doxycycline inducible lentiviral expression vector pcw cas9 puro  (Addgene inc)

Journal: Cancer Discovery

Article Title: The FBXO45–GEF-H1 Axis Controls Germinal Center Formation and B-cell Lymphomagenesis

doi: 10.1158/2159-8290.CD-24-0442

Figure Lengend Snippet: FBXO45 deficiency sensitizes BCL to MAPK pathway inhibition in vitro and in vivo . A, FBXO45 WT or CRISPR-Cas9–mediated FBXO45 KO iBJAB (“i”, doxycycline-inducible Cas9 expression) cells were treated with cycloheximide (CHX), and WCE was analyzed by WB with indicated antibodies. B, FBXO45 WT and KO iBJAB cells were treated with trametinib as indicated, and WCEs were analyzed by WB with the indicated antibodies. C, FBXO45 WT and KO iBJAB cells were xenografted in NSG mice and treated with trametinib as detailed in the “Methods” section. Tumor volumes of mice treated with trametinib for both FBXO45 WT and KO ( n = 6 each) were individually measured every 3 days. Experiments were performed in triplicate. D, In vivo imaging studies at day 25 show decreased tumor sizes in response to trametinib treatment in FBXO45 KO xenografts. At the end of the study (day 25), tumors were surgically removed for imaging analysis, as shown in the right panel. E, The top panel shows a schematic illustration of the experimental plan to evaluate trametinib treatment on xenograft tumors originated by FL518-CBG luciferase cells. The bottom panel shows representative BLI at days 1,6, 8 and 13. F, Relative bioluminescence (RLU or relative luminescence units) for each group of mice was measured and plotted for day 13. Two-tailed Mann–Whitney unpaired Student’s t test was used for analysis (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001). G, Kaplan–Meier survival curves of NS shRNA and FBXO45 shRNA expressing FL518-derived lymphoma xenografts in SCID-BEIGE mice. Log-rank (Mantel–Cox) test was used for analysis. WB, Western blotting; WCE, whole cell extract. Dox, doxycycline; ns, not significant.

Article Snippet: Lentiviral packaging plasmids pMD2.G (RRID: Addgene_12259), psPAX2 (RRID: Addgene_12260), and inducible Cas9 vector (RRID: Addgene_83481) were obtained from Addgene.

Techniques: Inhibition, In Vitro, In Vivo, CRISPR, Expressing, In Vivo Imaging, Imaging, Luciferase, Two Tailed Test, MANN-WHITNEY, shRNA, Derivative Assay, Western Blot

Journal: PLOS Pathogens

Article Title: MicroRNA-focused CRISPR/Cas9 screen identifies miR-142 as a key regulator of Epstein-Barr virus reactivation

doi: 10.1371/journal.ppat.1011970

Figure Lengend Snippet: A. Schematic of the miRNA-focused CRISPR/Cas9 loss-of-function screen carried out in EBV-positive MutuI-iCas9 BL cells. B. DrugZ-calculated normZ score plotted versus miRNA gene rank. Red indicates targets that were significantly enriched in gp350-positive population while black indicates targets significantly enriched in the gp350-negative population (p-value < 0.05). Highlighted is miR-142 which showed the most significant changes. C. EBV lytic transcripts are upregulated in miR-142 mutant cells. Four EBV lytic genes were measured by qRT-PCR in MutuI-iCas9 cells transduced with either a control gRNA (Neg3) or the top two scoring guide-RNAs (gRNAs) against miR-142 (10024 or 10033). Cells were either mock or anti-IgM treated for 48 hrs. Values are normalized to GAPDH and shown relative to anti-IgM treated Neg3 control cells. Shown is the average of six independent experiments. By Student’s t-test, *p<0.05. D. and E. miR-142-3p and miR-142-5p expression in multiple B cell lines. Total RNA was extracted from DLBCLs (IBL1, IBL4, BCKN1), LCLs (LCL16.1, 17.1, 17.2), EBV-positive BL (KemI, RaeI, Raji, MutuI, Akata), EBV-positive BL treated with anti-Ig for 24 hr (MutuI, Akata) and EBV-negative BL (BJAB, BL41, Ramos). miRNAs were assessed by Taqman qRT-PCR. Values are normalized to miR-16 and reported relative to IBL1.

Article Snippet: MutuI cells were transduced with lentiparticles derived from the doxycycline inducible Cas9 vector pCW-Cas9 (Addgene Plasmid #50661) and selected for stable integration with 2 μg/mL puromycin.

Techniques: CRISPR, Mutagenesis, Quantitative RT-PCR, Transduction, Control, Expressing