Biology subjects

Reeves, G. T.

Publications and source records attributed to Reeves, G. T..

At least 19 recordsLinked to original sources

bioRxiv2026

Enhancer Activity-informed Gap GEne Regulatory Network (EAGER) to model Drosophila gap gene expression on the entire anterior-posterior (A-P) axis

Across metazoa, morphogen gradients differentially regulate gene expression and activate a spatially distinct program to specify body axis development. The Drosophila gap gene network, initiated by maternal morphogen Bicoid, is one of the most well-studied systems. Several regulatory interactions act synergistically to produce distinct gap gene expression patterns along the anterior-posterior (AP) axis of the blastoderm stage Drosophila embryo to ensure the proper segmentation of the larval and, eventually, adult stage fly. Several mathematical models have been proposed to summarize the interconnectivity of gap gene regulatory elements and predict expression in mutant systems. However, these models have not successfully predicted the gap gene expression profile over the entire AP axis. Here, we present an Enhancer Activity-informed Gap GEne Regulatory network (EAGER) model that incorporates enhancer activity-driven differential regulation along the AP axis, which successfully summarizes gap gene expression patterns over the entire AP axis. We validated the predictions of EAGER on Kr mutants and performed a comprehensive parametric sensitivity and identifiability analysis to evaluate the robustness of the EAGER model fits and predictions. We also propose a reduced version of the model, rEAGER, which identifies a minimal set of regulatory interactions and successfully summarizes the gap gene interactions over the entire AP axis. Our results suggest that the expression driven by individual enhancers must be accounted for in models of developmental pattern formation.

developmental biology↗

bioRxiv2026

Genome-wide mapping of Bicoid/DNA interactions reveals quantitative constraints on transcriptional regulation

During Drosophila embryogenesis, Bicoid (Bcd) forms a gradient that provides positional information to regulate target genes along the anteroposterior axis. To understand how the information provided by the Bcd gradient drives gene expression in a concentration-dependent manner, the subpopulations of Bcd participating in gene regulation need to be characterized. Therefore, to understand the mechanism of Bcd-mediated gene regulation, we quantified the absolute concentration of the nuclear subpopulations of Bcd, such as freely diffusing and DNA bound Bcd. These subpopulations have distinct diffusivities and DNA-binding properties and are crucial for gene regulation. This quantification allowed us to construct a global dose/response relationship between the free and DNA-bound concentration of Bcd. Our data show that Bcd/DNA binding is strongly correlated with the free concentration of Bcd, indicated by the dose/response relationship being in the linear regime, despite the barrier presented by nucleosomes. Our data are quantitatively consistent with a Monod-Wyman-Changeux model in which transcription factors passively compete with nucleosomes for DNA binding. We further apply this model to Bcd/DNA interactions at the enhancer/promoter for hunchback (hb), a Bcd target gene which has a steep posterior boundary, despite being driven primarily by the graded Bcd concentration, a conundrum which has been under scrutiny for decades. We show that, using parameters determined from the global dose/response relationship, a reversible multistate promoter model, in which the promoter activation rate is determined by Bcd binding to the hb enhancer, can successfully recapitulate features of hb transcriptional dynamics, including the sharp posterior boundary. Therefore, this work sheds light on mechanism of hb regulation by Bcd and provides a potential experimental/computational pipeline that bridges the input of global properties of transcription factors to the transcriptional output of specific target genes.

developmental biology↗

bioRxiv2025

MIDOE: Maximally-informed Design of Experiment to infer experimentally inaccessible transcription factors dynamics

1The TGF-{beta}/Smad signaling pathway regulates growth, development, and homeostasis of tissues across the animal kingdom. The pathway is activated when transforming growth factor -{beta} (TGF-{beta}) binds to its cognate transmembrane receptors to activate Smad2 by phosphorylation. The activated phosphor-Smad2 (PSmad2) undergoes a series of biochemical interactions with transcriptional activator Smad4 to form several oligomers, including the transcription factor (PSmad2)2/Smad4, which regulates target gene expression. Quantitative live cell imaging and mathematical modeling have been used to estimate the dynamics of (PSmad2)2/Smad4. However, due to the emergent nature of Smad2-Smad4 interactions, deconvolving the dynamics of the (PSmad2)2/Smad4 is challenging. We show that the Smad model is sloppy, has large parameter uncertainties (O[~]1015), and the eigenvalues of the Fisher Information Matrix span over several decades. As such, well-fit parameter sets generate highly under-constrained predictions. To overcome this, we employ Profile Log-Likelihood to guide maximally informed design of experiments (MIDOE) and infer the dynamics of (PSmad2)2/Smad4. We generate these experiments computationally and validate that MIDOE can optimally constrain model predictions. We demonstrate that such careful analysis would not only improve the predictive power of models in systems biology but also reduce the time and expense of performing non-optimal experiments.

