Journal:

Article Title: Preferential accessibility to specific genomic loci for the repair of double-strand breaks in human cells

doi: 10.1093/nar/gkh952

Figure Lengend Snippet: Possible repair products resulting from DSB induction at the I-SceI site of the integrated pL1HsSceI vector. (A) Structure of the integrated pL1HsSceI vector containing origin of replication (ori), ampicillin gene resistance (amp) and neomycin gene selection (neo). The 2.5 kb L1 portion corresponds to the 3′ segment (3390–5976 bp) of cD11 (cDNA of an active L1 element), inside which the 23 bp NheI–BamHI segment was replaced by an 18 bp I-SceI restriction site (black box). (B) Structure of a typical NHEJ repair product, characterized by the retention of the Sp (SpeI) and S (SacII) plasmidic sites, the loss of the I-SceI site and the absence of the N B (NheI–BamHI) segment. NHEJ junctions frequently showed deletion and/or insertion sequences at the break site. (C) Structure of a typical NHCR repair product, characterized by the loss of the SpeI and SacII plasmidic sites and the absence of the NheI–BamHI segment and the acquisition of a novel chromosomal junction (dotted line). (D) Structure of a typical gene conversion product, characterized by the retention of the SpeI and SacII plasmidic sites and the presence of the NheI–BamHI segment acquired from an endogenous L1 partner by HR. (E) Structure of a typical crossing-over/one sided invasion product, characterized by the presence of the NheI–BamHI segment acquired from an endogenous L1 partner by HR but extending beyond the homologous L1 sequences into adjacent chromosomal sequences (dotted line).

Article Snippet: PCBASceTcr expression vector was created by cloning the PCR-amplified pBR322 tetracycline-resistance gene (New England Biolabs) in the ScaI site of pCBASce ( 17 ), in order to inactivate the ampicillin-resistance gene. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 Possible repair products resulting from DSB induction at the I-SceI site of the integrated pL1HsSceI vector. ( A ) Structure of the integrated pL1HsSceI vector containing origin of replication (ori), ampicillin gene resistance (amp) and neomycin gene selection (neo).

Techniques: Plasmid Preparation, Selection

Journal: Frontiers in Oncology

Article Title: PALB2 deficiency may sensitize H3K27M-mutant pediatric HGG cells to BMN673/talazoparib

doi: 10.3389/fonc.2025.1589396

Figure Lengend Snippet: Analysis of NHEJ activity via the EJ5 reporter assay in SF188 and Res259 cells stably expressing the H3 WT or H3K27M mutation. (A) Schematic representation of the EJ5 reporter for NHEJ repair. EJ5-GFP is shown along with two classes of NHEJ repair products that can restore a GFP expression cassette. In the starting construct, the GFP gene is inactive. GFP expression is only activated when I-Sce1-induced DSBs are successfully repaired via NHEJ. (B) H3K27M-mutant SF188 cells and WT SF188 cells containing the reporter cassette were transfected with the I-SceI and DsRed plasmids. Left panel: Immunofluorescence images of GFP-positive cells and DsRed-positive cells (scale bars, 100 µm). Right panel: NHEJ activity in H3K27M-mutant SF188 cells and WT SF188 cells. (C) H3K27M-mutant Res259 cells and WT Res259 cells containing reporter cassettes were transfected with I-SceI and DsRed plasmids. Left panel: Immunofluorescence images of GFP-positive cells and DsRed-positive cells (scale bars, 100 µm). Right panel: NHEJ activity in H3K27M-mutant Res259 cells and WT Res259 cells. NHEJ activity was determined by normalizing the percentage of eGFP-positive cells to the percentage of DsRed-positive cells. The data are shown as the mean ± SD from three independent experiments. Student’s t test was used to calculate p values. ** p < 0.01 versus the corresponding control.

Article Snippet: The indicated EJ5 reporter cell lines were transfected with two plasmids, namely, the I-SceI expression vector pCBASce (Addgene #26477) and the DsRed expression vector (Addgene #54493), 48 h later, the percentage of GFP-positive cells among DsRed-positive cells was evaluated by imaging with a Carl Zeiss LSM 710 confocal fluorescence microscope and quantified via FACS on a BD AccuriTM C6 flow cytometer (BD Biosciences, USA) using BD AccuriTM software.

Techniques: Activity Assay, Reporter Assay, Stable Transfection, Expressing, Mutagenesis, Construct, Transfection, Immunofluorescence, Control