scholarly journals Generating complex patterns of gene expression without regulatory circuits

2020 ◽  
Author(s):  
Sahil B. Shah ◽  
Alexis M. Hill ◽  
Claus O. Wilke ◽  
Adam J. Hockenberry
Keyword(s):  
Gene Expression ◽  
Genome Engineering ◽  
Expression Patterns ◽  
Fine Tuning ◽  
Genetic Circuits ◽  
Viral Genomes ◽  
Regulatory Circuits ◽  
Degradation Rates ◽  
Changes Over Time ◽  
Genetic Constructs

AbstractSynthetic biology has successfully advanced our ability to design complex, time-varying genetic circuits executing precisely specified gene expression patterns. However, such circuits usually require regulatory genes whose only purpose is to regulate the expression of other genes. When designing very small genetic constructs, such as viral genomes, we may want to avoid introducing such auxiliary gene products. To this end, here we demonstrate that varying only the placement and strengths of promoters, terminators, and RNase cleavage sites in a computational model of a bacteriophage genome is sufficient to achieve solutions to a variety of basic expression patterns. We discover these solutions by computationally evolving genomes to reproduce desired target expression patterns. Our approach shows non-trivial patterns can be evolved, including patterns in which the relative ordering of genes by abundance changes over time. We find that some patterns are easier to evolve than others, and different genomes that express comparable expression patterns may differ in their genetic architecture. Our work opens up a novel avenue to genome engineering via fine-tuning the balance of gene expression and gene degradation rates.

scholarly journals High-performance chemical- and light-inducible recombinases in mammalian cells and mice

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Benjamin H. Weinberg ◽  
Jang Hwan Cho ◽  
Yash Agarwal ◽  
N. T. Hang Pham ◽  
Leidy D. Caraballo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
High Performance ◽  
Genome Engineering ◽  
Genetic Circuits ◽  
Control Of Gene Expression ◽  
Expression Control ◽  
Gene Expression Control ◽  
High Performance Systems ◽  
Spatiotemporal Control

Abstract Site-specific DNA recombinases are important genome engineering tools. Chemical- and light-inducible recombinases, in particular, enable spatiotemporal control of gene expression. However, inducible recombinases are scarce due to the challenge of engineering high performance systems, thus constraining the sophistication of genetic circuits and animal models that can be created. Here we present a library of >20 orthogonal inducible split recombinases that can be activated by small molecules, light and temperature in mammalian cells and mice. Furthermore, we engineer inducible split Cre systems with better performance than existing systems. Using our orthogonal inducible recombinases, we create a genetic switchboard that can independently regulate the expression of 3 different cytokines in the same cell, a tripartite inducible Flp, and a 4-input AND gate. We quantitatively characterize the inducible recombinases for benchmarking their performances, including computation of distinguishability of outputs. This library expands capabilities for multiplexed mammalian gene expression control.

scholarly journals Characterization, modelling and mitigation of gene expression burden in mammalian cells

2019 ◽  
Author(s):  
T Frei ◽  
F Cella ◽  
F Tedeschi ◽  
J Gutierrez ◽  
GB Stan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Genome Engineering ◽  
Host Cells ◽  
Genetic Circuits ◽  
Circuit Performance ◽  
Resource Requirements ◽  
Synthetic Circuit ◽  
Resource Aware

AbstractDespite recent advances in genome engineering, the design of genetic circuits in mammalian cells is still painstakingly slow and fraught with inexplicable failures. Here we demonstrate that competition for limited transcriptional and translational resources dynamically couples otherwise independent co-expressed exogenous genes, leading to diminished performance and contributing to the divergence between intended and actual function. We also show that the expression of endogenous genes is likewise impacted when genetic payloads are expressed in the host cells. Guided by a resource-aware mathematical model and our experimental finding that post-transcriptional regulators have a large capacity for resource redistribution, we identify and engineer natural and synthetic miRNA-based incoherent feedforward loop (iFFL) circuits that mitigate gene expression burden. The implementation of these circuits features the novel use of endogenous miRNAs as integral components of the engineered iFFL device, a versatile hybrid design that allows burden mitigation to be achieved across different cell-lines with minimal resource requirements. This study establishes the foundations for context-aware prediction and improvement of in vivo synthetic circuit performance, paving the way towards more rational synthetic construct design in mammalian cells.

scholarly journals Regulostat Inferelator: a novel network biology platform to uncover molecular devices that predetermine cellular response phenotypes

