plasmid ppd005  (Addgene inc)

Journal: bioRxiv

Article Title: Conversion of natural cytokine receptors into orthogonal synthetic biosensors

doi: 10.1101/2024.03.23.586421

Figure Lengend Snippet: (a) Schematic of generalized VEGFR signaling mechanism highlighting receptor interactions (top), and schematic of the proposed VEGF NatE MESA signaling mechanism (bottom). (b) Surface expression of each chain (transfected HEK293FTs, anti-3xFLAG stain). Histograms show data for transfected (fluorescent) cells and the gray histograms in each column are transfection controls (no receptor). All receptors express well on the cell surface, with VEGFR2 constructs showing higher expression than VEGFR1 constructs. Mean fluorescence intensities are listed. (c) Functional evaluation of VEGF NatE MESA receptors (transfected HEK293FTs) in response to autocrine human VEGF (two isoforms). Many receptor configurations exhibit ligand-inducible signaling, with fold induction (FI: induced signal divided by background signal) shown below the plot (two-tailed Welch’s t-test results indicated above each bar pairing for p < 0.05). Some receptors signal more in response to one VEGF isoform over another. All receptors include the native signal sequence that matches the ECD. (d) Modulating VEGFA-165 expression by varying co-transfected plasmid masses yields dose-dependent receptor signaling. All plasmid masses used confer ligand-induced signaling compared to the no-ligand case (single-factor ANOVA, p < 0.001). (e) Functional evaluation of VEGF NatE MESA receptors incorporating different split TEVp mutants in response to co-expressed (left) and recombinant (right) human VEGF. Only receptors including the 190K CTEVp mutant show inducible signaling with both ligand formats, with choice of NTEVp mutant modulating overall signal magnitude (two-tailed Welch’s t-test, ** p < 0.01, *** p < 0.001). (f) Functional evaluation VEGF NatE MESA receptors expressed stably from the genome. HEK293FTs were modified with PiggyBac transposons encoding receptors and a reporter in the transposon (top) or encoding just receptors and introduced into a HEK293FT cell line containing a reporter integrated in the AAVS1 locus (bottom). Two receptor configurations were investigated: A CTEVp (190K) chain with a VEGFR1 signal sequence, ECD, and TMD paired with a NTEVp (75S or WT) chain with a VEGFR2 signal sequence, ECD, and TMD. Both transposons constitutively express mNeonGreen and puromycin resistance to facilitate selection and fluorescence-based identification of engineered cells (not shown here but included in schematics in Supplementary Figure 6h ). Data shown include all mNeonGreen+ cells. Results for two-tailed Welch’s t-test shown for p < 0.05. For all bar graphs, bars represent the mean of three biological replicates and error bars depict standard error of the mean (S.E.M.). Abbreviations: VEGFR, vascular endothelial growth factor receptor; PRS, protease recognition sequence; TF, transcription factor; ECD, ectodomain; TMD, transmembrane domain; TEVp, Tobacco Etch Virus protease; NTEVp, N-terminal component of split, mutant TEVp; CTEVp, C-terminal component of split, mutant TEVp; APC FI, allophycocyanin fluorescence intensity; MEPTRs, molecules of equivalent PE-TexasRed; Rep, reporter; FI, fold induction; R1, VEGFR1; R2, VEGFR2; REU, Rosetta energy units (dimensionless).

Article Snippet: Ligands were cloned into a pcDNA backbone to confer high expression in HEK293FT cells (Addgene #138749) .

Techniques: Expressing, Transfection, Staining, Construct, Fluorescence, Functional Assay, Two Tailed Test, Sequencing, Plasmid Preparation, Recombinant, Mutagenesis, Stable Transfection, Modification, Selection, Virus

