lenti guide mcherry plasmid  (Addgene inc)

Journal: Advanced Science

Article Title: CRISPR‐MI and scRNA‐Seq Reveal TREM2's Function in Monocyte Infiltration and Macrophage Apoptosis During Abdominal Aortic Aneurysm Development

doi: 10.1002/advs.202412227

Figure Lengend Snippet: Development of in vivo genome‐wide CRISPR Cas9 screen for monocyte infiltration (CRISPR‐MI). A) Flowchart of the adoptive transfer of BMDM labeled with VivoTrack680 fluorescent dye. B,C) The aortae from recipient mice in (A) were isolated and underwent in vivo imaging (n = 4). Scale bar = 1 cm. D) Flowchart of the adoptive transfer of BMDM from mTmG mice. E,F) The aortae from recipient mice in (D) were digested and analyzed by flow cytometry (n = 4). G, BMDMs were infected with lentivirus encoding EGFP (lenti‐EGFP, 50 MOI) 48 h after isolation with different adjuncts. 22 h after infection, cells were imaged by fluorescent microscopy. Scale bar = 100 µm. H) BMDMs in (G) were analyzed by flow cytometry to quantify EGFP+ cells (n = 3). I) BMDMs, were infected with lenti‐EGFP at different time points (0, 24, 48, 72 h) after isolation with or without F108. EGFP + cells were quantified by flow cytometry (n = 3). J) Rosa26‐Cas9 Tg mice were used as the BMDM donor, and the LentiGuide Cherry lentivirus vector was used to carry sgRNA. K) BMDMs from Rosa26‐Cas9 Tg mice were infected with lentivirus carrying different sgRNAs (50 MOI) 48 h after isolation. 72 h after infection, the genome editing efficiency was determined by CRISPR‐Seq. Data are presented as mean ± SEM. Unpaired t‐test was used for C, F. * , p < 0.05. CD, chow diet; WD, Western diet; AngII, angiotensin II; Tg, transgenic.

Article Snippet: We used the genome‐wide sgRNA library (Mouse Cherry Brie Pooled Library, from Addgene Pooled Library #170511) [ ] to perform the genome‐wide CRISPR knockout screen.

Techniques: In Vivo, Genome Wide, CRISPR, Adoptive Transfer Assay, Labeling, Isolation, In Vivo Imaging, Flow Cytometry, Infection, Microscopy, Plasmid Preparation, Western Blot, Transgenic Assay

Journal: Advanced Science

Article Title: CRISPR‐MI and scRNA‐Seq Reveal TREM2's Function in Monocyte Infiltration and Macrophage Apoptosis During Abdominal Aortic Aneurysm Development

doi: 10.1002/advs.202412227

Figure Lengend Snippet: CRISPR‐MI identified genes regulating monocyte infiltration in the murine AAA model. A) Flowchart of CRISPR‐MI. B) Number of sgRNA and corresponding genes identified by next‐generation sequencing (NGS) in BMDM donor and aorta, heart, and liver from recipient mice. C) Heat plot showing the Pearson correlation coefficient of gRNA counts from different organs and biological replicates. D) Principal component analysis gRNA counts from different organs and biological replicates. E) Scatter plots comparing gRNA targeting genes in aorta versus gDNA in donor BMDMs. Red dots indicated significantly enriched genes in the aorta. F) Scatter plot of overrepresented pathways of genes enriched in the aorta by GSEA.

Article Snippet: We used the genome‐wide sgRNA library (Mouse Cherry Brie Pooled Library, from Addgene Pooled Library #170511) [ ] to perform the genome‐wide CRISPR knockout screen.

Techniques: CRISPR, Next-Generation Sequencing