systems biology↗

bioRxiv2025

CalciumInsights: An Open-Source, Tissue-Agnostic Graphical Interface for High-Quality Analysis of Calcium Signals

Fluctuations and propagation of cytosolic calcium levels at both the cellular and tissue levels show complex patterns, referred to as calcium signatures, that regulate growth, organ development, damage responses, and survival. The quantitative analysis of calcium signatures at the cellular level is essential for identifying unique patterns that coordinate biological processes. However, a versatile framework applicable to multiple tissue types, allowing researchers to compare, measure, and validate diverse responses and recognize conserved patterns across model organisms, is missing. Here, we present a post-processing tool, CalciumInsights, which leverages the R packages Shiny and Golem. This tool has a graphical user interface and does not require software programming experience to perform calcium signal analysis. The open-source software has a modular framework with standardized functionalities that can be tailored for various research approaches. CalciumInsights provides descriptive statistical analysis through various metrics extracted from dynamic calcium transients and oscillations, such as peak amplitude, area under the curve, frequency, among others. The tool was evaluated with fluorescence imaging data from three model organisms: Danio rerio, Arabidopsis thaliana, and Drosophila melanogaster, demonstrating its ability to analyze diverse biological responses and models. Finally, the open-source nature of CalciumInsights enables community-driven improvements and developments for enabling new applications. Author SummaryThis manuscript introduces CalciumInsights, an open-source tool for calcium signature analysis. Designed to be a versatile tool that works with various tissue types and biological systems, CalciumInsights has an easy-to-use graphical user interface. Our program simplifies metrics extraction while maintaining the quality of the analysis by integrating several algorithms. CalciumInsights stands out for its user-friendliness, ease of use, and robust data exploration features, such as tunable filters for improved accuracy. These features promote inclusivity and lower barriers to scientific research by making calcium signature analysis accessible to users of all programming skill levels.

bioengineering↗

bioRxiv2025

Novel Fluorescent and Photoconvertible Fusions Reveal Dorsal Activator Dynamics

Over the last two decades, new in vivo and in cellulo imaging technologies have uncovered the inherently dynamic nature of transcriptional regulation in embryonic development and, in particular, in the fruit fly D. melanogaster. These technologies have made it possible to characterize the subnuclear and single-molecule dynamics of transcription factors. However, a lack of appropriate fluorescent protein fusions has, until now, limited these studies to only a few of the dozens of important transcription factors in the fruit fly gene regulatory network dictating early development. Here, we report the creation of four new fluorescent protein fusions to Dorsal, a member of the NF-{kappa}B/Rel family that initiates dorsal-ventral patterning. We generated and characterized two bright fluorescent protein fusions for Dorsal, meGFP and mNeonGreen, and two photoconvertible fluorescent protein fusions, mEos4a and Dendra2. We show that removal of the DsRed2 cassette commonly used to mark the CRISPR integration restores endogenous Dorsal mRNA and protein levels and enables the fusion allele to rescue a dorsal null allele, meeting the gold standard for endogenous function of a tagged protein in a fruit fly. We then demonstrate that our bright fluorescent protein fusions can be used to dissect the spatiotemporal dynamics of stable Dorsal clusters that traverse the nucleoplasm and uncovered that these clusters preferentially interact with active sites of Dorsal-modulated transcription. We further demonstrate that our photoconvertible fluorescent protein fusions make it possible to detect individual molecules of Dorsal in the nuclei of developing embryos. These new fluorescent protein fusions constitute a valuable resource for the community to elucidate the role of Dorsal activator dynamics in dictating fruit fly early embryonic development.

developmental biology↗

bioRxiv2025

Global maps of transcription factor properties reveal threshold-based formation of DNA-bound and mobile clusters

The relationship between bulk transcription factor concentration and DNA binding has been a central question in gene regulation for decades. Recent studies propose that DNA-bound transcription factor hubs, or clusters, aid in fast and precise transcriptional interpretation. Using live imaging techniques, we quantify the concentration, binding, and mobility of the morphogen Dorsal (Dl), both in bulk and in clusters, in Drosophila blastoderm embryo. Our experiments encompass multiple length and time scales, allowing us to obtain a nucleus-wide view of the mechanism connecting hub formation to bulk Dl concentration. Our results show that previously unobserved, slowly-moving clusters of Dl are present, in addition to the expected populations of freely mobile and DNA-bound Dl. Furthermore, both mobile clusters and DNA-bound Dl appear only once a threshold concentration in the nucleus is surpassed, a behavior consistent with liquid-liquid phase separation. Thus, our work elucidates how bulk transcription factor concentration dictates the formation and spatiotemporal changes of different populations needed for gene regulation.

