sgrna co expression plasmid empty backbone addgene  (Addgene inc)

Journal: eLife

Article Title: Co-regulation and function of FOXM1 / RHNO1 bidirectional genes in cancer

doi: 10.7554/eLife.55070

Figure Lengend Snippet: RHNO1 knockout was achieved using CRISPR-Cas9 with guide RNAs that targeted near the start and stop codon of RHNO1 . ( A, B ) RHNO1 homozygous knockout was confirmed by ( A ) genomic DNA PCR and ( B ) RHNO1 mRNA expression measured by end-point RT-PCR. NTC: no template control. ( C ) FT282 parental and RHNO1 clonal knockout cells were seeded in quadruplicate and grown in culture for a period of 1 week. Cell growth/viability was measured by the AlamarBlue assay every 24 hr. ( D ) Western blot analyses of two canonical phosphoprotein biomarkers of RS in FTE and HGSC cell lines. Cell line sample names are shown below.

Article Snippet: Genetic reagent ( Homo sapiens ) , PX458-WT-Cas9-empty , Addgene , 48138 , Plasmid.

Techniques: Knock-Out, CRISPR, Expressing, Reverse Transcription Polymerase Chain Reaction, Alamar Blue Assay, Western Blot

Journal: eLife

Article Title: Co-regulation and function of FOXM1 / RHNO1 bidirectional genes in cancer

doi: 10.7554/eLife.55070

Figure Lengend Snippet:

Article Snippet: Genetic reagent ( Homo sapiens ) , PX458-WT-Cas9-empty , Addgene , 48138 , Plasmid.

Techniques: Magnetic Beads, Purification, Single Cell Gel Electrophoresis, Bicinchoninic Acid Protein Assay, DNA Methylation Assay, Gel Extraction, TA Cloning, Reporter Assay, Reporter Gene Assay, Plasmid Preparation, Software

Journal: The EMBO Journal

Article Title: NR2F1 regulates regional progenitor dynamics in the mouse neocortex and cortical gyrification in BBSOAS patients

doi: 10.15252/embj.2019104163

Figure Lengend Snippet: A–B′ Pax6 (green; aRGs) and Nr2f1 (red) IF of E12.5 WT (A, A′) and KO (B, B′) lateral pallia. See Appendix Fig S4 for Pax6 and Nr2f1 staining and pixel intensity quantification in HET embryos. C Pax6 and Nr2f1 pixel intensity quantification at E12.5 and E14.5 indicating increased Pax6 levels upon Nr2f1 removal. n ≥ 3 brains. D Real‐time RT–PCR quantification of Nr2f1 and Pax6 expression in E12.5 cortices. n ≥ 3 cortices. E–G″ GFP (green; electroporated cells), Pax6 (red), and Nr2f1 (blue) IF of pCIG2‐Nr2f1‐IRES‐GFP electroporated brains at E13.5 (24 h after IUE). VZ (F‐F″) and CP (G‐G″) regions are shown at high magnification. Arrowheads point to GFP + electroporated cells overexpressing Nr2f1 (blue in F′, G′), down‐regulating Pax6 (F″), and rapidly migrating out of the VZ (G‐G″). H Scatter plot showing Pax6 and Nr2f1 pixel intensity of VZ electroporated progenitors, comparing Nr2f1 overexpressing cells (orange dots) with control pCIG2‐GFP electroporated cells (blue dots). Average Nr2f1 and Pax6 pixel intensities of the 2 populations were compared by two‐way ANOVA and resulted significantly different (*** P = < 0.0001). n = 2 electroporated brains. I Real‐time RT–PCR of cell cycle genes comparing WT (blue line) and KO (orange line) cortices. CyclinD1 and P21 transcripts are down‐regulated, whereas Dct is up‐regulated in mutants. n = 3 cortices. J–L GFP (green; electroporated cells) and P21 (red) IF of E14.5 cortex electroporated 48 h earlier with control PX458 plasmid (J) or CRISPR/Cas9‐expressing plasmid directed against Nr2f1 sequence ( PX458‐αNr2f1 ; K). Percentage of P21/GFP double‐positive cells (L). Arrowheads in (J, K) point to P21 + cells. n = 2 electroporated brains. M Schematic model of Nr2f1 action on NP cell cycle and neural differentiation. Nr2f1 promotes neurogenesis by repressing Pax6 and Dct expression and thus cell cycle progression, and by activating P21‐mediated cell cycle exit. Both actions modulate the G1‐phase length. N Graph showing the number of differentially expressed genes (DEG; see ) up‐regulated (UP) or down‐regulated (DOWN) in KO compared to WT , as detected by RNA‐Seq of E15.5 neocortices. O Gene ontology (GO) categories significantly enriched among DEG genes ( DAVID Gene Ontology software ; see ). P Hierarchical clustering and heatmap of the expression level of DEGs belonging to the “neuron differentiation” GO category in (O). Heat map color scale indicates normalized gene expression from high (red) to low (white) level. Data information: In (A, B, J, K), nuclei (blue) were stained with DAPI. In (C), the pixel intensity was quantified in 100 μm‐width boxes, randomly placed across the lateral pallium. Data are represented as means ± SEM. Student's t ‐test (L; * P < 0.05) and two‐way ANOVA (C, D, H, I) (* P < 0.05, ** P < 0.01, *** P < 0.001). For statistical analysis of RNA‐Seq data (N‐P), see . Scale bars: 50 μm. SVZ: subventricular zone; VZ: ventricular zone.

