crispr cas9 cdna  (Integrated DNA Technologies)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Role for the EWS domain of EWS/FLI in binding GGAA-microsatellites required for Ewing sarcoma anchorage independent growth

doi: 10.1073/pnas.1701872114

Figure Lengend Snippet: Deletion of the NR0B1 microsatellite reduces NR0B1 expression, impairs A673 cell growth, and inhibits colony formation. (A) Sequencing results validating knockout of the NR0B1 GGAA-microsatellite about 1.5 kb upstream of the NR0B1 TSS in A673 cells. The sgRNAs targeted to either side of this region are underlined. GGAA-microsatellite is highlighted red, and CRISPR/Cas9 deleted region is highlighted blue. Gel shows deletion of NR0B1 microsatellite region compared with control (nondeleted), with densitometry quantification on Right (P < 0.01). Data are represented as mean ± SEM (n = 2). (B) NR0B1 mRNA (P < 0.05) and protein expression levels in control and CRISPR/Cas9-mediated knockout of NR0B1 microsatellite in A673 Ewing sarcoma cells, with Western blot densitometry quantification on Right. Control CRISPR/Cas9 plasmids do not contain sgRNAs. Data are represented as mean ± SEM (n = 3). (C) Growth and colony formation assay quantification of CRISPR/Cas9 control vs. NR0B1 microsatellite knockout in A673 cells (P < 0.05). Data are represented as mean ± SEM (n = 3).

Article Snippet: Mammalian expression constructs included the following: Lentiviral vectors containing CRISPR/Cas9 cDNA and sgRNA ( SI Materials and Methods ); retroviral vectors encoding Luc-RNAi and EF-2–RNAi and cDNAs for EWS/FLI, Δ22, R2L2, Mut9, and NR0B1 are previously described ( 4 , 13 , 22 , 23 ); the Mut9/R2L2 construct was ordered as a gene block (IDT) and cloned into the pMSCV hygro vector between EcoRI and HindIII restriction sites.

Techniques: Expressing, Sequencing, Knock-Out, CRISPR, Western Blot, Colony Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Role for the EWS domain of EWS/FLI in binding GGAA-microsatellites required for Ewing sarcoma anchorage independent growth

doi: 10.1073/pnas.1701872114

Figure Lengend Snippet: Deletion of the NR0B1 microsatellite in other cell lines. (A) NR0B1 mRNA and protein expression levels in control and CRISPR/Cas9-mediated knockout of the NR0B1 microsatellite in TC-71 and EWS/502 Ewing sarcoma cells (P < 0.05). Data are represented as mean ± SEM (n = 3). (B) Growth curves and soft agar assay quantification for NR0B1 microsatellite deletion in two other Ewing sarcoma cell lines (TC-71 cells and EWS/502 cells). Growth curve data are represented as mean ± SEM (n = 4). Control vs. CRISPR for TC-71 and EWS/502 cells are each statistically significant (P < 0.05). Soft agar data are represented as mean ± SEM (n = 2). (C) Densitometry quantification of PCR-amplified NR0B1-microsatellite–containing region for A673 control (wild-type NR0B1-microsatellite) allele vs. CRISPR-Cas9 knockout (deleted NR0B1-microsatellite) allele at different time points for up to 3 wk postlentiviral infection. (D) NR0B1 mRNA and protein expression levels in control and CRISPR/Cas9-mediated knockout of the NR0B1 microsatellite in non-Ewing sarcoma HEK293 cells. Data are represented as mean ± SEM (n = 3). n.s., not statistically significant. (E) NR0B1 protein levels and colony formation assay quantification for A673 cells with NR0B1 cDNA rescue in CRISPR/Cas9 control vs. microsatellite knockout. n.s., not statistically significant. Data are represented as mean ± SEM (n = 2).

Article Snippet: Mammalian expression constructs included the following: Lentiviral vectors containing CRISPR/Cas9 cDNA and sgRNA ( SI Materials and Methods ); retroviral vectors encoding Luc-RNAi and EF-2–RNAi and cDNAs for EWS/FLI, Δ22, R2L2, Mut9, and NR0B1 are previously described ( 4 , 13 , 22 , 23 ); the Mut9/R2L2 construct was ordered as a gene block (IDT) and cloned into the pMSCV hygro vector between EcoRI and HindIII restriction sites.

