dsred gene  (New England Biolabs)

Journal: Nature Communications

Article Title: RNA-mediated double-strand break repair by end-joining mechanisms

doi: 10.1038/s41467-024-51457-9

Figure Lengend Snippet: a Schemes of sense and antisense-genetic constructs expressing RNA transcripts that differ by sequence and transcription level. (left) Sense constructs with sgRNA A and B: Sense, BranchΔ, and pCMVΔ (right) Sense constructs with sgRNA E and J: Sense/ BranchΔ/pCMVΔ, Antisense constructs with sgRNA C(C’) and D: Antisense and 5′-SplicingΔ. All these constructs contain the DsRed gene (blue-framed box) with an intron (green-framed box) in the sense or antisense orientation, respectively. The sense and antisense transcript RNAs (in red) are depicted after intron splicing (thin green mark) or carrying the intron (thick green line) for the splicing-mutant constructs. The single-guide RNA (sgRNA) A (orange line), sgRNA B (purple line), sgRNA E (brown line) and sgRNA J (blue line) with Cas9 endonuclease (light gray ovals) bind to the complementary DNA of the sense constructs to generate a DSB. The sgRNA C (orange line) or C’ (dark orange line) and D (purple line) with Cas9 endonuclease bind to the complementary DNA of the antisense constructs to generate a DSB. Black arrows: CMV , cytomegalovirus promoter, or EF1α , human eukaryotic translation elongation factor 1 alpha promoter. Transcription activity by a cryptic promoter is indicated with a yellow arrow. Dark green box: branch site of the intron or 5′-splice site for the sense and antisense constructs, respectively. b Scheme of DNA products for the Sense construct obtained following DSB repair by different DSB repair mechanisms. NHEJ non-homologous end joining, MMEJ microhomology-mediated end joining, DSB (black parallel lines); an example of a microhomology pair (light blue lines); DNA repaired by NHEJ (blue zigzag line).

Article Snippet: The plasmid having the antisense orientation of the artificial intron in the DsRed gene was digested by PciI and SspI restriction enzymes (New England BioLabs).

Techniques: Construct, Expressing, Sequencing, Mutagenesis, Activity Assay, Non-Homologous End Joining

Journal: Nature Communications

Article Title: Enhancing extracellular vesicle cargo loading and functional delivery by engineering protein-lipid interactions

doi: 10.1038/s41467-024-49678-z

Figure Lengend Snippet: a Schematic of workflow for evaluating the loading into EVs and functional activity of lipidated synthetic transcription factors (synTFs). Reporter cells express a red fluorescent protein, dsRed-Express2 (dsRedExp2), when activated. b Activity of synTF variants when plasmids were directly transfected into reporter cells. Left, log transform of reporter expression measured by flow cytometry in absolute units of mean molecules of equivalent phycoerythrin-texas red (PE-TR) (MEPTRs). Each symbol is an independent biological replicate, and error bars are the SEM ( n = 3). A one-way ANOVA was performed, and comparisons were evaluated using Dunnett’s multiple comparisons correction (**** p < 0.0001). synTFs were compared to the Sol condition; comparisons not shown were not significant. Right, scatter plot for the experiment on the left plotted against band intensity of synTF expression in those cells as measured by a single western blot ( n = 1) (Supplementary Fig. ). c Western blot band intensities of synTFs loaded into EVs, normalized to each individual blot’s PPF condition. Each symbol represents a biologically independent EV preparation and western blot, and the error bars represent SEM ( n = 3). The numbers above each bar are the average fold increase of synTF loading compared to the Sol synTF condition. A one-way ANOVA was performed, and comparisons were evaluated using Dunnett’s multiple comparisons correction (**** p < 0.0001). SynTFs were compared to the Sol condition; comparisons not shown were not significant. d Representative micrograph of reporter cells activated via EV-mediated delivery of lipidated synTFs (here, the PM variant). Scale bar is 100 µm. e Flow cytometry dot plots for a representative sample of reporter cells treated with either EVs isolated from Mock-transfected cells (no synTF) or from cells expressing PM-tagged synTF. Annotations state the percentage of reporter cells that expressed dsRedExp2. f Activation of reporter cells treated with synTF-containing EVs (evaluated by flow cytometry). Each symbol represents an independent biological replicate, and error bars represent SEM ( n = 3). A one-way ANOVA was performed, and comparisons were evaluated using Dunnett’s multiple comparisons correction (**** p < 0.0001). SynTFs were compared to the Mock condition; comparisons not shown were not significant. Throughout the figure, “Mock” refers to conditions where an empty backbone plasmid was transfected into a cell in lieu of a SynTF-encoding plasmid. Data in ( c , e , and f ) are representative of two independent experiments. Data in panel ( b ) were collected from one experiment. Source data are provided as a Source Data file.

Article Snippet: The reaction also included six transcription units of the synTF (ZF6) promoter with 12 compact binding sites upstream of a YB_TATA minimal promoter , and a DsRed-Express2 reporter gene (pHIE324–329), a seventh transcription unit containing a constitutive hEF1a promoter and a blasticidin resistance gene (pHIE280), and a linker plasmid to close the assembly (pPD1157, Addgene #139245).

Techniques: Functional Assay, Activity Assay, Transfection, Expressing, Flow Cytometry, Western Blot, Variant Assay, Isolation, Activation Assay, Plasmid Preparation