gfp reporter  (Addgene inc)

Journal: Cells

Article Title: 5' DREDGE: Direct Repeat-Enabled Downregulation of Gene Expression via the 5' UTR of Target Genes.

doi: 10.3390/cells14120866

Figure Lengend Snippet: Figure 2. Comparative efficacy of 5′ DREDGE implemented with five different Cas RNases and their cognate DRs. (A) Overview of the construction of the vectors encoding GFPd2 with individual cognate DRs (green) in the 5′ UTR (top) and vectors co-expressing different Cas RNases (red) together with mCherry (bottom). Constructs lacking a DR or RNase served as controls. (B) Cell cytometry results from MEFs transiently transfected with the constructs in (A). Depicted are the percentages of cells in Q2 relative to controls (top), which were quantified from log-log plots of GFP vs. mCherry RFU values (n = 3 replicates), with representative plots for each condition provided (bottom). (C) GFP intensity in mCherry-positive cells, normalized to controls, derived from the RFU plots shown in (B), together with representative images of GFP fluorescence in cells from the various conditions (bottom), which were acquired immediately prior to cytometry.

Article Snippet: Vectors for Transient Transfection Experiments The vector expressing destabilized GFP (GFPd2) was generated by modifying Addgene plasmid #14760 [13] to include a puromycin resistance cassette, as described [4].

Techniques: Expressing, Construct, Cytometry, Transfection, Derivative Assay, Fluorescence

Journal: Cells

Article Title: 5' DREDGE: Direct Repeat-Enabled Downregulation of Gene Expression via the 5' UTR of Target Genes.

doi: 10.3390/cells14120866

Figure Lengend Snippet: Figure 3. Characterization of 5′ DREDGE using an inducible system. (A) Cartoon depicting the generation of clonal cell lines constitutively expressing GFPd2 with a single Cas12a or Csy4 DR in the 5′ UTR (or no DR as a control). (B) Generation of double-stable lines for inducible Cas RNase expression. The lines in (A) were used to generate lines that also stably express either dCas12a or Csy4 (or no RNase as a control) in a Dox-regulatable manner. (C,D) Performance of the double- stable cell lines from (B), assessed using the percentage of cells in Q2 (C) and mean GFP RFU (D) in the absence or presence of Dox (1 µg/mL). Data are shown as the mean ± SEM normalized to Dox-treated No-RNase controls; n = 2–3 per condition. (E,F) Dox dose–response experiments with double-stable cell lines inducibly expressing dCas12a or Csy4. Graphs depict responsiveness to a range of concentrations of Dox, quantified in terms of the percentage mCherry-positive cells that were also GFP-positive (E) or the mean GFP RFU in all cells (F). Mean IC50 values are shown. Data are shown as the mean ± SEM for 2–3 replicates per condition, normalized to values in the absence of Dox for each line. (G) Temporal dynamics of GFPd2 expression in cell lines inducibly expressing either dCas12a or Csy4 following addition (solid lines) or withdrawal (dashed lines) of Dox (1 µg/mL). Data are shown as the mean ± SEM for 2–3 independent experiments, normalized to No-Dox controls. Mean t1/2 values are indicated. (H,I) Relative expression of GFPd2 mRNA in the absence versus the presence of Dox (1 µg/mL) in double-stable cell lines instantiating 5′ DREDGE with Cas12a (H) and Csy4 (I). Note the dramatic increase in GFPd2 mRNA levels triggered by dCas12a expression (H). Data are mean ± SEM for 3 independent experiments, expressed as a percentage of No-Dox controls.

Article Snippet: Vectors for Transient Transfection Experiments The vector expressing destabilized GFP (GFPd2) was generated by modifying Addgene plasmid #14760 [13] to include a puromycin resistance cassette, as described [4].

