of fixed interphase U2OS cells expressing Halo-MDC1 from its endogenous locus, MDC1 knock out cells (ΔMDC1), and U2OS ΔMDC1 cells stably expressing HaloTagged MDC1 variants untreated or treated with zeocin to induce DNA breaks. Cells were stained with JFX650 HaloTag-ligand and Hoechst, and immuno-labeled with an antibody targeting 53BP1. (F) Quantification of the number of 53BP1 foci of the experiment in (E). An individual replicate is plotted on the left (red line = median, >100 cells per condition) and 3 biological replicates are plotted on the right (Median ± S.D., Two-way ANOVA). (G-H) Quantification of the accumulation of (G) Halo-MDC1 and Halo-MDC1 ΔPST and (H) ubiquitin Reader1.0 at laser micro-irradiation induced DNA lesions in U2OS ΔMDC1 cells stably expressing indicated HaloTagged MDC1 variants (N = 10-19 cells, Mean ± 95% CI). (H) Quantification of the maximal accumulation of ubiquitin Reader1.0 at laser micro-irradiation induced DNA lesions in U2OS ΔMDC1 cells stably expressing indicated HaloTagged MDC1 variants (N = 10-19 cells, Mean ± 95% CI). (G) Quantification of HR efficiency using the DR-GFP reporter stably integrated into the AAVS1 locus of U2OS ΔMDC1 cells. I-Sce and MDC1 variants were transiently transfected (N = 3-4 biological replicates, Mean ± S.D., Two-way ANOVA).

Article Snippet: To knock-in inducible WT and T>A GFP-PST-LacI fusion proteins into the AAVS1 locus in U2OS 2-6-3 cells, we PCR amplified GFP and LacI from a GFP-LacI expression plasmid (a kind gift from Iain Cheeseman) 68 and codon-optimized WT and T>A PST repeat sequences and cloned them by Gibson assembly into MluI and SalI-linearized AAVS1-TRE3G-EGFP (a gift from Su-Chun Zhang (Addgene plasmid # 52343)) .

Techniques: Expressing, Staining, Labeling, Knock-Out, Stable Transfection, Irradiation, Transfection

Journal: Neuron

Article Title: SYNGAP1 deficiency disrupts synaptic neoteny in xenotransplanted human cortical neurons in vivo

doi: 10.1016/j.neuron.2024.07.007

Figure Lengend Snippet:

Article Snippet: pLenti-TRE3G-GCaMP7b-WPRE: The TRE3G fragment (pLenti CMVTRE3G eGFP Puro (w819-1) was a gift from Eric Campeau (Addgene plasmid # 27570; http://n2t.net/addgene:27570 ; RRID:Addgene_27570)) and the GCaMP7b fragment (pGP-AAV- syn -jGCaMP7b-WPRE was a gift from Douglas Kim & GENIE Project (Addgene plasmid # 104489; http://n2t.net/addgene:104489 ; RRID:Addgene_104489)) was transferred to lentiviral backbone by restriction enzyme digestion and ligation. pLenti-hSynI-TetON3G-WPRE: The DNA fragment of TetON3G was amplified from AAVS1-TRE3G-EGFP was a gift from Su-Chun Zhang (Addgene plasmid # 52343; http://n2t.net/addgene:52343 ; RRID:Addgene_52343) using following primers pair: 5′-GTCGACatgtctagactggacaagag-3’/5′-ACGCGTttacccggggagcatgtcaa-3’.

Techniques: Virus, Recombinant, Knock-Out, Transfection, Software