crispr cas9 knockout target genes  (Addgene inc)

Journal: bioRxiv

Article Title: Stable lentiviral-mediated expression of Cytochrome P450 2D6 in HepaRG cells: New means for in vitro assessment of xenobiotic biotransformation and cytotoxicity

doi: 10.1101/2025.01.25.634872

Figure Lengend Snippet: A) Parental (WT) and CYP2D6 + progenitor cells were thawed and plated to monitor the cell morphology in phase contrast microscopy at days 3, 7 14 and 30 days after plating. B) Immunoblotting of CYP2D6 and CYP3A4 in parental (WT) and CYP2D6 + cells at different times after plating until days 30 in absence (-) or presence (+) of DMSO. HSC70 protein was used as loading control. C) Dextrometorphan O -demethylase activity (Cl int values expressed in μL.min -1 .mg -1 ) measured in human hepatocytes (PHH), HepaSH cells, parental (WT) and CYP2D6 + HepaRG cells. Statistics: *p < 0.05 and **p< 0.01 between Parental and CYP2D6 + HepaRG cells. D) Inhibition of dextrometorphan O -demethylase activity in CYP2D6 + HepaRG cells by quinidine at 1 and 10 μM. Statistics: *p < 0.05 between parental and CYP2D6 + HepaRG cells (a), and quinidine-treated cells versus untreated CYP2D6 + HepaRG cells (b).

Article Snippet: The Gene Knockout kit (CRISPR-Cas9 kit) targeting human CYP2D6 purchased form Synthego (Redwood, CA, USA) contained three different sgRNA ( Supporting Information-Table 1 ) and a Cas9 protein batch. sgRNA were resuspended at 30μM in buffer R while Cas9 was ready to use at 20μM.

Techniques: Microscopy, Western Blot, Control, Activity Assay, Inhibition

Journal: bioRxiv

Article Title: Stable lentiviral-mediated expression of Cytochrome P450 2D6 in HepaRG cells: New means for in vitro assessment of xenobiotic biotransformation and cytotoxicity

doi: 10.1101/2025.01.25.634872

Figure Lengend Snippet: In situ detection of GFP by fluorescence microscopy (A) and by flow cytometry (B) in hepatocyte- and cholangiocyte-enriched cell populations. For flow cytometry data (B), results are expressed in percentages of GFP positive (Pos) cells and means of fluorescence (FITC-A mean) in both hepatocyte- and cholangiocyte-enriched cell populations. C) Dextrometorphan O -demethylase, phenacetin O-deethylase, bupropion hydroxylase and midazolam 1’-hydroxylase activities probing CYP2D6, CYP1A2, CYP2B66 and CYP3A4/5, respectively, in both hepatocyte- and cholangiocyte-enriched cell populations. All data were significantly different between the two cell populations: ***p < 0.001, ****p < 0.0001. D) In situ detection of CYP2D6 (red staining) and ERAP2 (green staining) by immunofluorescence and mitochondria staining using mitotracker.

Article Snippet: The Gene Knockout kit (CRISPR-Cas9 kit) targeting human CYP2D6 purchased form Synthego (Redwood, CA, USA) contained three different sgRNA ( Supporting Information-Table 1 ) and a Cas9 protein batch. sgRNA were resuspended at 30μM in buffer R while Cas9 was ready to use at 20μM.

Techniques: In Situ, Fluorescence, Microscopy, Flow Cytometry, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: Stable lentiviral-mediated expression of Cytochrome P450 2D6 in HepaRG cells: New means for in vitro assessment of xenobiotic biotransformation and cytotoxicity

doi: 10.1101/2025.01.25.634872

Figure Lengend Snippet: A) Schematic representation of the biotransformation of tramadol via CYP2D6, CYP2B6 and CYP3A4 enzymatic activities and the main metabolites of tramadol: M1: O -desmethyltramadol, M2: N -desmethyltramadol, M3: N , N -bidesmethyltramadol, M4: O -desmethyl- N , N -bisdesmethyltramadol, M5: N , O -didesmethyltramadol (M1, M2, M3, M4 and M5). B) Chromatograms of M1-M2 metabolites detected in parental (HepaRG WT) and CYP2D6 + transgenic HepaRG cells. C) Table presenting the quantification LC–MS/MS analysis of metabolites M1 to M5 in progenitor and differentiated parental (WT) and CYP2D6 + HepaRG cells. Results were expressed in µg/L.

Article Snippet: The Gene Knockout kit (CRISPR-Cas9 kit) targeting human CYP2D6 purchased form Synthego (Redwood, CA, USA) contained three different sgRNA ( Supporting Information-Table 1 ) and a Cas9 protein batch. sgRNA were resuspended at 30μM in buffer R while Cas9 was ready to use at 20μM.

