cas9 expressing vectors  (Addgene inc)

Journal: bioRxiv

Article Title: DROSHA, DICER and Damage-Induced long ncRNA control BMI1-dependent transcriptional repression at DNA double-strand break

doi: 10.1101/2024.08.07.606960

Figure Lengend Snippet: A. RT-qPCR analysis of GFP mRNA levels in HeLa cells bearing an integrated GFP gene (HeLa-GFP) treated with control siRNAs (siCTRL) or siRNAs against DROSHA and DICER transcripts (siDROSHA/siDICER). Data are relative to uncut cells for each knockdown condition. Error bars represent SEM from three independent experiments. B. RT-qPCR analysis of GFP mRNA levels in cut and uncut HeLa-GFP cells treated with siCTRL or siRNAs against BMI1 transcript (siBMI1). Data are relative to uncut cells for each knockdown condition. Error bars represent SEM from three independent experiments. C. RT-qPCR analysis of GFP mRNA levels in cut and uncut cells treated with control ASOs or ASOs targeting dilncRNAs generated at the GFP locus. Error bars represent SEM from three independent experiments. Data are relative to uncut cells for each condition. Statistical test used was paired Student t-test. D. RT-qPCR analysis of GFP mRNA levels in cut and uncut HeLa-GFP stably expressing an inducible Cas13d. Cells were treated with a control Cas13 RNA guide (CTRL Cas13g) or dilncRNAs-targeting guides (anti-dilncRNAs Cas13g). Error bars represent SEM from four independent experiments. E. ChIP-qPCR analysis for BMI1 at the GFP locus and an unrelated region, performed in uncut (scramble-guide-Cas9) and cut (GFP-guide-Cas9) HeLa-GFP cells treated with control ASOs or dilncRNAs-targeting ASOs. Data are relative to uncut cells after the subtraction of mock values for each treated condition. Error bars represent SEM from three independent experiments. F. RT-qPCR analysis of LIN54 (DSB at Chr4) mRNA levels in cut and uncut DIvA cells stably expressing an inducible Cas13d. Cells were treated with a control Cas13 RNA guide (CTRL Cas13g) or guides targeting dilncRNAs generated downstream the AsI cut site on Chr4 (Chr4 RNA Cas13g). Error bars represent SEM from three independent experiments. Statistical analyses in panels B, D, E and F were performed by One-Way ANOVA.

Article Snippet: For experiments in HeLa-GFP cells, Cas9 expressing vectors (Addgene) bearing either a non-targeting scrambled gRNA or a cut-inducing GFP-targeting guide gRNA (see for sequences of scramble gRNA and GFP-targeting gRNA) were transfected using Lipofectamine 2000 (Invitrogen; #11668-027) for 24 hours before cell fixation or RNA extraction.

Techniques: Quantitative RT-PCR, Control, Knockdown, Generated, Stable Transfection, Expressing, ChIP-qPCR

Journal: Nature Communications

Article Title: MYCN amplification and ATRX mutations are incompatible in neuroblastoma

doi: 10.1038/s41467-020-14682-6

Figure Lengend Snippet: a Immunoblot of 12 cell lines. Boxes indicate ATRX -mutant lines. b Immunoblot for HeLa cells transfected with sc-shRNA or ATRX -specific shRNAs. The fraction of ATRX relative to sc-shRNA is indicated. c Bar plot of the number of colonies/well (mean of duplicates) after transfection of shRNAs, normalized to the sc-shRNA (dashed line). d Photograph of cresyl violet-stained colonies after transfection with shRNAs and bar plots of the average number of colonies/well. e Map of two gRNAs targeting ATRX . f Plot of the mutation frequency for gRNA-2, deletions (green) and insertions (red). g Bar plot of the proportion of ATRX -mutant alleles (100,000× coverage/sample). h Immunoblot for the cell lines with the doxycycline-inducible MYCN expression construct. Numbers indicate MYCN/GAPDH fluorescence intensity. The experiment was done three times with similar results. i Line graphs of the growth curves (mean ± SD) for each cell line ± doxycycline, n = 3. j Colony assay for SKNBE2 MYCN and SKNMM MYCN cells ± doxycycline, the experiment was repeated twice with similar results. k Brightfield micrograph of SKNMM MYCN cells after 8 days in culture ± doxycycline, the experiment was repeated twice with similar results. l Brightfield micrographs of three individual SKNMM MYCN cells ± doxycycline at indicated timepoints, relative to the starting point of the movies (6 days + doxycycline), 44 cells were analyzed. m Immunoblots of SKNMM MYCN cells without doxycycline or after 2, 4, or 45 days in culture. The escapers are pools of cells that grew in the presence of doxycycline ( n = 4). PCR for MYCN transgene and a control locus ( RPL0 ) is indicated in the lower portion. n Xenogen image of a mouse with an orthotopic neuroblastoma tumor (photograph), that arose from a 1:1 mixture of SKNMM MYCN cells and SKNMM CONT –Luc:YFP cells ( n = 5). o Line graph of xenogen image data described in n . p Xenogen image of a mouse with an orthotopic neuroblastoma tumor (photograph), that arose from a 1:1 mixture of SKNMM MYCN –Luc:YFP cells and SKNMM CONT cells ( n = 5). q Line graph of xenogen image data described in p . Scale bars: k , 10-μm. DOX doxycycline, IFD in-frame deletion, sc scrambled.

Article Snippet: To test for the NHEJ efficiency after CRISPR targeting in IMR32 cells, we transfected the cells with the same mix of plasmids but added a control plasmid expressing gRNA targeting the hAAVS1 locus (Addgene # 41818) in a ratio of 4:4:6:1 for hAAVS1 .gRNA: ATRX .gRNA: Cas-9: GFP expressing-plasmids, respectively.

Techniques: Western Blot, Mutagenesis, Transfection, shRNA, Staining, Expressing, Construct, Fluorescence, Colony Assay, Control