Article Snippet: The anti‐Nr2f1 sgRNA probe for the CRISPR/Cas9 construct (cloning primers: CACCgcgagatccgcaggacgacg; AAACcgtcgtcctgcggatctcgc) was selected and checked for specificity in silico with Crispor ( http://crispor.tefor.net/ ) and then cloned in the PX458 empty vector (Addgene; pSpCas9(BB)‐2A‐GFP).

Techniques: Staining, Quantitative RT-PCR, Expressing, Control, Plasmid Preparation, CRISPR, Sequencing, RNA Sequencing, Software, Gene Expression

Journal: The EMBO Journal

Article Title: NR2F1 regulates regional progenitor dynamics in the mouse neocortex and cortical gyrification in BBSOAS patients

doi: 10.15252/embj.2019104163

Figure Lengend Snippet: A–C NR2F1 expression in cerebral organoids during neural induction and differentiation in vitro (day 40 and day 70, respectively), together with the NP markers PAX6 (green in A), SOX2 (green in B, B′), and DCX as a neural marker (blue in A, B, B″). Quantification of NR2F1 levels in NPs is shown in (C). n ≥ 4 organoids from n = 2 batches. D–G NR2F1 (red) and PAX6 (green) IF in day 40 organoids. Neuroepithelia with high NR2F1 levels show weak PAX6 expression (E, E′), and vice versa (F, F′). Quantification by pixel intensity analysis (G). n ≥ 4 organoids from n = 2 batches. H Schematic representation of an organoid electroporation. PX458 plasmids were injected in ventricular‐like cavities, and organoids were electroporated and processed after 7 days of in vitro culture. I, J NR2F1 (red) and GFP (green) IF in day 40 human brain organoids upon electroporation of the PX458‐αNR2F1 plasmid. In (I, I′), GFP + NR2F1 − cells (empty arrowheads) and a GFP + NR2F1 + cell (white arrowhead) are shown; quantification in (J). n ≥ 6 organoids from n = 2 batches. K–M TUJ1 (red; differentiating neurons) and GFP (green) IF 7 days after electroporation of control (K) and PX458‐αNr2f1 plasmids (L). Co‐expression of GFP with SOX2 or TUJ1 distinguishes neural progenitors (NPs) from neurons (Ns), as quantified in (M). Arrowheads in (K, L) point to TUJ1 + GFP + cells. n ≥ 6 organoids from n = 2 batches. N Red and green arrowheads in (N) point to a SOX2 + (red) progenitor and a TUJ1 + (blue) neuron, respectively. Data information: Nuclei (blue in D, I, K, L) stained with DAPI. In (C, G, J), the pixel intensity or the number of positive cells was quantified in 100 μm‐width boxes, randomly placed across the cortex/organoid neuroepithelia, while in (M) the number of positive cells was normalized over the total number of GFP + cells. In graphs, data are represented as means ± SEM. Two‐way ANOVA test (* P < 0.05, ** P < 0.01, *** P < 0.001). Scale bars: 50 μm.

Article Snippet: The anti‐Nr2f1 sgRNA probe for the CRISPR/Cas9 construct (cloning primers: CACCgcgagatccgcaggacgacg; AAACcgtcgtcctgcggatctcgc) was selected and checked for specificity in silico with Crispor ( http://crispor.tefor.net/ ) and then cloned in the PX458 empty vector (Addgene; pSpCas9(BB)‐2A‐GFP).

Techniques: Expressing, In Vitro, Marker, Electroporation, Injection, Plasmid Preparation, Control, Staining