Journal: American Journal of Human Genetics

Article Title: Therapeutic validation of MMR-associated genetic modifiers in a human ex vivo model of Huntington disease

doi: 10.1016/j.ajhg.2024.04.015

Figure Lengend Snippet: Construction and validation of the 125Q HD CRISPRi iPSC lines (A) Schematic showing TALEN-mediated knockin of a dCas9-tagBFP ZIM3 KRAB construct into the CLYBL safe harbor. (B) Single-cell clones were isolated and screened for TagBFP expression and pluripotency-associated marker expression. Scale bars, 50 μm. (C) Accurate knockin was verified by PCR across homology arm junctions (5′ and 3′ junctions) and insert site spanning (5′ to 3′ spanning) PCR alongside an internal dCas9 PCR. (D) Zygosity and off-target integration was assessed by copy-number qPCR. (E) Modal CAG length was determined by fragment analysis. (F) Expression of pluripotency-associated genes was further assessed by qPCR for OCT3/4 and NANOG relative to the parental iPSC line. Clone 1B12 ( ∗ ) used for further work. Mean values ± SEM in triplicate.

Article Snippet: The KOX1 KRAB domain of the dCas9-KRAB fusion in the CLYBL-targeting vector pC13 dCas9-TagBFP-KRAB (addgene #127968 ) was swapped for ZIM3 KRAB domain from pLX303-ZIM3-KRAB-dCas9 (addgene #331490 ).

Techniques: Biomarker Discovery, Knock-In, Construct, Clone Assay, Isolation, Expressing, Marker

Journal: American Journal of Human Genetics

Article Title: Therapeutic validation of MMR-associated genetic modifiers in a human ex vivo model of Huntington disease

doi: 10.1016/j.ajhg.2024.04.015

Figure Lengend Snippet: Homogeneous lowering of MMR factor expression in CRISPRi pools leads to characteristic MMR deficiencies (A) Immunostaining for indicated target proteins in red channel shows knockdown is consistent cell-to-cell. Representative images for a non-targeting control guide and a targeting guide. EGFP from the guide vectors and TagBFP from the dCas9-KRAB knock-in. Scale bars, 50 μm. (B and C) Quantification of fluorescence intensity for each target normalized to NTC1 levels for MutS and LIG1 (B) and MutL (C). Line indicate median values, 1–2,000 cells per plot from four random positions in three independent experiments. (D and E) iPSC colony formation after treatment with the indicated concentration of methylnitronitrosoguanidine (MNNG) for CRISPRi lowered MutS and LIG1 (D) and MutL (E). Mean colony frequency ± SEM in triplicate as a proportion of untreated cells of the same genotype, normalized to non-targeting control iPSCs (NTC) and shown alongside MSH6 knockout (MSH6 KO) iPSC survival. Dotted lines 95% CIs, ∗ p < 0.05.

Article Snippet: The KOX1 KRAB domain of the dCas9-KRAB fusion in the CLYBL-targeting vector pC13 dCas9-TagBFP-KRAB (addgene #127968 ) was swapped for ZIM3 KRAB domain from pLX303-ZIM3-KRAB-dCas9 (addgene #331490 ).

Techniques: Expressing, Immunostaining, Knockdown, Control, Knock-In, Fluorescence, Concentration Assay, Knock-Out

Journal: BMC Microbiology

Article Title: Identification and whole-genome sequencing analysis of Vibrio vulnificus strains causing pearl gentian grouper disease in China

doi: 10.1186/s12866-022-02610-1

Figure Lengend Snippet: Physiological and biochemical characteristics of pathogenic strain EPL 0201 and other V. vulnificus strains

Article Snippet: The code of the EPL 0201 strain, according to the API 20E coding manual is 500600557, which is consistent with the physiological and biochemical characteristics of V. vulnificus strain ATCC 33,149.

Techniques:

Journal: BMC Microbiology

Article Title: Identification and whole-genome sequencing analysis of Vibrio vulnificus strains causing pearl gentian grouper disease in China

doi: 10.1186/s12866-022-02610-1

Figure Lengend Snippet: Symptoms of fish tested for recursive infection. A Body chart of experimental fish in control group. B Abdominal cavity of fish in control group (white arrow). C Phosphorus loss in a large area of the skin (white arrow). D Hemorrhagic necrosis of the liver (aa white arrow); a large amount of fluid in the abdominal cavity (ab white arrow); and intestinal inflammation (ac white arrow). E Agarose gel electrophoresis of PCR products of vvhA and rtxA from the suspected V. vulnificus isolates. M, Marker (unit, bp); 1–4, PCR products of vvhA from V. vulnificus in samples from four diseased fishes in the 1 × 10 7 –1 × 10 4 CFU mL −1 groups; 5–8, PCR products of rtxA from V. vulnificus in samples from 4 diseased fishes in the 1 × 10 7 –1 × 10 4 CFU mL. −1 groups; 9, negative control; 10–11, positive control (PCR products of vvhA and rtxA from EPL 0201 isolate). Original agarose gel electropherogram (Fig. S )

Article Snippet: The code of the EPL 0201 strain, according to the API 20E coding manual is 500600557, which is consistent with the physiological and biochemical characteristics of V. vulnificus strain ATCC 33,149.

Techniques: Infection, Control, Agarose Gel Electrophoresis, Marker, Negative Control, Positive Control

Journal: BMC Microbiology

Article Title: Identification and whole-genome sequencing analysis of Vibrio vulnificus strains causing pearl gentian grouper disease in China

doi: 10.1186/s12866-022-02610-1

Figure Lengend Snippet: Results of recursive infection test following intraperitoneal injection

Article Snippet: The code of the EPL 0201 strain, according to the API 20E coding manual is 500600557, which is consistent with the physiological and biochemical characteristics of V. vulnificus strain ATCC 33,149.

Techniques: Infection, Bacteria, Concentration Assay, Control