Techniques: Transgenic Assay, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Stable lentiviral-mediated expression of Cytochrome P450 2D6 in HepaRG cells: New means for in vitro assessment of xenobiotic biotransformation and cytotoxicity

doi: 10.1101/2025.01.25.634872

Figure Lengend Snippet: A) Relative ATP contents (left panel) and LDH release (right panel) and B) IC 50 in parental (WT) and CYP2D6 + differentiated HepaRG cells exposed to various concentration of perhexiline. C) Oxygen Consumption Rate (OCR) in parental (WT) and CYP2D6 + differentiated HepaRG cells exposed to perhexiline at 10μM and quantifications of proton leak, ATP production and coupling efficiency (left panel) and D) levels of mitochondrial β-oxidation of linoleic acid. All data were significantly different between the two cell lines: *p < 0.05 and **p< 0.01

Article Snippet: The Gene Knockout kit (CRISPR-Cas9 kit) targeting human CYP2D6 purchased form Synthego (Redwood, CA, USA) contained three different sgRNA ( Supporting Information-Table 1 ) and a Cas9 protein batch. sgRNA were resuspended at 30μM in buffer R while Cas9 was ready to use at 20μM.

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Stable lentiviral-mediated expression of Cytochrome P450 2D6 in HepaRG cells: New means for in vitro assessment of xenobiotic biotransformation and cytotoxicity

doi: 10.1101/2025.01.25.634872

Figure Lengend Snippet: A) UMAP representation of parental (WT) and CYP2D6 + HepaRG cells at different time points after plating (2, 4, 7, 14, 32, 37 and 44 days). The first dimension is mainly associated to the time of culture. B) Heatmap of the top 100 most differentially expressed genes during differentiation, with consistent expression patterns observed between parental (WT and CYP2D6 + HepaRG cells. C) Volcano plot of genes differentially expressed at D37 highlighting significant downregulation of various CYP450 genes in transgenic cells compared to parental cells. D) Radar plot showing enrichment of gene ontology (GO) terms related to xenobiotic metabolism at D37 and D44. The size of the points is the ratio r/R and the scale corresponds to the log10 of the adjusted p-value, where r = number of genes in the list associated with the term of interest and R = total number of genes associated with one term of interest.

Article Snippet: The Gene Knockout kit (CRISPR-Cas9 kit) targeting human CYP2D6 purchased form Synthego (Redwood, CA, USA) contained three different sgRNA ( Supporting Information-Table 1 ) and a Cas9 protein batch. sgRNA were resuspended at 30μM in buffer R while Cas9 was ready to use at 20μM.

Techniques: Expressing, Transgenic Assay

Journal: bioRxiv

Article Title: Stable lentiviral-mediated expression of Cytochrome P450 2D6 in HepaRG cells: New means for in vitro assessment of xenobiotic biotransformation and cytotoxicity

doi: 10.1101/2025.01.25.634872

Figure Lengend Snippet: A) Detection of GFP by fluorescence microscopy and by flow cytometry in CYP2D6 + and KO CYP2D6/GFP HepaRG cells. Results are expressed in percentages of GFP positive (Pos) or negative (Neg) cells and means of fluorescence (FITC-A mean). B) RT-qPCR evaluating the relative mRNA expression of CYP2D6, 3A4, 2C8, 2C9, 2A7, AhR, FXR, PXR, HNF4α and SHP in parental (HRP WT), CYP2D6 + and KO CYP2D6/GFP (HRP KO) HepaRG cells. Statistics: *p < 0.05 between parental (HRP WT = a), CYP2D6 + (b) and KO CYP2D6/GFP (HRP KO = c) HepaRG cells.

Article Snippet: The Gene Knockout kit (CRISPR-Cas9 kit) targeting human CYP2D6 purchased form Synthego (Redwood, CA, USA) contained three different sgRNA ( Supporting Information-Table 1 ) and a Cas9 protein batch. sgRNA were resuspended at 30μM in buffer R while Cas9 was ready to use at 20μM.

