scholarly journals Spectral recording of gene expression history by fluorescent timer protein

BioTechniques ◽  
2019 ◽  
Vol 67 (4) ◽  
pp. 154-164
Author(s):  
Anna R Tröscher ◽  
Barbara Werner ◽  
Nadia Kaouane ◽  
Wulf Haubensak
Keyword(s):  
Gene Expression ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Temporal Patterns ◽  
Spectral Shift ◽  
Early Gene ◽  
Proof Of Concept ◽  
Spatio Temporal ◽  
Living Organisms ◽  
Post Hoc

Monitoring spatio-temporal patterns of gene expression by fluorescent proteins requires longitudinal observation, which is often difficult to implement. Here, we fuse a fluorescent timer (FT) protein with an immediate early gene (IEG) promoter to track live gene expression in single cells. This results in a stimulus- and time-dependent spectral shift from blue to red for subsequent monitoring with fluorescence activated cell sorting (FACS) and live cell imaging. This spectral shift enables imputing the time point of activity post-hoc to dissociate early and late responders from a single snapshot in time. Thus, we provide a tool for tracking stimulus-driven IEG expression and demonstrate proof of concept exploiting promoter::FT fusions, adding new dimensions to experiments that require reconstructing spatio-temporal patterns of gene expression in cells, tissues or living organisms.

scholarly journals Novel tunable spatio-temporal patterns from a simple genetic oscillator circuit

2020 ◽  
Author(s):  
Guillermo Yáñez Feliú ◽  
Gonzalo Vidal ◽  
Macarena Muñoz Silva ◽  
Timothy J. Rudge
Keyword(s):  
Gene Expression ◽  
Growth Rate ◽  
Protein Degradation ◽  
Rational Design ◽  
Single Cells ◽  
Temporal Patterns ◽  
Ring Oscillator ◽  
Genetic Circuits ◽  
Growing Cell ◽  
Spatio Temporal

AbstractMulticellularity, the coordinated collective behaviour of cell populations, gives rise to the emergence of self-organized phenomena at many different spatio-temporal scales. At the genetic scale, oscillators are ubiquitous in regulation of multicellular systems, including during their development and regeneration. Synthetic biologists have successfully created simple synthetic genetic circuits that produce oscillations in single cells. Studying and engineering synthetic oscillators in a multicellular chassis can therefore give us valuable insights into how simple genetic circuits can encode complex multicellular behaviours at different scales. Here we develop a study of the coupling between the repressilator synthetic genetic ring oscillator and constraints on cell growth in colonies. We show in silico how mechanical constraints generate characteristic patterns of growth rate inhomogeneity in growing cell colonies. Next, we develop a simple one-dimensional model which predicts that coupling the repressilator to this pattern of growth rate via protein dilution generates travelling waves of gene expression. We show that the dynamics of these spatio-temporal patterns are determined by two parameters; the protein degradation and maximum expression rates of the repressors. We derive simple relations between these parameters and the key characteristics of the travelling wave patterns: firstly, wave speed is determined by protein degradation and secondly, wavelength is determined by maximum gene expression rate. Our analytical predictions and numerical results were in close quantitative agreement with detailed individual based simulations of growing cell colonies. Confirming published experimental results we also found that static ring patterns occur when protein stability is high. Our results show that this pattern can be induced simply by growth rate dilution and does not require transition to stationary phase as previously suggested. Our method generalizes easily to other genetic circuit architectures thus providing a framework for multi-scale rational design of spatio-temporal patterns from genetic circuits. We use this method to generate testable predictions for the synthetic biology design-build-test-learn cycle.

scholarly journals Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy

2014 ◽  
Vol 25 (22) ◽  
pp. 3699-3708 ◽  
Author(s):  
Anyimilehidi Mazo-Vargas ◽  
Heungwon Park ◽  
Mert Aydin ◽  
Nicolas E. Buchler
Keyword(s):  
Gene Expression ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Time Lapse ◽  
Photon Flux ◽  
Luminescence Microscopy ◽  
Cell Cycle Genes ◽  
Light Excitation ◽  
Better Than