Techniques: Expressing, Control, Stable Transfection

Journal: bioRxiv

Article Title: Targeted control of gene expression using CRISPR-associated endoribonucleases

doi: 10.1101/2025.03.17.643761

Figure Lengend Snippet: Screening of candidate Cas RNases for 3’ DREDGE. A , Mechanism of 3’ DREDGE. Cleavage of the DR(s) in the 3’ UTR of mRNA (green) by a Cas RNase (red) removes the poly(A) tail, triggering rapid degradation. B , DRs for the five Cas RNases investigated in this study, with cleavage sites indicated (red arrows). Note the “synSeparator” adjacent to the 5’ end of the Cas12a DR. C , Designs of constructs expressing GFPd2 with different DRs (or no DR) in the 3’ UTR (green) and constructs expressing individual Cas RNases (or no RNase) together with mCherry (red). D , Cell cytometry data for MEFs expressing different Cas RNases and GFPd2 constructs with their cognate DRs. Graph of the percentage of cells in Q2 relative to controls (top) within log-log plots of GFP vs. mCherry RFU values (n=3 replicates), with representative plots shown (bottom). E , Graph of GFP intensity in mCherry+ cells derived from RFU plots shown in D and normalized to controls, along with representative images of GFP fluorescence in cells prior to cytometry (bottom).

Article Snippet: The parent vector expressing destabilized GFP (GFPd2) under the control of the CAG promoter, pCAG-GFPd2 (Addgene plasmid #14760 [ ]) was modified by inserting an open-reading frame (ORF) encoding a puromycin resistance cassette (Puro r )-T2A-TagBFP fusion protein (amplified from Addgene Plasmid #155307 [ ]) into an AvrII restriction site between the SV40 promoter and SV40 poly(A) signal within the pCAG-GFPd2 vector.

Techniques: Construct, Expressing, Cytometry, Derivative Assay, Fluorescence

Journal: bioRxiv

Article Title: Targeted control of gene expression using CRISPR-associated endoribonucleases

doi: 10.1101/2025.03.17.643761

Figure Lengend Snippet: Dox-regulatable control of gene expression by 3’ DREDGE. A , Design of constructs constitutively expressing GFPd2 with zero, one, or three Cas12a DRs in the 3’ UTR used to create three different stable cell lines. B , Design of constructs with Dox-regulatable co-expression of dCas12a (or No RNase) and mCherry used to create double-stable cell lines from the lines in A. C , Percentage of cells in Q2 for double-stable cell lines expressing GFPd2 with zero, one, or three DRs and also conditionally expressing either Cas12a or no RNase, tested in the absence or presence of Dox (top) derived from log-log plots of GFP vs. mCherry RFU (bottom). D , Mean GFP RFU in the cell lines in C in the absence or presence of Dox derived from cell cytometry (top) with representative images of cells in the different conditions (bottom). Data in C and D are normalized to Dox-treated No-DR and No-RNase controls; n=2-3 per condition. E , F , Dose-response curves showing ( E ) the percent of mCherry+ cells (i.e., Q2+Q4) that were also GFP+ (i.e., in Q2) and ( F ) mean GFP RFU as a function of Dox dose in stable cell lines with one or three DRs conditionally expressing cDas12a. Red columns in F show the mean mCherry RFU as a function of Dox dose for both cell lines, normalized to the maximum at 1000 ng/mL. Mean IC50 values for all dose-responses are indicated. Data are mean ± SEM, normalized to values in the absence of Dox for each line; n=2-3 per condition. G , Time courses of GFP RFU in response to addition (solid lines) or withdrawal (dashed lines) of Dox in the cell lines in E and F normalized to No-Dox controls. Mean half-life values (t1/2) are shown. Data are mean ± SEM for 2-3 independent experiments.

Article Snippet: The parent vector expressing destabilized GFP (GFPd2) under the control of the CAG promoter, pCAG-GFPd2 (Addgene plasmid #14760 [ ]) was modified by inserting an open-reading frame (ORF) encoding a puromycin resistance cassette (Puro r )-T2A-TagBFP fusion protein (amplified from Addgene Plasmid #155307 [ ]) into an AvrII restriction site between the SV40 promoter and SV40 poly(A) signal within the pCAG-GFPd2 vector.

Techniques: Control, Gene Expression, Construct, Expressing, Stable Transfection, Derivative Assay, Cytometry

Journal: eLife

Article Title: SAFB regulates hippocampal stem cell fate by targeting Drosha to destabilize Nfib mRNA

doi: 10.7554/eLife.74940

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pCAG::GFPd2 (plasmid) , , RRID: Addgene_14760 , pCAG expression vector.

Techniques: Protease Inhibitor, Recombinant, Clone Assay, Bicinchoninic Acid Protein Assay, Mutagenesis, Transfection, Sequencing, esiRNA, Blocking Assay, Plasmid Preparation, Expressing, Software