Journal: bioRxiv

Article Title: A tunable dual-input system for ‘on-demand’ dynamic gene expression regulation

doi: 10.1101/404699

Figure Lengend Snippet: (a-d) Dual-input regulation system consisting of a reverse transactivator (rtTA) and stable (a) or conditionally destabilised (c) mCherry fluorescent protein. mRNA (b, d, left panels) and protein levels (b, d, right panels) measured in EF1a-rtTA_TRE3G-mCherry (b) and EF1a-rtTA_TRE3G-DDmCherry (d) mESCs treated for 24hrs with Doxy (1000ng/mL) or Doxy/TMP (1000ng/mL and 100nM, respectively). (e) Experimental scheme of protein half-life measurement experiments. Following 14hrs of treatment with Doxy (1000ng/mL) or Doxy/TMP (1000ng/mL and 100nM, respectively), EF1a-rtTA_TRE3G-mCherry and EF1a-rtTA_TRE3G-DDmCherry mESCs were cultured in presence of the protein synthesis inhibitor cycloheximide (CHX, 25μg/mL) and combination of Doxy (1000ng/mL), TMP (100nM) and Doxy/TMP. Protein half-life was measured by SDS-PAGE and western-blotting after the indicated times of treatment. (f, g) Western-blot densitometric quantification of EF1a-rtTA_TRE3G-mCherry (f) and EF1a-rtTA_TRE3G-DDmCherry (g) mESCs. Data are means ± SEM (n = 2, b and d ; n=3, f and g ). p > 0.1, *p < 0.05, **p < 0.01,***p < 0.0001.

Article Snippet: Following manufacturer’s instructions, fragments were assembled into pLVX_TRE3G Puro (Clontech) and linearized with SmaI (NEB) using HiFi-DNA assembly cloning kit (NEB). pLVX_TRE3G-DDmCherry is available on Addgene (Plasmid #108679).

Techniques: Cell Culture, SDS Page, Western Blot

Journal: bioRxiv

Article Title: A tunable dual-input system for ‘on-demand’ dynamic gene expression regulation

doi: 10.1101/404699

Figure Lengend Snippet: (a-d) mCherry and DDmCherry protein (a, b) and mRNA (c, d) levels measured by western-blot and qPCR, respectively, in EF1a-rtTA_TRE3G-mCherry (a, c) and EF1a-rtTA_TRE3G-DDmCherry (b, d) . mESCs were treated as indicated in and relative legend. (e) EF1a-rtTA_TRE3G-DDmCherry mESCs were incubated with Doxy (1000ng/mL) and either TMP (10μM) or MG132 (5μM) for 16hrs. Blocking proteosomal degradation enabled to effectively mimic the stabilising effect of TMP specifically on DDmCherry but not on endogenous β-catenin. Western-blot densitometric quantifications are shown. (f) Ubiquitination status analysis. EF1a-rtTA_TRE3G-DDmCherry mESCs were incubated for 24hrs with Doxy (1000ng/mL) and TMP (100nM), washed and incubated with or without TMP (100nM) for additional 12hrs. Samples were processed as indicated in the Materials and methods section, immunoprecipitated with an anti-ubiquitin antibody and blotted with the mCherry antibody to analyse the ubiquitination status of the DDmCherry protein. Note the decrease in poly-ubiquitinated species of DDmCherry when TMP is present. Data are means ± SEM (n=3). p > 0.1, *p < 0.05, **p < 0.01, ***p < 0.0001.

Article Snippet: Following manufacturer’s instructions, fragments were assembled into pLVX_TRE3G Puro (Clontech) and linearized with SmaI (NEB) using HiFi-DNA assembly cloning kit (NEB). pLVX_TRE3G-DDmCherry is available on Addgene (Plasmid #108679).

Techniques: Western Blot, Incubation, Blocking Assay, Immunoprecipitation

Journal: bioRxiv

Article Title: A tunable dual-input system for ‘on-demand’ dynamic gene expression regulation