Time-lapse fluorescence microscopy is an important tool for measuring in vivo gene dynamics in single cells. However, fluorescent proteins are limited by slow chromophore maturation times and the cellular autofluorescence or phototoxicity that arises from light excitation. An alternative is luciferase, an enzyme that emits photons and is active upon folding. The photon flux per luciferase is significantly lower than that for fluorescent proteins. Thus time-lapse luminescence microscopy has been successfully used to track gene dynamics only in larger organisms and for slower processes, for which more total photons can be collected in one exposure. Here we tested green, yellow, and red beetle luciferases and optimized substrate conditions for in vivo luminescence. By combining time-lapse luminescence microscopy with a microfluidic device, we tracked the dynamics of cell cycle genes in single yeast with subminute exposure times over many generations. Our method was faster and in cells with much smaller volumes than previous work. Fluorescence of an optimized reporter (Venus) lagged luminescence by 15–20 min, which is consistent with its known rate of chromophore maturation in yeast. Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression.

scholarly journals Shaping development by stochasticity and dynamics in gene regulation

Open Biology ◽  
2017 ◽  
Vol 7 (5) ◽  
pp. 170030 ◽  
Author(s):  
Peng Dong ◽  
Zhe Liu
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Single Cell ◽  
Cell Fate ◽  
Single Molecule ◽  
Large Scale ◽  
Single Cells ◽  
Individual Gene ◽  
Functional Specification ◽  
Spatio Temporal

Animal development is orchestrated by spatio-temporal gene expression programmes that drive precise lineage commitment, proliferation and migration events at the single-cell level, collectively leading to large-scale morphological change and functional specification in the whole organism. Efforts over decades have uncovered two ‘seemingly contradictory’ mechanisms in gene regulation governing these intricate processes: (i) stochasticity at individual gene regulatory steps in single cells and (ii) highly coordinated gene expression dynamics in the embryo. Here we discuss how these two layers of regulation arise from the molecular and the systems level, and how they might interplay to determine cell fate and to control the complex body plan. We also review recent technological advancements that enable quantitative analysis of gene regulation dynamics at single-cell, single-molecule resolution. These approaches outline next-generation experiments to decipher general principles bridging gaps between molecular dynamics in single cells and robust gene regulations in the embryo.

scholarly journals Characterizing the temporal dynamics of gene expression in single cells with sci-fate

2019 ◽  
Author(s):  
Junyue Cao ◽  
Wei Zhou ◽  
Frank Steemers ◽  
Cole Trapnell ◽  
Jay Shendure
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Temporal Dynamics ◽  
Single Cells ◽  
Receptor Activation ◽  
State Transitions ◽  
Proof Of Concept ◽  
Transcriptional Dynamics ◽  
A New Technique ◽  
Normal Differentiation

AbstractGene expression programs are dynamic, e.g. the cell cycle, response to stimuli, normal differentiation and development, etc. However, nearly all techniques for profiling gene expression in single cells fail to directly capture the dynamics of transcriptional programs, which limits the scope of biology that can be effectively investigated. Towards addressing this, we developed sci-fate, a new technique that combines S4U labeling of newly synthesized mRNA with single cell combinatorial indexing (sci-), in order to concurrently profile the whole and newly synthesized transcriptome in each of many single cells. As a proof-of-concept, we applied sci-fate to a model system of cortisol response and characterized expression dynamics in over 6,000 single cells. From these data, we quantify the dynamics of the cell cycle and glucocorticoid receptor activation, while also exploring their intersection. We furthermore use these data to develop a framework for inferring the distribution of cell state transitions. We anticipate sci-fate will be broadly applicable to quantitatively characterize transcriptional dynamics in diverse systems.

scholarly journals The Gene Expression Signature Modulated by Dupilumab is Correlated with Histological Severity and Endoscopic Features of Mucosal Inflammation and Remodelling in Eosinophilic Oesophagitis

2020 ◽  
Keyword(s):  
Gene Expression ◽  
Monoclonal Antibody ◽  
Gene Expression Signature ◽  
Mucosal Inflammation ◽  
Proof Of Concept ◽  
Eosinophilic Oesophagitis ◽  
Post Hoc Analysis ◽  
Post Hoc ◽  
Multiple Type