Journal: bioRxiv

Article Title: Conversion of natural cytokine receptors into orthogonal synthetic biosensors

doi: 10.1101/2024.03.23.586421

Figure Lengend Snippet: (a) Schematic of generalized IL-10R signaling mechanism highlighting receptor interactions (top). Schematic of the proposed converted IL-10R-based NatE MESA signaling mechanism (bottom). (b) Surface expression of each single chain (transfected HEK293FT cells, anti-3xFLAG stain). Histograms represent transfected (fluorescent) cells and gray histograms are transfection controls (no receptor). All receptors express well on the cell surface. Mean fluorescence intensities are listed. (c) Functional evaluation of IL-10 NatE MESA receptor candidates (transfected HEK293FT cells) in response to autocrine human IL-10. Heteroassociative pairs exhibit ligand-inducible signaling, with fold inductions (FI: induced signal divided by background signal) shown below the plot (two-tailed Welch’s t-test results indicated above each bar pairing for * p < 0.05, ** p < 0.01, *** p < 0.001). Inclusion of the CD28 TMD increased background signal. All receptors include the native signal sequence that matches the ECD. (d) Functional evaluation of response to recombinant, exogenous ligand added at early (14 h post-transfection) and/or late (38 h post-transfection) time points (transfected HEK293FT cells). Ligand induced a significant increase in reporter expression over the non-treated condition for late treatments (two-tailed Welch’s t-test, * p < 0.05, ** p < 0.01). (e) Functional evaluation of IL-10 NatE MESA receptors expressed stably from the genome. HEK293FTs were modified with PiggyBac transposons encoding receptors and a reporter in the transposon (top) or encoding just receptors and introduced into a HEK293FT cell line containing a reporter integrated in the AAVS1 locus (bottom). Two receptor configurations were investigated: A CTEVp receptor with an IL-10Ra signal sequence, IL-10Ra ECD, and IL-10Ra TMD paired with a NTEVp receptor with an IL-10Rb ECD, IL-10Rb TMD, and either a hIgG VH or hCD8a signal sequence. The CD8a signal sequence NTEVp pairing exhibits more inducibility across both transposon designs. Both transposons constitutively express mNeonGreen and puromycin resistance to facilitate selection and fluorescence-based identification of engineered cells (not shown here but included in schematics in Supplementary Figure 7f ). Data shown include all mNeonGreen+ cells. Ligand induced a significant increase in reporter expression over the non-treated condition for all receptor-expressing cell lines except for the cell line containing the inert receptors (receptors containing rapamycin-binding domains in place of IL-10R ECDs) (two-tailed Welch’s t-test indicated above each bar pairing for * p < 0.05, ** p < 0.01). (f) IL-10 NatE MESA dose response to recombinant human IL-10 (HEK293FT cells with genomically integrated receptor configuration: CTEVp chain with an IL-10Ra signal sequence, IL-10Ra ECD, and IL-10Ra TMD paired with an NTEVp chain with an IL-10Rb ECD, IL-10Rb TMD and hCD8a signal sequence). A dose-dependent increase in reporter expression is observed until 250 ng/mL, after which reporter expression plateaus, potentially due to saturation of receptor sites or toxicity. Ligand-induced reporter expression is significantly different from the untreated condition down to 16 ng/mL (single factor ANOVA, ** p < 0.01). Analogous dose responses for the same receptor configuration implemented via Sleeping Beauty transposon and when implemented via PiggyBac transposon followed by sorting for top mNeonGreen expressers are shown in Supplementary Figure 7r . For all bar graphs and scatter plots, bars and points represent the mean across transfected cells or transposon modified (mNeonGreen+) cells and error bars depict standard error of the mean (S.E.M.). (g) Microscopy images of the cell line used in panel (f) incubated with 250 ng/mL IL-10 for 48 h (top) and quantification of mean reporter expression over time (in engineered cells; green pixels) (bottom). After 22 h, the ligand-treated conditions are significantly different than the non-ligand treated conditions (multi-factor ANOVA, p < 0.05). Error bars represent the standard error across three fields of view for each time point. Abbreviations: IL-10R, interleukin-10 receptor; PRS, protease recognition sequence; TF, transcription factor; ECD, ectodomain; TMD, transmembrane domain; TEVp, Tobacco Etch Virus protease; NTEVp, N-terminal component of split, mutant TEVp; CTEVp, C-terminal component of split, mutant TEVp; APC FI, allophycocyanin fluorescence intensity; MEPTRs, molecules of equivalent PE-TexasRed; Rep, reporter; MFI, mean fluorescence intensity; FI, fold induction; Ra, IL-10Rα; Rb, IL-10Rβ.

