Spatiotemporal Control of Gene Expression in Mammalian Cells and in Mice Using the LightOn System

Chen, Xianjun; Wang, Xue; Du, Zengmin; Ma, Zhengcai; Yang, Yi

doi:10.1002/9780470559277.ch120267

Cited by 28 publications

(24 citation statements)

References 54 publications

(115 reference statements)

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“…Optimization and characterization of TAEL, an optogenetic gene expression system for zebrafish Two LOV-based optogenetic expression systems, LightOn and EL222, were recently developed (Wang et al, 2012;Chen et al, 2013;Motta-Mena et al, 2014). We previously showed that the EL222 system could function in zebrafish embryos (Motta-Mena et al, 2014).…”

Section: Resultsmentioning

confidence: 99%

“…The LightOn system is based on a synthetically constructed transcriptional activator, GAVPO, consisting of the LOV domain from the Neurospora protein Vivid fused to both a Gal4 DNA-binding domain and a p65 transactivating domain. Upon light-induced dimerization, GAVPO binds to its corresponding UAS promoter and induces transcription of a gene of interest (Wang et al, 2012;Chen et al, 2013). The EL222 system relies on similar principles but is based on a naturally occurring light-responsive transcription factor, EL222, found in Erythrobacter litoralis.…”

Section: Introductionmentioning

confidence: 99%

“…Several optogenetic gene expression systems have been developed (Shimizu-Sato et al, 2002;Yazawa et al, 2009;Kennedy et al, 2010;Ye et al, 2011;Ohlendorf et al, 2012;Gersbach, 2012, 2015;Liu et al, 2012;Wang et al, 2012;Chen et al, 2013;Motta-Mena et al, 2014), but so far, these systems are non-optimal for use in zebrafish. An ideal zebrafish optogenetic system would be genetically encoded, not require complicated optics or exogenous small molecules, have a large range of induction, be reversible with fairly rapid kinetics and, importantly, have little to no toxicity.…”

Section: Introductionmentioning

confidence: 99%

“…Two recently reported light-gated transcriptional systems, LightOn (Wang et al, 2012;Chen et al, 2013) and EL222 (Motta-Mena et al, 2014), are promising candidates for spatiotemporal control of transcription in zebrafish. Both systems make use of an engineered light-oxygen-voltage (LOV) protein that dimerizes when illuminated with blue light (Crosson et al, 2003;Zoltowski et al, 2013;Fig.…”

Section: Introductionmentioning

confidence: 99%

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TAEL: A zebrafish-optimized optogenetic gene expression system with fine spatial and temporal control

et al. 2016

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Here, we describe an optogenetic gene expression system optimized for use in zebrafish. This system overcomes the limitations of current inducible expression systems by enabling robust spatial and temporal regulation of gene expression in living organisms. Because existing optogenetic systems show toxicity in zebrafish, we re-engineered the blue-light-activated EL222 system for minimal toxicity while exhibiting a large range of induction, fine spatial precision and rapid kinetics. We validate several strategies to spatially restrict illumination and thus gene induction with our new TAEL (TA4-EL222) system. As a functional example, we show that TAEL is able to induce ectopic endodermal cells in the presumptive ectoderm via targeted sox32 induction. We also demonstrate that TAEL can be used to resolve multiple roles of Nodal signaling at different stages of embryonic development. Finally, we show how inducible gene editing can be achieved by combining the TAEL and CRISPR/Cas9 systems. This toolkit should be a broadly useful resource for the fish community.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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TAEL: A zebrafish-optimized optogenetic gene expression system with fine spatial and temporal control

et al. 2016

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“…Second, we tested whether the ability to spatially focus activation can improve the method. Stimulation of optogenetic tools is often realized using custom-built hardware and continuous illumination [15][16][17] . However, microplate readers commonly part of screening platforms employ discontinuous flash lamps.…”

