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: Nature chemical biology

Article Title: Controllable genome editing with split-engineered base editors.

doi: 10.1038/s41589-021-00880-w

Figure Lengend Snippet: Fig. 2 | Split-engineered BEs represent a generalizable strategy to enable small-molecule-controlled editing. a, Schematics of a traditional intact BE in the BE4max scaffold and the seBE strategy, including chemically induced dimerization of FRB and FKBP12 by rapamycin. b, Editing efficiency can be evaluated in a HEK293T cell line containing a single copy of integrated, constitutively expressed d2gfp. The presence of d2gfp-targeting sgRNA can introduce a stop codon (Q158*) and abrogate fluorescence to generate GFPoff cells, which can be tracked by either flow cytometry or deep sequencing of the locus. c, Representative flow cytometry histograms associated with transfection of intact or seBE constructs in the presence or absence of rapamycin. d, Mean and standard deviation for quantification of GFPoff cells by flow cytometry, with individual data points shown. A two-sided Mann–Whitney test was performed to compare intact and seBE GFPoff% (*P ≤ 0.05; **P ≤ 0.01). Exact P values provided as statistical source data files. NA, not applicable. e, Left shows deep-sequencing results demonstrating C to T conversion efficiency of the Q158 target cytosine under conditions identical to d. The mean and standard deviation are noted, with individual data points shown. Fold change (FC) is the ratio of mean values for the higher versus the lower condition in each comparison. Two-sided Mann–Whitney test was performed (NS, not significant; *P ≤ 0.05; **P ≤ 0.01). Right shows the editing footprints across the d2gfp locus for each condition. The full targeting sequencing is provided with the sgRNA protospacer (black) starting 20 bp from the protospacer adjacent motif (yellow) and the target C highlighted in red. In the editing footprint, the target cytosine base within the Q158 codon is noted with a blue arrow. Data represent position-wise averages of three or more biological replicates, with individual replicate data provided in Supplementary Table 1. Exact P values are provided as statistical source data files.

Article Snippet: HEK293T cells were lentivirally transduced with a constitutively expressed destabilized GFP (d2GFP) reporter (derived from Addgene no. 14760) and selected for individual clones that contained a single copy of integrated d2gfp.

Techniques: Introduce, Fluorescence, Flow Cytometry, Sequencing, Transfection, Construct, Standard Deviation, MANN-WHITNEY, Comparison