Simultaneous Examination of Cellular Pathways using Multiplex Hextuple Luciferase Assaying

Sarrión-Perdigones, Alejandro; Chang, Lyra; Gonzalez, Yezabel; Gallego-Flores, Tatiana; Young, Damian W.

doi:10.1002/cpmb.122

Cited by 6 publications

(5 citation statements)

References 25 publications

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“…Others have previously described the application of two or more spectrally distinct bioluminescent colors to simultaneously image multiple independent biological parameters in mouse models. 14,30,31 Although technically impressive, none of the multi-parametric measures are internally controlled, and so the signal from each one is potentially prone to influence from any of the various factors that affect light production. Unlike the well-defined and spectrally tight light emission typical of fluorophores, the emission wavelengths of CBG99 and PRE9 are broad and overlap considerably.…”

Section: ■ Discussionmentioning

confidence: 99%

“…Named GemLuc and OxiLuc (for detection of changes in the cell cycle or in levels of OS, respectively), the functional signal is corrected from substantial experimental variability by a constitutively expressed control color that metabolizes the same substrate. Others have previously described the application of two or more spectrally distinct bioluminescent colors to simultaneously image multiple independent biological parameters in mouse models. ,, Although technically impressive, none of the multi-parametric measures are internally controlled, and so the signal from each one is potentially prone to influence from any of the various factors that affect light production.…”

Section: Discussionmentioning

confidence: 99%

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Internally Controlled and Dynamic Optical Measures of Functional Tumor Biology

et al. 2023

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Imaging defined aspects of functional tumor biology with bioluminescent reporter transgenes is a popular approach in preclinical drug development as it is sensitive, relatively high-throughput and low cost. However, the lack of internal controls subject functional bioluminescence to a number of unpredictable variables that reduce this powerful tool to semi-quantitative interpretation of large-scale effects. Here, we report the generation of sensitive and quantitative live reporters for two key measures of functional cancer biology and pharmacologic stress: the cell cycle and oxidative stress. We developed a two-colored readout, where two independent enzymes convert a common imaging substrate into spectrally distinguishable light. The signal intensity of one color is dependent upon the biological state, whereas the other color is constitutively expressed. The ratio of emitted colored light corrects the functional signal for independent procedural variables, substantially improving the robustness and interpretation of relatively low-fold changes in functional signal intensity after drug treatment. The application of these readouts in vitro is highly advantageous, as peak cell response to therapy can now be readily visualized for single or combination treatments and not simply assessed at an arbitrary and destructive timepoint. Spectral imaging in vivo can be challenging, but we also present evidence to show that the reporters can work in this context as well. Collectively, the development and validation of these internally controlled reporters allow researchers to robustly and dynamically visualize tumor cell biology in response to treatment. Given the prevalence of bioluminescence imaging, this presents significant and much needed opportunities for preclinical therapeutic development.

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Section: ■ Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Internally Controlled and Dynamic Optical Measures of Functional Tumor Biology

et al. 2023

View full text Add to dashboard Cite

show abstract

“…Named GemLuc and OxiLuc (for detection of changes in the cell cycle or in levels of oxidative stress respectively), the functional signal is corrected from substantial experimental variability by a constitutively expressed control color that metabolizes the same substrate. Others have previously described the application of two or more spectrally distinct bioluminescent colors to simultaneously image multiple independent biological parameters in mouse models [14,28,29]. Although technically impressive, none of the multi-parametric measures are internally controlled and so the signal from each one is potentially prone to influence from any of the various factors that influence light production.…”

Section: Discussionmentioning

confidence: 99%

Internally-controlled and dynamic optical measures of functional tumor biology

Chung

Garcia

Swamynathan

et al. 2022

Preprint

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Imaging defined aspects of functional tumor biology with bioluminescent reporter transgenes is a popular approach amongst the research community in drug development, as it is sensitive, relatively high-throughput and low cost. However, the lack of internal controls subject functional bioluminescence to a number of unpredictable variables that reduce this powerful tool to semi-quantitative interpretation of large-scale effects. Here we report the generation of sensitive and quantitative live reporters for two key measures of functional cancer biology and pharmacologic stress: the cell cycle and oxidative stress. We developed a two-colored readout, where two independent enzymes convert a common imaging substrate into spectrally distinguishable light. The signal intensity of one color is dependent upon biological state, whereas the other color is constitutively expressed. The ratio of emitted colored light corrects the functional signal for independent procedural variables, substantially improving the robustness and interpretation of relatively low-fold changes in functional signal intensity after drug treatment. The application of these readouts in vitro is highly advantageous, as peak cell response to therapy can now be readily visualized for single or combination treatments and not simply assessed at an arbitrary and destructive timepoint. Spectral imaging in vivo can be challenging, but we also present evidence to show that the reporters can work in this context as well. Collectively, the development and validation of these internally controlled reporters allow researchers to robustly and dynamically visualize tumor cell biology in response to treatment. Given the prevalence of bioluminescence imaging, this presents significant and much needed opportunities for preclinical therapeutic development.

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“…The fluorescent proteins are limited to about four to five colors for a simultaneous measurement due to their broad emission spectra 14 . As for luciferases, although emission filters allow multiple measurements up to around six 15,16 , the number of available substrates without cross-reactivity is low. The most commonly used substrates are D-luciferin (for Firefly luciferase) and coelenterazine (for Renilla luciferase).…”

Section: Introductionmentioning

confidence: 99%

Expanding luciferase reporter systems for cell-free protein expression

Sato

Rasmussen

Deich

et al. 2022

Preprint

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Luciferases are often used as a sensitive, versatile reporter in cell-free transcription-translation (TXTL) systems, for research and practical applications such as engineering genetic parts, validating genetic circuits, and biosensor outputs. Currently, only two luciferases (Firefly and Renilla) are commonly used without substrate cross-talk. Here we demonstrate expansion of the cell-free luciferase reporter system, with two orthogonal luciferase reporters: N. nambi luciferase (Luz) and LuxAB. These luciferases do not have cross-reactivity with the Firefly and Renilla substrates. We also demonstrate a substrate regeneration pathway for one of the new luciferases, enabling long-term time courses of protein expression monitoring in the cell-free system. Furthermore, we reduced the number of genes required in TXTL expression, by engineering a cell extract containing part of the luciferase enzymes. Our findings lead to an expanded platform with multiple orthogonal luminescence translation readouts for in vitro protein expression.

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Simultaneous Examination of Cellular Pathways using Multiplex Hextuple Luciferase Assaying

Cited by 6 publications

References 25 publications

Internally Controlled and Dynamic Optical Measures of Functional Tumor Biology

Internally Controlled and Dynamic Optical Measures of Functional Tumor Biology

Internally-controlled and dynamic optical measures of functional tumor biology

Expanding luciferase reporter systems for cell-free protein expression

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