developmental biology↗

bioRxiv2024

Elucidating the role of multiple feedback loops in regulating stem cell decisions

Stem cell decisions are regulated by a complex network of gene regulatory pathways that determine the reproductive health of the tissue. The Drosophila ovarian germline is a well-characterized model system which facilitates visualizing stem cell behavior in its native environment to attain a systems-level understanding of the stem cell dynamics. The asymmetric division of the Germline Stem Cells (GSCs) forms two daughter cells-a self-renewed GSC and a differentiated Cystoblast (CB). The highly conserved Bone Morphogenetic Protein (BMP) pathway ensures growth and maintenance of the GSCs, but is downregulated in the CBs, to allow for differentiation. BMP signal transduction upregulates dad and represses Fused, both of which are negative regulators of the BMP pathway. Moreover, these regulatory mechanisms operate on a system of two cells which remain connected during a portion of the cell cycle. We developed a biologically-informed mathematical model of multi-compartment GSC division to investigate the dynamic roles Dad and Fused play in determining cell fate. We found that Dad optimally controls the BMP signal transduction to enable GSC homeostasis and differentiation. In dadKO mutants, GSCs were more likely to divide symmetrically. Our work identifies the synergistic role of Dad and Fused rendering robustness to stem cell division.

systems biology↗

bioRxiv2024

Bulk-level maps of pioneer factor binding dynamics during Drosophila maternal-to-zygotic transition

Gene regulation by transcription factors (TFs) binding cognate sequences is of paramount importance. For example, the TFs Zelda (Zld) and GAGA factor (GAF) are widely acknowledged for pioneering gene activation during zygotic genome activation (ZGA) in Drosophila. However, quantitative dose/response relationships between bulk TF concentration and DNA binding, an event tied to transcriptional activity, remain elusive. Here, we map these relationships during ZGA: a crucial step in metazoan development. To map the dose/response relationship between nuclear concentration and DNA binding, we performed raster image correlation spectroscopy, a method that can measure biophysical parameters of fluorescent molecules. We found that, although Zld concentration increases during nuclear cycles (ncs) 10 to 14, its binding in the transcriptionally active regions decreases, consistent with its function as an activator for early genes. In contrast, GAF-DNA binding is nearly linear with its concentration, which sharply increases during the major wave, implicating it in the major wave. This study provides key insights into the properties of the two factors and puts forward a quantitative approach that can be used for other TFs to study transcriptional regulation.

systems biology↗

bioRxiv2024

Spatiotemporal dynamics of NF-κB/Dorsal inhibitor IκBα/Cactus in Drosophila blastoderm embryos

The NF-{kappa}B signaling pathway is a key regulatory network in mammals that controls many cellular processes, including immunity and inflammation. Of particular note is the relationship between NF-{kappa}B and its inhibitor I{kappa}B, which sequesters NF-{kappa}B to the cytoplasm of cells until needed. It is also known that I{kappa}B can enter nuclei, disrupt NF-{kappa}B binding to DNA, and shuttle it out to again sequester NF-{kappa}B in the cytoplasm. In Drosophila melanogaster, a homologous system between the proteins Dorsal (homologous to NF-{kappa}B) and Cactus (homologous to I{kappa}B) is important in embryo development, specifically in establishment of the Dorsal nuclear concentration gradient. Previous work suggests Cactus also enters the nucleus; mathematical models of the Dorsal gradient fail to accurately predict the normal range of the gradient without nuclear Cactus. However, direct, in vivo visualization of Cactus spatiotemporal dynamics, including its localization to the nuclei, has been difficult to gather. Previously, imaging Cactus in live embryos was complicated by rapid protein turnover, preventing fluorescent protein fusions from fully maturing. To address this, we used the CRISPR/Cas9 system to tag Cactus with the recently developed "LlamaTag" (LT), a genetically encodable nanobody from llamas that dynamically binds to GFP in vivo. We then employed standard confocal imaging, as well as advanced optical techniques such as raster image correlation spectroscopy (RICS) and fluorescent recovery after photobleaching (FRAP) to investigate the spatiotemporal distribution of Cactus-LlamaTag in Drosophila embryos at the blastoderm stage. Our results demonstrate that Cactus can be found in the nuclei of early embryos, consistent with its role as a transcription factor regulator. Moreover, by using the data from FRAP and RICS, we were able to estimate biophysical parameters of Cactus dynamics in vivo, including its nuclear transport rate constants and fraction bound to GFP. These data were further used to constrain a mathematical model that allowed us to infer experimentally inaccessible biophysical parameters, such as the concentration of Cact protein and the dissociation constant of LT and GFP. Our study provides new insights into the regulation of the NF-{kappa}B pathway in early Drosophila embryos and highlights the power of advanced optical techniques for investigating complex biological dynamics.