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confidence: 99%

Light-assisted small-molecule screening against protein kinases

et al. 2015

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High-throughput live-cell screens are intricate elements of systems biology studies and drug discovery pipelines. Here, we demonstrate an optogenetics-assisted method that obviates the addition of chemical activators and reporters, reduces the number of operational steps and increases information content in a cell-based small molecule screen against human protein kinases including an orphan receptor tyrosine kinase. This blueprint for all-optical screening can be adapted to many drug targets and cellular processes.Over the past decades, many chemical processes have been improved by replacing additives, such as catalysts, initiators or emulsifiers, with physical stimuli, such as light or ultrasound [1][2][3][4] . Although replacement results in reduced cost, increased robustness and improved sustainability, this general principle has not found many adaptations in chemical biology. Automated screens using living cells are essential in the identification and characterization of small molecules that act on disease-related proteins and cellular pathways. However, in many cell-based screens the need to add reagents that alter or report on cell activity results in complex operational design, high cost and sources of error. Furthermore, mammalian cells are sensitive to environmental perturbations (e.g. temperature or ionic strength) and subject to inherent variability. In neurobiology and cell biology, optogenetics and photopharmacology have recently harnessed the power of light to Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use

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Near‐Infrared Nano‐Optogenetic Activation of Cancer Immunotherapy via Engineered Bacteria

Zhu

Chen

et al. 2022

Advanced Materials

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Certain obligate and facultative anaerobic microbes preferentially grow within this region, [2,3] which makes them ideal platforms for delivering antitumor therapeutics. [4,5] Different from conventional nano-drug delivery systems, bacteria-based drug carriers can be designed to generate and release drugs, which avoids the need for cumbersome post-purification or delivery protection. [6] However, bacteria sometimes can grow outside their natural niches, leading to offtarget therapeutics release that may incur severe toxicities toward normal tissues. [7,8] Therefore, it is crucial to "tune" the bacteria for precise and on-demand release of the therapeutics at the tumor site so as to minimize the side effects as well as promote antitumor therapeutic efficacy.Spatial and temporal activation of inducible or repressible gene expression systems facilitate precise gene expression as required. Ideally, programming bacteria with the capability to rapidly and precisely switch between "ON" and "OFF" states at will, would sense and respond to the physiological or pathological conditions upon utilization of certain stimuli. [9] Typically, exogenous chemical inducers, such as antibiotics, were used to achieve artificial control of gene expression in live cells. [10] However, it is very difficult to remove the residual chemical inducers, which limited the precise control of gene expression at desired levels. Light, as an external inducer, possesses high spatiotemporal control ability, non-invasiveness, and minimal cytotoxicity. Therefore, light-regulated modules have attracted wide attention for controlling molecular or cellular behavior. Among them, optogenetics combines optics and genetics in technology to rapidly activate/deactivate photo-sensitive proteins, which shows great potential in the modulation of neural activities, gene transcription, and the regulation of cellular processes within organisms. [11] Most of these applied optogenetics are blue light (BL)-based control systems, including CRY2/CIB, [12] VVD, [13] EL222, [14,15] and magnet systems. [16] However, a major hurdle in these optogenetics systems stems from poor BL penetration to turbid human tissues and potential phototoxicity. It was also noted that some red/far-red light-responsive optogenetic devices, phyB/PIF1/SPA1, [17] BphP1/PpsR2 [18] and BphS, [19] have been developed to control transgene expression. However, the in Certain anaerobic microbes with the capability to colonize the tumor microenvironment tend to express the heterologous gene in a sustainable manner, which will inevitably compromise the therapeutic efficacy and induce off-tumor toxicity in vivo. To improve the therapeutic precision and controllability of bacteriabased therapeutics, Escherichia coli Nissle 1917 (EcN), engineered to sense blue light and release the encoded flagellin B (flaB), is conjugated with lanthanide upconversion nanoparticles (UCNPs) for near-infrared (NIR) nano-optogenetic cancer immunotherapy. Upon 808 nm photoirradiation, UCNPs emit at the blue region to photoactivat...

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Spatiotemporal Control of Gene Expression in Mammalian Cells and in Mice Using the LightOn System

Cited by 28 publications

References 54 publications

TAEL: A zebrafish-optimized optogenetic gene expression system with fine spatial and temporal control

TAEL: A zebrafish-optimized optogenetic gene expression system with fine spatial and temporal control

Light-assisted small-molecule screening against protein kinases

Near‐Infrared Nano‐Optogenetic Activation of Cancer Immunotherapy via Engineered Bacteria

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