Techniques: Fluorescence, Microscopy, Flow Cytometry, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Stable lentiviral-mediated expression of Cytochrome P450 2D6 in HepaRG cells: New means for in vitro assessment of xenobiotic biotransformation and cytotoxicity

doi: 10.1101/2025.01.25.634872

Figure Lengend Snippet: A) Schematic representation of the lentiviral derived mRNA transcribed from the transgene inserted into the genome of HepaRG cells and CRISPR/Cas9 mediated alteration of the lentiviral transgene in KO CYP2D6/GFP (HRP KO) HepaRG cells. B) RT-qPCR evaluating the relative mRNA expression of the CYP2D6-GFP-WPRE lentiviral derived transcript and NXF3 and TRIM63 in CYP2D6 + and KO CYP2D6/GFP HepaRG cells. Statistics: *p < 0.05 between parental (HRP WT = a), CYP2D6 + (b) and KO CYP2D6/GFP (HRP KO = c) HepaRG cells.

Article Snippet: The Gene Knockout kit (CRISPR-Cas9 kit) targeting human CYP2D6 purchased form Synthego (Redwood, CA, USA) contained three different sgRNA ( Supporting Information-Table 1 ) and a Cas9 protein batch. sgRNA were resuspended at 30μM in buffer R while Cas9 was ready to use at 20μM.

Techniques: Derivative Assay, CRISPR, Quantitative RT-PCR, Expressing

Journal: Microbiology Spectrum

Article Title: S. aureus virulence factors decrease epithelial barrier function and increase susceptibility to viral infection

doi: 10.1128/spectrum.01684-23

Figure Lengend Snippet: Establishment of CD40 KO in N/TERT-2G keratinocytes. ( A and B ) Undifferentiated (Undiff) and differentiated keratinocytes at day 1 and 2 post-differentiation ( D1, D2 ) were stained for CD40 expression and analyzed by flow cytometry. The fourth exon of CD40 was targeted through triple gRNA-mediated CRISPR/Cas9. The clonal selection was utilized to isolate individual clones. ( C ) Undifferentiated CD40 KO clones (1–4) and the WT clone were stained for CD40 expression and analyzed by flow cytometry. ( D ). KO was confirmed by gel electrophoresis for CD40 deletions and by sequencing. Statistics were calculated using the nonparametric unpaired ANOVA with Dunn’s multiple comparison test. Error bars denote mean ± standard deviation. * P < 0.05

Article Snippet: A CRISPR/Cas9 knockout kit targeting CD40 (Gene Knockout Kit v2) was obtained through Synthego.

Techniques: Staining, Expressing, Flow Cytometry, CRISPR, Selection, Clone Assay, Nucleic Acid Electrophoresis, Sequencing, Comparison, Standard Deviation

Journal: Microbiology Spectrum

Article Title: S. aureus virulence factors decrease epithelial barrier function and increase susceptibility to viral infection

doi: 10.1128/spectrum.01684-23

Figure Lengend Snippet: CD40 KO decreases proinflammatory responses to S. aureus enterotoxins/SE l toxins. The WT clone and CD40 KO clone 1 were treated with purified S. aureus toxins (0.25 µg/mL) for 6 h while undifferentiated. Proinflammatory cytokine mRNA expression was determined by qPCR and displayed as “fold change over media” for both WT clone and CD40 KO clone. Statistical analysis was performed using the mixed-effects ANOVA model with Dunnett’s multiple comparisons test comparing the delta cq values of each toxin treatment to media within each clone population ( A-C ). “Fold reduction in expression compared to WT” was calculated by the difference in fold change over media for CD40 KO clone 1 compared to fold change over media for the WT clone. Statistical analysis was performed using the ratio paired t -test on the delta cq values comparing WT and CD40 KO ( D-F ). Error bars denote mean ± standard error the mean. n = 5 experiments * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: A CRISPR/Cas9 knockout kit targeting CD40 (Gene Knockout Kit v2) was obtained through Synthego.

Techniques: Purification, Expressing

Journal: Microbiology Spectrum

Article Title: S. aureus virulence factors decrease epithelial barrier function and increase susceptibility to viral infection

doi: 10.1128/spectrum.01684-23

Figure Lengend Snippet: CD40 KO decreases basal tight junction barrier function but does not prevent the barrier disruption caused by SE l Q. The WT clone and CD40 KO clone 1 were treated with high Ca 2+ media [1.8 mM] to initiate differentiation and treated with media alone ( A ) or media with 0.25 µg/mL of SE l Q ( B ). TEER was measured for 6 d following differentiation, and media with treatment groups was resupplied every 2 d. Data are shown as adjusted TEER values (ohms × cm 2 ) ( A ) or percent TEER of media (matched clone) ( B ). Ratio paired t -test, n = 3 ( A ). Paired t -test, n = 3 ( B ). Error bars denote mean ± standard error the mean. * P < 0.05, ** P < 0.01

Article Snippet: A CRISPR/Cas9 knockout kit targeting CD40 (Gene Knockout Kit v2) was obtained through Synthego.

Techniques: Disruption