Article Snippet: Ligands were cloned into a pcDNA backbone to confer high expression in HEK293FT cells (Addgene #138749) .

Techniques: Expressing, Transfection, Staining, Fluorescence, Functional Assay, Two Tailed Test, Sequencing, Recombinant, Stable Transfection, Modification, Selection, Binding Assay, Microscopy, Incubation, Virus, Mutagenesis

Journal: bioRxiv

Article Title: Conversion of natural cytokine receptors into orthogonal synthetic biosensors

doi: 10.1101/2024.03.23.586421

Figure Lengend Snippet: (a) OR gate schematic, implementation, and desired behavior. Each receptor releases the same synTF upon ligand binding, which can then bind to a cognate promoter and induce output gene expression when either of the inputs (IL-10 and/or VEGF) are present. (b) OR gate functional evaluation. IL-10 and VEGF NatE MESA receptors were expressed by co-transfection of HEK293FT reporter cells along with co-expressed ligands or empty vector DNA. The heatmap and bar graph show the same data (desired OR gate behavior) (two-tailed Welch’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001; insignifcant results are not displayed). See Supplementary Figure 9b for all configurations evaluated. (c) Hybrid promoter AND gate schematic, implementation, and desired behavior. Each receptor releases a different synTF upon ligand binding, which can then bind to a cognate hybrid promoter and induces output gene expression when both inputs are present (IL-10 and VEGF). (d) Hybrid promoter AND gate functional evaluation. Two promoter architectures were integrated genomically into HEK293FT-LP cells and receptors were expressed by transfection of multi-gene expression vectors ( Supplementary Figure 9c-d ), along with co-expressed ligands or empty vector. Heatmaps and bar graphs show the same data, and each heatmap scale is normalized to the maximum value within that heatmap. Synergy values are shown below the plot and this metric is defined in Supplementary Note 7 . Some receptor-synTF pairings showed better synergy than others, although all displayed AND gate behavior (two-tailed Welch’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001; insignificant results are not displayed). See Supplementary Figure 9e for all configurations evaluated. (e) Split-intein synTF AND gate schematic, implementation, and desired behavior. Each receptor releases a different synTF upon ligand binding, which induces expression of one half of a split-intein synTF (activation domain fused to intN, DNA-binding domain fused to intC ), which then reconstitutes and induces output gene expression when both inputs are present (IL-10 and VEGF). (f) Split-intein synTF AND gate functional evaluation. Reporter setups were integrated genomically into HEK293FT-LP cells and receptors were expressed by transfection of multi-gene expression vectors ( Supplementary Figure 9c-d ), along with co-expressed ligands or empty vector. Heatmaps and bar graphs show the same data, and each heatmap scale is normalized to the maximum value within that heatmap. Some configurations showed better synergy than others, and most configurations displayed AND gate behavior (two-tailed Welch’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001; insignificant results are not displayed). See Supplementary Figure 9f for all configurations evaluated. The VEGF receptor pair used in this figure is a NTEVp receptor chain with VEGFR2 signal sequence, ECD and TMD, 75S NTEVp and a CTEVp receptor chain with VEGFR1 signal sequence, ECD and TMD, 190K CTEVp. The IL-10 receptor pair used in this figure is a NTEVp receptor chain with IgGVH signal sequence, IL-10Rb ECD and TMD, 75S NTEVp and a CTEVp receptor chain with IL-10Ra signal sequence, IL-10Ra ECD and TMD, 190K CTEVp. Bar graphs represent the mean of three biological replicates of transfected cells, and error bars depict standard error of the mean (S.E.M.). Abbreviations: MEPTRs, molecules of equivalent PE-TexasRed; Rep, reporter; synTF, synthetic transcription factor; PX, promoter design X; intN, N-terminal split intein fragment; intC, C-terminal split intein fragment; VEGF, vascular endothelial growth factor; IL-10, interleukin-10.

Article Snippet: Ligands were cloned into a pcDNA backbone to confer high expression in HEK293FT cells (Addgene #138749) .

Techniques: Ligand Binding Assay, Gene Expression, Functional Assay, Cotransfection, Plasmid Preparation, Two Tailed Test, Transfection, Expressing, Activation Assay, Binding Assay, Sequencing