doi: 10.1101/404699

Figure Lengend Snippet: (a) mCherry protein levels in EF1a-rtTA_TRE3G-mCherry mESCs following 24hrs of treatment with increasing concentration of Doxy (0; 1; 5; 50; 100; 500; 1000ng/mL). (b, c) mCherry protein levels measured in EF1a-rtTA_TRE3G-DDmCherry following 24hrs of treatment with increasing concentration of Doxy (0; 1; 5; 50; 100; 500; 1000ng/mL) and saturated TMP (10μM) (b), or increasing concentration of TMP (0; 1; 10; 100nM; 1; 10μM) and saturated Doxy (1000ng/mL) (c) . (d, e) Dynamic response of EF1a-rtTA_TRE3G-mCherry (d) and EF1a-rtTA_TRE3G-DDmCherry (e) mESCs to Doxy (1000ng/mL) or Doxy/TMP (1000ng/mL and 100nM, respectively) in a time-course experiment of 38hrs, in which inducers were washed out after incubation in the first 14hrs. The dots are experimental data of MFI measured by flow cytometry and normalised over the maximum activation point. Dashed lines represent model fitting of steady-state data (a-c) or model prediction of switch-off dynamics (d, e) . (f) Simulated steady-state of EF1a-rtTA_TRE3G-DDmCherry mESCs upon combined treatment with Doxy and TMP at various concentrations. mCherry values are shown as heatmaps of scaled values across the entire dynamic range of expression levels. (g) Experimental validation of simulations in (f) , measuring EF1a-rtTA_TRE3G-DDmCherry mESC steady-state following 24hrs induction with constant concentration of Doxy (100ng/mL) and varying TMP (0; 10nM; 10μM), or constant TMP (10nM) and varying Doxy (0; 50; 1000ng/mL). Maximum concentrations of Doxy and TMP (1000mg/mL and 10μM, respectively) are used as control. Data are means ± SD (n=3, a-f ); ± SEM (n=3, g ).

Article Snippet: Following manufacturer’s instructions, fragments were assembled into pLVX_TRE3G Puro (Clontech) and linearized with SmaI (NEB) using HiFi-DNA assembly cloning kit (NEB). pLVX_TRE3G-DDmCherry is available on Addgene (Plasmid #108679).

Techniques: Concentration Assay, Incubation, Flow Cytometry, Activation Assay, Expressing

Journal: bioRxiv

Article Title: A tunable dual-input system for ‘on-demand’ dynamic gene expression regulation

doi: 10.1101/404699

Figure Lengend Snippet: (a-d, m-p) Median Fluorescence Intensity (MFI) and % of mCherry+ cells measured by flow cytometry in EF1a-rtTA_TRE3G-mCherry (a, c) , EF1a-rtTA_TRE3G-DDmCherry (b, d) , EF1a-DDrtTA_TRE3G-mCherry (m, o) and EF1a-DDrtTA_TRE3G-DDmCherry (n, p) mESCs. Inducer titration and dynamic response were performed using concentrations and incubation times indicated in the tables. (e,f) Cartoon of the designed dual input system consisting of conditionally destabilised transactivator (DDrtTA) driving a stable (e) or a conditionally destabilised (f) mCherry fluorescent protein. Protein expression following 24hrs Doxy/TMP treatment (1000ng/mL and 100nM, respectively) in EF1a-DDrtTA_TRE3G-mCherry ( e, right panel) and EF1a-DDrtTA_TRE3G-DDmCherry (f, right panel) mESCs. (g-j) Fitted model simulations (dashed lines) and experimental data (dots) of EF1a-DDrtTA_TRE3G-mCherry (g, h) and EF1a-DDrtTA_TRE3G-DDmCherry (i, j) mESC steady-state response. Dots represent experimental data of MFI in , normalised over the maximum activation point. (k, l) Model predicted dynamic response (dashed lines) and experimental data (dots) of EF1a-DDrtTA_TRE3G-mCherry (k) and EF1a-DDrtTA_TRE3G-DDmCherry (l) mESCs. Dots represent experimental data of MFI in , normalised over the maximum activation point. (q-t) Simulations of the model (q, s) and experimental validation (r, t) of EF1a-DDrtTA_TRE3G-mCherry (q, r) and EF1a-DDrtTA_TRE3G-DDmCherry (s, t) mESC steady-state response following 24hrs induction with constant concentration of Doxy (100ng/mL) and varying TMP (0; 10nM; 10μM) or constant TMP (10nM) and varying Doxy (0; 50; 1000ng/mL). Maximum concentrations of Doxy and TMP (1000mg/mL and 10μM, respectively) are used as control. mCherry values are shown as heatmaps of scaled values across the entire dynamical range of expression levels. Data are means ±SEM (n=3, a-d, m-p, r, t); ±SD (n=3, g-l, q, s).

Article Snippet: Following manufacturer’s instructions, fragments were assembled into pLVX_TRE3G Puro (Clontech) and linearized with SmaI (NEB) using HiFi-DNA assembly cloning kit (NEB). pLVX_TRE3G-DDmCherry is available on Addgene (Plasmid #108679).