2019 ◽  
Vol 47 (14) ◽  
pp. e82-e82
Author(s):  
Choong Yong Ung ◽  
Mehrab Ghanat Bari ◽  
Cheng Zhang ◽  
Jingjing Liang ◽  
Cristina Correia ◽  
...  
Keyword(s):  
Cellular Response ◽  
Expression Patterns ◽  
Fine Tuning ◽  
Molecular Devices ◽  
Proof Of Concept ◽  
Genetic Circuits ◽  
Continuous Response ◽  
Context Specific ◽  
First Time ◽  
Phenotypic Responses

Abstract With the emergence of genome editing technologies and synthetic biology, it is now possible to engineer genetic circuits driving a cell's phenotypic response to a stressor. However, capturing a continuous response, rather than simply a binary ‘on’ or ‘off’ response, remains a bioengineering challenge. No tools currently exist to identify gene candidates responsible for predetermining and fine-tuning cell response phenotypes. To address this gap, we devised a novel Regulostat Inferelator (RSI) algorithm to decipher intrinsic molecular devices or networks that predetermine cellular phenotypic responses. The RSI algorithm is designed to extract gene expression patterns from basal transcriptomic data in order to identify ‘regulostat’ constituent gene pairs, which exhibit rheostat-like mode-of-cooperation capable of fine-tuning cellular response. Our proof-of-concept study provides computational evidence for the existence of regulostats and that these networks predetermine cellular response prior to exposure to a stressor or drug. In addition, our work, for the first time, provides evidence of context-specific, drug–regulostat interactions in predetermining drug response phenotypes in cancer cells. Given RSI-inferred regulostat networks offer insights for prioritizing gene candidates capable of rendering a resistant phenotype sensitive to a given drug, we envision that this tool will be of great value in bioengineering and medicine.

scholarly journals The Circadian NAD+Metabolism: Impact on Chromatin Remodeling and Aging

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Yasukazu Nakahata ◽  
Yasumasa Bessho
Keyword(s):  
Gene Expression ◽  
Circadian Clock ◽  
Cell Fate ◽  
Chromatin Remodeling ◽  
Expression Patterns ◽  
Fine Tuning ◽  
Cellular Functions ◽  
Nad Metabolism ◽  
Structure Changes ◽  
Age Related

Gene expression is known to be a stochastic phenomenon. The stochastic gene expression rate is thought to be altered by topological change of chromosome and/or by chromatin modifications such as acetylation and methylation. Changes in mechanical properties of chromosome/chromatin by soluble factors, mechanical stresses from the environment, or metabolites determine cell fate, regulate cellular functions, or maintain cellular homeostasis. Circadian clock, which drives the expression of thousands of genes with 24-hour rhythmicity, has been known to be indispensable for maintaining cellular functions/homeostasis. During the last decade, it has been demonstrated that chromatin also undergoes modifications with 24-hour rhythmicity and facilitates the fine-tuning of circadian gene expression patterns. In this review, we cover data which suggests that chromatin structure changes in a circadian manner and that NAD+is the key metabolite for circadian chromatin remodeling. Furthermore, we discuss the relationship among circadian clock, NAD+metabolism, and aging/age-related diseases. In addition, the interventions of NAD+metabolism for the prevention and treatment of aging and age-related diseases are also discussed.

scholarly journals Modelling co-translational dimerisation for programmable nonlinearity in synthetic biology

2020 ◽  
Author(s):  
Ruud Stoof ◽  
Ángel Goñi-Moreno
Keyword(s):  
Nonlinear Dynamics ◽  
Biological Networks ◽  
Rational Design ◽  
Fine Tuning ◽  
Genetic Circuits ◽  
On Demand ◽  
Regulatory Circuits ◽  
Protein Dimers ◽  
The Impact

AbstractNonlinearity plays a fundamental role in the performance of both natural and synthetic biological networks. Key functional motifs in living microbial systems, such as the emergence of bistability or oscillations, rely on nonlinear molecular dynamics. Despite its core importance, the rational design of nonlinearity remains an unmet challenge. This is largely due to a lack of mathematical modelling that accounts for the mechanistic basics of nonlinearity. We introduce a model for gene regulatory circuits that explicitly simulates protein dimerization—a well-known source of nonlinear dynamics. Specifically, our approach focusses on modelling co-translational dimerization: the formation of protein dimers during—and not after—translation. This is in contrast to the prevailing assumption that dimer generation is only viable between freely diffusing monomers (i.e., post-translational dimerization). We provide a method for fine-tuning nonlinearity on demand by balancing the impact of co- versus post-translational dimerization. Furthermore, we suggest design rules, such as protein length or physical separation between genes, that may be used to adjust dimerization dynamics in-vivo. The design, build and test of genetic circuits with on-demand nonlinear dynamics will greatly improve the programmability of synthetic biological systems.

