e coli dhfr  (Addgene inc)

Journal: Chemical Science

Article Title: Destabilized reporters for background-subtracted, chemically-gated, and multiplexed deep-tissue imaging †

doi: 10.1039/d4sc00377b

Figure Lengend Snippet: Biochemical modulation of Aqp1 for background-subtracted imaging. (a) Percentage change in the diffusivities of CHO cells engineered to express ligand-activated Aqp1-DDs, following a 24 hour incubation with the respective ligand. Ligands include indole-3-acetic acid (IAA), dTAG-13, and shield-1 (shld1) which bind to degrons based on full-length (228 amino acids) and truncated (68 amino acids) forms of the plant protein, IAA17; the F36V mutant of the mammalian prolyl isomerase, FKBP12 (denoted as FK12 for brevity); and FKBP12 F36V modified to incorporate a 19-amino acid cryptic degron (denoted as FK12 (ref. )). (b) Percentage change in the diffusivities of CHO cells that have been engineered to express ligand-stabilized Aqp1-DDs, following a 24 hour incubation with the respective ligand. Ligands include 4-hydroxytamoxifen (4HT), trimethoprim (TMP), and shield-1, which bind to DDs based on the estrogen receptor ligand-binding domain (ER), e. coli dihydrofolate reductase (DH), and the F36V/L106P double mutant of FKBP12 (FK12). (c) Time-dependent increase in the diffusivity of Aqp1-FKBP12-DD transduced CHO and U87 cells upon exposure to shield-1. (d) Relative viability of cells following 24 hour incubation with 1 μM shield-1. (e) Percent change in the diffusivities of various cell lines engineered to express Aqp1-FKBP12-DD, following a 24 hour incubation with 1 μM shield-1. (f) Percent change in the diffusivities of cell lines engineered to express Aqp1 N-terminally fused to FKBP12-DD, following a 24 hour incubation with 1 μM shield-1. (g) Western blotting of cell extracts obtained from Aqp1-FKBP12-DD expressing CHO cells in the absence or presence of shield-1 treatment. Aqp1 was detected using antibodies targeting a FLAG epitope inserted at its N -terminus. The Na + /K + -ATPase pump (100 kDa) served as a loading control. (h) Immunofluorescence imaging of Aqp1-FKBP12-DD expressing CHO cells in the presence and absence of shield-1 treatment. Scale bar is 5 μm. Detection was performed using antibodies targeting a FLAG epitope inserted at the N -terminus of Aqp1. (i) Representative image of CHO cells expressing DD-free Aqp1 and Aqp1-FKBP12-DD that have undergone background subtraction. The difference image was produced by subtracting voxel-wise diffusion-weighted datasets (effective b -value ∼3 ms μm −2 ) acquired with and without shield-1 incubation, denoising the resulting image using a median filter, and displaying it as a pseudo colored “hotspot.” Error bars represent the standard deviation ( n ≥ 3). * denotes P < 0.05, ** denotes P < 0.01, *** denotes P < 0.001, and n.s. is non-significant ( P ≥ 0.05). P -values were determined using unpaired, 2-sided t -test. For the time-series data, P -values were computed through one-way ANOVA followed by Tukey's HSD test.

Article Snippet: Plasmids harboring the various degron sequences – DHFR-DD (Addgene 29326), ER-DD (Addgene 37261), miniIAA7 (Addgene 129721), and FKBP12 (Addgene 17416) were amplified using Q5 High-Fidelity 2× Master Mix and cloned by Gibson assembly in a lentiviral transfer vector at the C or N -terminus of the aquaporin-1 reporter (Aqp1) under the control of either a constitutive promoter, EF1α (Addgene 60058) or a doxycycline-inducible minimal CMV promoter (Addgene 26431).

Techniques: Imaging, Incubation, Mutagenesis, Modification, Ligand Binding Assay, Western Blot, Expressing, FLAG-tag, Control, Immunofluorescence, Produced, Diffusion-based Assay, Standard Deviation

Journal: bioRxiv

Article Title: Engineering ligand stabilized aquaporin reporters for magnetic resonance imaging