Techniques: Fluorescence, Flow Cytometry, Titration, Incubation, Expressing, Activation Assay, Concentration Assay

Journal: bioRxiv

Article Title: A tunable dual-input system for ‘on-demand’ dynamic gene expression regulation

doi: 10.1101/404699

Figure Lengend Snippet: ( a, left panel ) Cartoon of the designed dual-input control system consisting of a reverse transactivator (rtTA) and conditionally destabilised and constitutively active β-catenin (β-catenin S33Y ), fused to the mCherry fluorescent protein. (a, right panel) Representative flow cytometry profile of Doxy and/or TMP treated EF1a-rtTA_TRE3G-DDmCherryβ-catenin S33Y mESCs. (b) DDmCherryβ-catenin S33Y protein levels measured by western-blot in EF1a-rtTA_TRE3G-DDmCherryβ-catenin S33Y mESCs treated with Doxy and/or TMP; mCherry ( left panel ) and β-catenin ( right panel ) antibodies were used. * indicates a non-specific band. (c, d) β-catenin immunostaining in EF1a-rtTA_TRE3G-DDmCherryβ-catenin S33Y mESCs treated for 24hrs with TMP (c) or Doxy/TMP (d) , using mCherry (red signal) and β-catenin (green signal) antibodies. DAPI was used to stain the nuclei. Zoomed pictures of selected clones are shown. Scale bars 25μm. (a-d) Doxy and TMP were used at 1000ng/mL and 100nM, respectively. (e) Set-point control experiment, using inducers (Doxy 100ng/mL and TMP 10nM) as control inputs. In red the measured output (normalised mCherry fluorescence), in blue the reference fluorescence, set at 50% of the average values measured during the calibration phase (120mins with continuous Doxy/TMP administration). The time-lapse sampling time was set at 60mins. Cells received either media with both inducers when the measured fluorescence is below the control reference or plain media when the measured fluorescence is above the control reference. Details about microfluidics device, segmentation and control algorithms, and live cell imaging setting, are provided in Supplementary Information.

Article Snippet: Following manufacturer’s instructions, fragments were assembled into pLVX_TRE3G Puro (Clontech) and linearized with SmaI (NEB) using HiFi-DNA assembly cloning kit (NEB). pLVX_TRE3G-DDmCherry is available on Addgene (Plasmid #108679).

Techniques: Flow Cytometry, Western Blot, Immunostaining, Staining, Clone Assay, Fluorescence, Sampling, Live Cell Imaging

Journal: bioRxiv

Article Title: A tunable dual-input system for ‘on-demand’ dynamic gene expression regulation

doi: 10.1101/404699

Figure Lengend Snippet: (a, b) Exogenous β-catenin mRNA levels measured by qPCR in EF1a-rtTA_TRE3G-DDmCherryβ-catenin S33Y mESCs induced with Doxy (1000ng/mL) and/or TMP (100nM) for 24hrs. mCherry (a) and β-catenin (b) specific primers were used. (c, d) β-catenin immunostaining in EF1a-rtTA_TRE3G-DDmCherryβ-catenin S33Y mESCs treated for 24hrs with Doxy (1000ng/mL) and/or TMP (100nM); mCherry (c) and β-catenin (d) antibodies were used. DAPI was used to stain the nuclei. Zoomed pictures of selected cells are shown. Scale bars 25μm. (e) Western-blot of nuclear and cytosolic fractions from Doxy/TMP (1000ng/mL and 100nM, respectively) treated EF1a-rtTA_TRE3G-DDmCherryβ-catenin S33Y mESCs, blotted with an anti-mCherry antiboby. (f) Set-point control experiment using inducers (Doxy 1000ng/mL and TMP 100nM) as control inputs. In red the measured output (normalised mCherry fluorescence), in blue the control reference fluorescence, set at 50% of the average value measured during the calibration phase (120mins with continuous Doxy/TMP administration). Time-lapse sampling time of 60mins. Cells received either media with both inducers when the measured fluorescence is below the reference, or plain media when the measured fluorescence is above the reference. (g) Median Fluorescence Intensity (MFI) and % of mCherry+ cells measured by flow cytometry in EF1a-rtTA_TRE3G-DDmCherryβ-catenin S33Y mESCs after 24hrs of treatment with indicated concentration of Doxy and TMP. Data are means ± SEM (n=2). p > 0.1, *p < 0.05, **p < 0.01, ***p < 0.0001.

Article Snippet: Following manufacturer’s instructions, fragments were assembled into pLVX_TRE3G Puro (Clontech) and linearized with SmaI (NEB) using HiFi-DNA assembly cloning kit (NEB). pLVX_TRE3G-DDmCherry is available on Addgene (Plasmid #108679).

Techniques: Immunostaining, Staining, Western Blot, Fluorescence, Sampling, Flow Cytometry, Concentration Assay