scholarly journals Hepatitis E rORF2p Stimulated and Unstimulated Peripheral Expression Profiling in Patients with Self-Limiting Hepatitis E Infection

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Sanjay B. Rathod ◽  
Anuradha S. Tripathy
Keyword(s):  
Gene Expression ◽  
Hepatitis E Virus ◽  
T Regulatory Cells ◽  
Current Knowledge ◽  
Expression Patterns ◽  
Hepatitis E ◽  
Fine Tuning ◽  
Regulatory Cells ◽  
Expression Array ◽  
Acute Patients

To improve the current knowledge on the involvement of peripheral lymphocytes in hepatitis E virus (HEV) associated pathogenesis, we analyzed alterations in (1) immunophenotypic expressions (by flow cytometry) and (2) gene expression patterns (by TaqMan Low Density Array) of activatory, inhibitory, integrin, homing, ectonucleotidase machinery, costimulatory, inflammatory markers, and T regulatory cells (Treg) associated cytokines on HEV rORF2p stimulated and unstimulated PBMCs of 43 acute HEV patients, 30 recovered individuals, and 43 controls. The phenotypic expressions of key molecules CTLA-4, GITR, CD103, CD25, CD69, IL10 and TGF-β1in the acute patients and TGF-β1in the recovered individuals were significantly elevated on both unstimulated and stimulated PBMCs. Gene expression array data revealed upregulations of CD25, PD1, CD103, CCR4, IL10, and TGF-β1on both unstimulated and HEV rORF2p stimulated PBMCs of acute patients. The observed upregulations of inhibitory, integrin, activatory, and Treg-associated cytokine genes on the PBMCs of acute HEV patients complemented by their frequency data suggest them as the major players in the fine-tuning of immune response in self-limiting hepatitis E infection.

scholarly journals Chromatin Alterations in Neurological Disorders and Strategies of (Epi)Genome Rescue

2021 ◽  
Vol 14 (8) ◽  
pp. 765
Author(s):  
Marcin Janowski ◽  
Małgorzata Milewska ◽  
Peyman Zare ◽  
Aleksandra Pękowska
Keyword(s):  
Gene Expression ◽  
Neurological Disorders ◽  
Genome Engineering ◽  
New Technologies ◽  
Expression Patterns ◽  
Clinical Practices ◽  
Modifying Factors ◽  
Functional Consequences ◽  
Altered Gene ◽  
Regulatory Dna

Neurological disorders (NDs) comprise a heterogeneous group of conditions that affect the function of the nervous system. Often incurable, NDs have profound and detrimental consequences on the affected individuals’ lives. NDs have complex etiologies but commonly feature altered gene expression and dysfunctions of the essential chromatin-modifying factors. Hence, compounds that target DNA and histone modification pathways, the so-called epidrugs, constitute promising tools to treat NDs. Yet, targeting the entire epigenome might reveal insufficient to modify a chosen gene expression or even unnecessary and detrimental to the patients’ health. New technologies hold a promise to expand the clinical toolkit in the fight against NDs. (Epi)genome engineering using designer nucleases, including CRISPR-Cas9 and TALENs, can potentially help restore the correct gene expression patterns by targeting a defined gene or pathway, both genetically and epigenetically, with minimal off-target activity. Here, we review the implication of epigenetic machinery in NDs. We outline syndromes caused by mutations in chromatin-modifying enzymes and discuss the functional consequences of mutations in regulatory DNA in NDs. We review the approaches that allow modifying the (epi)genome, including tools based on TALENs and CRISPR-Cas9 technologies, and we highlight how these new strategies could potentially change clinical practices in the treatment of NDs.

scholarly journals An optimized pipeline for parallel image-based quantification of gene expression and genotyping after in situ hybridization

2017 ◽  
Author(s):  
Tomasz Dobrzycki ◽  
Monika Krecsmarik ◽  
Florian Bonkhofer ◽  
Roger Patient ◽  
Rui Monteiro
Keyword(s):  
Gene Expression ◽  
Image Analysis ◽  
In Situ Hybridization ◽  
Quantitative Methods ◽  
Genome Engineering ◽  
Expression Patterns ◽  
Computer Based ◽  
Molecular Phenotypes ◽  
Mutant Lines

