lentiviral vector encoding s pyogenes cas9  (Addgene inc)

Journal: Hepatology Communications

Article Title: Genome-wide CRISPR screen identifies ACSL3 as a regulator of lipotoxicity and progression of MASLD

doi: 10.1097/HC9.0000000000000884

Figure Lengend Snippet: Genome-wide CRISPR-Cas9 screen identifies mediators of lipotoxic stress. (A) Schematic of the screening workflow in HLF cells stably expressing Cas9 and transduced with a genome-wide lentiviral sgRNA library; cultures were exposed for 3 weeks to 200 μM palmitic acid (PA) or control medium. (B) Bar plot of log2-normalized sgRNA counts for each group showing comparable library representation. (C) Distribution of sgRNA enrichment in PA versus control. (D) Gene-level scatter plot of −log10 p values; x -axis, PA versus vehicle; y -axis, day 14 versus day 0. (E) Log2 fold changes of individual sgRNAs in PA-treated versus control cells. Abbreviations: ACSL3, acyl-CoA synthetase long-chain family member 3; HLF, human liver cancer cell line; sgRNA, single-guide RNA.

Article Snippet: HLF cells were transduced with a lentiviral vector encoding S. pyogenes Cas9 (lentiCas9-Blast, Addgene #52962) and selected with blasticidin for 14 days.

Techniques: Genome Wide, CRISPR, Stable Transfection, Expressing, Transduction, Control

Journal: Nature Communications

Article Title: Murine hematopoietic progenitor cell lines with erythroid and megakaryocyte potential

doi: 10.1038/s41467-025-62668-z

Figure Lengend Snippet: a RBC (left) and platelet (right) numbers in the PB of mice that were adoptively transferred with GFP + Hoxa7-TPO cells 7 days after transfer. Hoxa7-TPO cells were transduced with expression vectors for Cas9 and sgRNAs against Nfe2 , Gp1ba or Klf1 as indicated. Cell numbers were determined by flow cytometry and CBC analysis. Data represent mean ± SD for n mice: Ctrl n = 3, Nfe2-sg1 n = 3, Nfe2-sg2 n = 3, Gp1ba-sg1 n = 4, Gp1ba-sg2 n = 3, Klf1-sg1 n = 4, Klf1-sg2 n = 4. * P < 0.05, ** P < 0.01, *** P < 0.001; Two-tailed unpaired t test. Source data are provided as a Source Data file. b Immuno-fluorescence analysis of washed platelets derived from PF4-Cre-iDTR mice that were transferred with Hoxa7-TPO cells transduced with expression vectors for Cas9 and control sgRNA (sgRNA-ctrl) or sgRNA targeting Gp1ba (Gp1ba-sg2). Platelets were seeded on chamber slides coated with poly-L-lysine and stained with antibodies against β-tubulin, VWF and GFP, followed by confocal microscopy. Arrows indicate GFP + , Hoxa7-TPO-derived platelets, arrow heads indicate residual endogenous (GFP - ) platelets. Scale bars = 10 μm. c The cell size of platelets derived from the PB of mice that were adoptively transferred with Hoxa7-TPO cells transduced with Gp1ba-sgRNA (and control-sgRNA) was determined by flow cytometry. Data represent mean ± SD for n mice: Ctrl n = 3, Gp1ba-sg1 n = 4, Gp1ba-sg2 n = 3. * P = 0.0136, **** P < 0.0001. Two-tailed unpaired t test. Source data are provided as a file.

Article Snippet: Cas9-mediated deletion of genes in Hoxa7-TPO cells was done on the basis of cells transduced with the lentiviral CAS9 expression vector lentiCas9-Blast (Addgene, no. 52962) .

Techniques: Transduction, Expressing, Flow Cytometry, Two Tailed Test, Fluorescence, Derivative Assay, Control, Staining, Confocal Microscopy