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Biochemically-gated imaging of transcriptional activity. (a) Gated reporters leverage genetic AND logic to generate an output only when transcriptional activity ( T x ) is accompanied by an externally added drug (in this case, shield-1). (b) Doxycycline-dependent change in diffusivities of CHO cells expressing conventional DD-free Aqp1 under the control of a doxycycline-inducible minimal CMV promoter. (c) Doxycycline-driven changes in diffusivities of CHO cells expressing Aqp1-FKBP12-DD under the control of a doxycycline-inducible minimal CMV promoter following incubation with shield-1, doxycycline, and both ligands. (d) Representative difference image showing shield-1 gated imaging of transcriptional activity. The difference image was obtained through voxel-wise subtraction of diffusion-weighted datasets (effective b -value ∼1.6 ms μm −2 ) acquired in the presence of shield-1, doxycycline, or both from a dataset acquired in their absence. The resulting image was subsequently denoised using a median filter and presented as a pseudo-colored “hotspot.” Error bars represent the standard deviation ( n = 3–6). *** denotes P < 0.001. P -values were computed based on 2-sided, unpaired t -test (2b) or using one-way ANOVA followed by Tukey's HSD test (2c).

Journal: Chemical Science

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

doi: 10.1039/d4sc00377b

Figure Lengend Snippet: Biochemically-gated imaging of transcriptional activity. (a) Gated reporters leverage genetic AND logic to generate an output only when transcriptional activity ( T x ) is accompanied by an externally added drug (in this case, shield-1). (b) Doxycycline-dependent change in diffusivities of CHO cells expressing conventional DD-free Aqp1 under the control of a doxycycline-inducible minimal CMV promoter. (c) Doxycycline-driven changes in diffusivities of CHO cells expressing Aqp1-FKBP12-DD under the control of a doxycycline-inducible minimal CMV promoter following incubation with shield-1, doxycycline, and both ligands. (d) Representative difference image showing shield-1 gated imaging of transcriptional activity. The difference image was obtained through voxel-wise subtraction of diffusion-weighted datasets (effective b -value ∼1.6 ms μm −2 ) acquired in the presence of shield-1, doxycycline, or both from a dataset acquired in their absence. The resulting image was subsequently denoised using a median filter and presented as a pseudo-colored “hotspot.” Error bars represent the standard deviation ( n = 3–6). *** denotes P < 0.001. P -values were computed based on 2-sided, unpaired t -test (2b) or using one-way ANOVA followed by Tukey's HSD test (2c).

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, Activity Assay, Expressing, Control, Incubation, Diffusion-based Assay, Standard Deviation