Dupilumab is a monoclonal antibody that inhibits IL-4 and IL-13 signalling in multiple Type 2 inflammatory disorders, including eosinophilic oesophagitis (EoE). This article reviews the oral presentation given by Dr Collins at the United European Gastroenterology (UEG) Week Virtual 2020 and describes the results of a post hoc analysis of a Phase II proof-of-concept study of dupilumab in adults with active EoE. The aim of the analysis was to ascertain whether there were any correlations between gene expression and disease severity in patients enrolled in the study.

scholarly journals A CCaMK/Cyclops response element in the promoter of L. japonicus Calcium-Binding Protein 1 (CBP1) mediates transcriptional activation in root symbioses

2021 ◽  
Author(s):  
Xiaoyun Gong ◽  
Simone Bucerius ◽  
Elaine Jensen ◽  
Martin Parniske
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Calcium Binding ◽  
Regulatory Element ◽  
Expression Patterns ◽  
Lotus Japonicus ◽  
Gus Expression ◽  
Early Gene ◽  
Dna Hypermethylation ◽  
Spatio Temporal

Early gene expression in arbuscular mycorrhiza (AM) and the nitrogen-fixing root nodule symbiosis (RNS) is governed by a shared regulatory complex. Yet many symbiosis-induced genes are specifically activated in only one of the two symbioses. The Lotus japonicus T-DNA insertion line T90, carrying a promoterless uidA (GUS) gene in the promoter of Calcium Binding Protein1 (CBP1) is exceptional as it exhibits GUS activity in both root endosymbioses. To identify the responsible cis- and trans-acting factors, we subjected deletion/modification series of CBP1 promoter:reporter fusions to transactivation and spatio-temporal expression analysis and screened EMS-mutagenized T90 populations for aberrant GUS expression. We identified one cis-regulatory element required for GUS expression in the epidermis and a second element, necessary and sufficient for transactivation by the Calcium and Calmodulin-dependent protein kinase (CCaMK) in combination with the transcription factor Cyclops and conferring gene expression during both AM and RNS. Lack of GUS expression in T90 white mutants could be traced to DNA hypermethylation detected in and around this element. We concluded that the CCaMK/Cyclops complex can contribute to at least three distinct gene expression patterns on its direct target promoters NIN (RNS), RAM1 (AM), and CBP1 (AM and RNS), calling for yet-to-be identified specificity-conferring factors.

scholarly journals In Vivo Simultaneous Analysis of Gene Expression by Dual-Color Luciferases in Caenorhabditis elegans

2020 ◽  
Vol 22 (1) ◽  
pp. 119
Author(s):  
Motomichi Doi ◽  
Megumi Sato ◽  
Yoshihiro Ohmiya
Keyword(s):  
Gene Expression ◽  
Fluorescent Proteins ◽  
Morphological Changes ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Gene Expression Profiles ◽  
Simultaneous Analysis ◽  
Soil Nematode ◽  
Living Organisms

Both fluorescent and luminescent observation are widely used to examine real-time gene expression patterns in living organisms. Several fluuorescent and luminescent proteins with specific optical properties have been developed and applied for simultaneous, multi-color observation of more than two gene expression profiles. Compared to fluorescent proteins, however, the application of multi-color luminescent imaging in living organisms is still limited. In this study, we introduced two-color luciferases into the soil nematode C. elegans and performed simultaneous analysis of two gene expression profiles. Using a green-emitting luciferase Eluc (emerald luciferase) and red-emitting luciferase SLR (stable luciferase red), the expression patterns of two genes were simultaneously observed in single animals from embryonic to adult stages over its whole life span. In addition, dual gene activities were observed at the single embryo level, with the simultaneous observation of morphological changes. These are the first application of a two-color luciferase system into a whole animal and suggest that precise relationship of expression patterns of multiple genes of interest can be analyzed over the whole life of the animal, dependent on the changes in genetic and/or environmental conditions.

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