doi: 10.1101/2023.06.02.543364

Figure Lengend Snippet: a , Fusing ligand-induced degradation (LID) tags to the C-terminus of Aqp1 generates modest changes in the diffusion coefficients of ligand-treated CHO cells relative to vehicle-treated controls. b , In contrast, fusing ligand-induced stabilization (LIS) tags to the C-terminus generates substantial changes in the diffusivities of ligand-treated CHO cells relative to controls. The largest response was observed in cells expressing Aqp1 fused to the FKBP12F36V/L106P degron (hereafter, the chimeric construct is referred to as LSAqp1), which was stabilized by a small-molecule, shield-1 (denoted as sh-1 in figures). c , Time-dependent increase in diffusion rate of LSAqp1-transduced cells following treatment with shield-1 (1 µM). d , Treatment of LSAqp1-transduced cells with shield-1 did not result in acute toxicity, as determined by MTT and ATP-based viability assays. e , Shield-1 modulation is specific to LSAqp1 and does not affect the baseline diffusivity of wild type cells or the enhanced diffusivity of red blood cells or cells engineered to express aquaporin-1. f , LSAqp1 permits small-molecule modulation of diffusivity in multiple cell types. g , Immunoblotting with anti-FLAG antibodies reveals a band of the expected size of LSAqp1 in membrane fractions extracted from shield-1 treated CHO cells (lane 2), which was only faintly observed in extracts obtained from vehicle-treated cells (lane 1). h , Confocal microscopy using anti-FLAG antibodies revealed membrane-localized LSAqp1 in shield-1 treated, but not vehicle-treated CHO cells. Images of both cells under both treatment conditions were acquired using identical optical settings (see Methods) and were displayed on the same color-scale. Scale bar is 5 µm. i , Background-free MRI with LSAqp1. Differential imaging was performed by voxel-wise subtraction of diffusion-weighted images acquired with and without shield-1 treatment. The ensuing image was denoised using a median filter and displayed as a pseudo-colored “hotspot”. Error bars represent the standard deviation ( n ≥ 3 biological replicates). * denotes P < 0.05, ** denotes P < 0.01, *** denotes P < 0.005, and n.s. is non-significant ( P ≥ 0.05). P -values were computed based on 2-sided Student’s t-test (unpaired).

Article Snippet: Plasmids harboring the various degron sequences - DHFR (Addgene 29326), ER (Addgene 37261), miniIAA7 (Addgene 129721), SMASh (Addgene 68853), and FKBP12 (Addgene 17416) were amplified using Q5 High-Fidelity 2X Master Mix and cloned by Gibson assembly in a lentiviral transfer vector at the C or N-terminus of the aquaporin-1 reporter (Aqp1) under the control of either a constitutive promoter, EF1α (Addgene 60058) or a doxycycline-inducible minimal CMV promoter (Addgene 26431).

Techniques: Diffusion-based Assay, Expressing, Construct, Western Blot, Membrane, Confocal Microscopy, Imaging, Standard Deviation

Journal: bioRxiv

Article Title: Engineering ligand stabilized aquaporin reporters for magnetic resonance imaging

doi: 10.1101/2023.06.02.543364

Figure Lengend Snippet: a , LSAqp1 (responsive to shield-1, sh-1), Aq-dhfr (stabilized by trimethoprim, TMP), and Aq-ER (responsive to 4-hydroxytamoxifen, HT) are mutually orthogonal, because treatment with non-cognate ligands does not increase the diffusion coefficients of CHO cells transduced with the respective reporter construct. b , Ligand-dependent increase in diffusion (relative to vehicle-treated cells) in a mixed population comprising equal numbers of LSAqp1- and Aq-dhfr transduced cells. Ligand addition permits the stepwise modulation of diffusivity by stabilizing one or both degron-tagged Aqp1 constructs. c , Components of the mixed-cell population were resolved by differential imaging. Subtraction of diffusion-weighted images of untreated cells from images acquired after treatment with shield-1 reveals the LSAqp1 population. Likewise, subtracting diffusion-weighted images of shield-1 treated cells from those obtained after treatment with shield-1 and TMP revealed the Aq-dhfr population. The images were denoised by median filtering and pseudo-colored to distinguish the LSAqp1- and Aq-dhfr-expressing sub-populations. d , Ligand-dependent increase in the diffusion coefficient of a mixed population comprising U87 and Jurkat cells transduced respectively with LSAqp1 and Aq-ER. As before, ligand addition permits the stepwise modulation of diffusion. e , The two cell types can be distinguished by differential imaging after sequential treatment with ligands. The images were denoised by median filtering and pseudo-colored to distinguish between the two cell-types. Error bars represent standard deviation ( n ≥ 4 biological replicates).

Article Snippet: Plasmids harboring the various degron sequences - DHFR (Addgene 29326), ER (Addgene 37261), miniIAA7 (Addgene 129721), SMASh (Addgene 68853), and FKBP12 (Addgene 17416) were amplified using Q5 High-Fidelity 2X Master Mix and cloned by Gibson assembly in a lentiviral transfer vector at the C or N-terminus of the aquaporin-1 reporter (Aqp1) under the control of either a constitutive promoter, EF1α (Addgene 60058) or a doxycycline-inducible minimal CMV promoter (Addgene 26431).

Techniques: Diffusion-based Assay, Transduction, Construct, Imaging, Expressing, Standard Deviation