Techniques: Expressing, CRISPR, Knock-Out, Soft Agar Assay, Amplification, Infection, Colony Assay

Journal: Cell reports

Article Title: Structural and Functional Studies of the RBPJ-SHARP Complex Reveal a Conserved Corepressor Binding Site

doi: 10.1016/j.celrep.2018.12.097

Figure Lengend Snippet: (A) The wild-type SHARP RBPID (RBPID WT ), but not the RBPJ-interacting defective SHARP RBPID (RBPID LI/AA ) mutant, causes upregulation of Notch target genes in mouse mature T (MT) cells by outcompeting endogenous SHARP for RBPJ binding. MT cells were infected with plasmids encoding GFP-tagged SHARP (2,776–2,833), either wild-type (GFP-SHARP/RBPID WT , black bars) or the RBPJ-interacting defective mutant L2791A/I2811A (GFP-SHARP/RBPID LI/AA , gray bars) or an empty vector control (Control, white bars). Left: total RNA from MT cells was analyzed by qPCR using primers specific for Tbp , Hes1 , or Hey1 . Data shown represent the mean ± SD of triplicate experiments (**p < 0.01, ***p < 0.001, unpaired Student’s t test). Right: nuclear extracts (NEs) were prepared from MT cells and analyzed by western blotting, with TBP used as a loading control. (B–D) GFP-SHARP/RBPID WT derepresses Notch target genes via histone deacetylation. MT cells were infected with plasmids encoding GFP-SHARP/RBPID WT , GFP-SHARP/RBPID LI/AA , or an empty vector control and analyzed by chromatin immunoprecipitation (ChIP) using antibodies against H3K9ac (B), H3K27ac (C), or H3 (D). The enrichment was analyzed by qPCR on the enhancers of Hes1 and Hey1 , located at approximately +0.6 kb and −0.8 kb relative to the transcription start site, respectively. Gene desert was used as a negative control. Data were normalized to the positive control ( Gapdh 0kb ), and, in the case of the H3K9ac and H3K27ac ChIP, data were further normalized to histone occupancy (H3). Shown is the mean ± SD of two experiments measured twice each (NS, not significant; *p < 0.05; ***p < 0.001; unpaired Student’s t test). See also . (E) RBPJ is required to repress the Notch target genes Hes1 and Hey1 in MT cells, as revealed by CRISPR/Cas9 depletion of RBPJ. Left: total RNA from wild-type (control) or RBPJ-depleted (clones sgRbpj 2–12 and sgRbpj 2–14 ) MT cells was analyzed by qPCR. Shown is the mean ± SD of triplicate experiments (*p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student’s t test). Right: whole-cell extracts (WCEs) were prepared from MT cells and analyzed by western blotting using an anti-RBPJ antibody. GAPDH was used as loading control. (F) Hes1 and Hey1 Notch target genes are upregulated upon shRNA-mediated Rbpj knockdown but not with the SCR control shRNA. Left: total RNA from MT cells infected with shRNAs targeting Rbpj ( Rbpj sh1 or Rbpj sh4 ) or scrambled shRNA control (SCR) was analyzed by qPCR using primers specific for Tbp , Hes1 , or Hey1 . Shown is the mean ± SD of quadruplicate experiments (**p < 0.01, ***p < 0.001, unpaired Student’s t test). Right: WCE was prepared from MT cells and analyzed by western blotting using an RBPJ antibody. GAPDH was used as a loading control. (G) Expression of RBPJ WT but not RBPJ FL/AA in the RBPJ-depleted background rescues the repression of Notch target genes. Left: total RNA from sgRbpj 2–12 MT cells infected with empty vector (control), RBPJ WT , or RBPJ FL/AA was analyzed by qPCR using primers specific for Tbp , Hes1 , or Hey1 . Shown is the mean± SD of three independent experiments measured twice each (*p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student’s t test). Right: NEs were prepared from sgRbpj 2–12 MT cells infected with empty vector (control), RBPJ WT , or RBPJ FL/AA and analyzed by western blotting using anti-RBPJ antibodies. Histone H3 was used as a loading control.

Article Snippet: An engineered CRISPR/Cas9 resistant mouse RBPJ cDNA was synthetized at GENEART/Life Technologies and inserted into the pcDNA3.1 Flag2 via NotI digestion.

Techniques: Mutagenesis, Binding Assay, Infection, Plasmid Preparation, Western Blot, Chromatin Immunoprecipitation, Negative Control, Positive Control, CRISPR, Clone Assay, shRNA, Expressing