ABSTRACTAdvances in genome engineering have resulted in the generation of numerous zebrafish mutant lines. A commonly used method to assess gene expression in the mutants is in situ hybridization (ISH). Because the embryos can be distinguished by genotype after ISH, comparing gene expression between wild type and mutant siblings can be done blinded and in parallel. Such experimental design reduces the technical variation between samples and minimises the risk of bias. This approach, however, requires an efficient method of genomic DNA extraction from post-ISH fixed zebrafish samples to ascribe phenotype to genotype. Here we describe a method to obtain PCR-quality DNA from 95-100% of zebrafish embryos, suitable for genotyping after ISH. In addition, we provide an image analysis protocol for quantifying gene expression of ISH-probed embryos, adaptable for the analysis of different expression patterns. Finally, we show that intensity-based image analysis enables accurate representation of the variability of gene expression detected by ISH and that it can complement quantitative methods like qRT-PCR. By combining genotyping after ISH and computer-based image analysis, we have established a high-confidence, unbiased methodology to assign gene expression levels to specific genotypes, and applied it to the analysis of molecular phenotypes of newly generated lmo4a mutants.SUMMARY STATEMENTOur optimized protocol to genotype zebrafish mutant embryos after in situ hybridization and digitally quantify the in situ signal will help to standardize existing experimental designs and methods of analysis.

scholarly journals High-performance chemical and light-inducible recombinases in mammalian cells and mice

2019 ◽  
Author(s):  
Benjamin H. Weinberg ◽  
Jang Hwan Cho ◽  
Yash Agarwal ◽  
N. T. Hang Pham ◽  
Leidy D. Caraballo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
High Performance ◽  
Genome Engineering ◽  
Dynamic Range ◽  
Signal To Noise Ratio ◽  
Genetic Circuits ◽  
Control Of Gene Expression ◽  
Gene Expression Control ◽  
High Performance Systems

ABSTRACTSite-specific DNA recombinases are some of the most powerful genome engineering tools in biology. Chemical and light-inducible recombinases, in particular, enable spatiotemporal control of gene expression. However, the availability of inducible recombinases is scarce due to the challenge of engineering high performance systems with low basal activity and sufficient dynamic range. This limitation constrains the sophistication of genetic circuits and animal models that can be created. To expand the number of available inducible recombinases, here we present a library of >20 orthogonal split recombinases that can be inducibly dimerized and activated by various small molecules, light, and temperature in mammalian cells and mice.Furthermore, we have engineered inducible split Cre systems with better performance than existing inducible Cre systems. Using our orthogonal inducible recombinases, we created a “genetic switchboard” that can independently regulate the expression of 3 different cytokines in the same cell. To demonstrate novel capability with our split recombinases, we created a tripartite inducible Flp and a 4-Input AND gate. We have performed extensive quantitative characterization of the inducible recombinases for benchmarking their performances, including computation of distinguishability of outputs in terms of signal-to-noise ratio (SNR). To facilitate sharing of this set of reagents, we have deposited our library to Addgene. This library thus significantly expands capabilities for precise and multiplexed mammalian gene expression control.

Laser Capture Microdissection, Amplified Antisense Rna, and Highdensity Microarrays: The New Frontier for Gene Expression Profiling From Limited Amounts of Tissue

2000 ◽  
Vol 6 (S2) ◽  
pp. 840-841
Author(s):  
Roger D. Madison, Ph.D.
Keyword(s):  
Gene Expression ◽  
Laser Capture Microdissection ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Mrna Levels ◽  
Functional Protein ◽  
Protein Levels ◽  
Degradation Rates ◽  
A Cell ◽  
Laser Capture

Because the genes in all somatic cells of an individual are identical, it is the differential expression of the proteins encoded by particular genes that determines a cell's phenotype. There is now increasing interest in relating the simultaneous expression patterns of multiple proteins to complex biological processes. The goal is to develop ‘expression profiles’ of the proteins that are active within a cell or tissue during a particular condition. The ultimate control of functional protein expression is quite complex and may involve transcriptional, combinatorial, and postranslational mechanisms, as well as differences in protein degradation rates that vary under different conditions. Although mRNA levels do not always predict functional protein levels, they are a useful first step in determining the expression profile.The evolution of three key technologies offers the promise of obtaining quantitative analysis of gene expression from restricted amounts of nervous system tissue. Laser capture microdissection can accomplish harvesting of identified cell populations from histologically processed tissue.

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