developmental biology↗

bioRxiv2024

Optimal Performance Objectives in the Highly Conserved Bone Morphogenetic Protein Signaling Pathway

Throughout development, complex networks of cell signaling pathways drive cellular decision-making across different tissues and contexts. The transforming growth factor {beta} (TGF-{beta}) pathways, including the BMP/Smad pathway, play crucial roles in these cellular responses. However, as the Smad pathway is used reiteratively throughout the life cycle of all animals, its systems-level behavior varies from one context to another, despite the pathway connectivity remaining nearly constant. For instance, some cellular systems require a rapid response, while others require high noise filtering. In this paper, we examine how the BMP- Smad pathway balances trade-offs among three such systems-level behaviors, or "Performance Objectives (POs)": response speed, noise amplification, and the sensitivity of pathway output to receptor input. Using a Smad pathway model fit to human cell data, we show that varying non-conserved parameters (NCPs) such as protein concentrations, the Smad pathway can be tuned to emphasize any of the three POs and that the concentration of nuclear phosphatase has the greatest effect on tuning the POs. However, due to competition among the POs, the pathway cannot simultaneously optimize all three, but at best must balance trade-offs among the POs. We applied the multi-objective optimization concept of the Pareto Front, a widely used concept in economics to identify optimal trade-offs among various requirements. We show that the BMP pathway efficiently balances competing POs across species and is largely Pareto optimal. Our findings reveal that varying the concentration of NCPs allows the Smad signaling pathway to generate a diverse range of POs. This insight identifies how signaling pathways can be optimally tuned for each context.

systems biology↗

bioRxiv2023

ISRES+: An improved evolutionary strategy for function minimization to estimate the free parameters of Systems Biology models

Model development is essential to gain a mathematical understanding of the underlying phenomena in systems biology. In most models, it is typically hard to estimate the values of the biophysical/phenomenological parameters that characterize the model. The parameters are estimated by minimizing a function that reduces a measure of the error between model predictions and experimental data. In this work, we build on an algorithm for function minimization proposed by Runnarson and Yao, named Improved Evolutionary Strategy by Stochastic Ranking (ISRES), that finds a best-fit individual by evolving a population in the direction of minimizing error by using information at most from a pair of individuals in any generation to create a new population. Our algorithm, named ISRES+, builds on it by combining information from all individuals across the population and across all generations to gain a better sense of direction to evolve the population. ISRES+ makes use of the additional information generated by the creation of a large population in the evolutionary methods to approximate the local neighborhood around the best-fit individual using linear least squares fit in one and two dimensions. We compared the performance of the two algorithms on three systems biology models with varying complexities and found that not only does the ISRES+ lead to fitter individuals, but it also leads to a tighter distribution of fittest individuals over successive runs.

systems biology↗

bioRxiv2022

Dynamics of BMP signaling in the early Drosophila embryo

In developing tissues, morphogen gradients are thought to initialize gene expression patterns. However, the relationship between the dynamics of morphogen-encoded signals and gene expression decisions are largely unknown. Here we examine the dynamics of the Bone Morphogenetic Protein (BMP) pathway in Drosophila blastoderm-stage embryos. In this tissue, the BMP pathway is highly dynamic: it begins as a broad and weak signal on the dorsal half of the embryo, then 20-30 min later refines into a narrow, intense peak centered on the dorsal midline. This dynamical progression of the BMP signal raises questions of how it stably activates target genes. Therefore, we performed live imaging of the BMP signal and found that dorsal-lateral cells experience only a short transient in BMP signaling, after which the signal is lost completely. Moreover, we measured the transcriptional response of the BMP target gene pannier in live embryos and found it to remain activated in dorsal-lateral cells, even after the BMP signal is lost. Our findings may suggest that the BMP pathway activates a memory, or "ratchet" mechanism that may sustain gene expression.

developmental biology↗

bioRxiv2022

Experimental and analytical framework for ''mix-and-read'' assays based on split luciferase

The use of immunodetection assays including the widely used enzyme-linked immunosorbent assay (ELISA) in applications such as point-of-care detection is often limited by the need for protein immobilization and multiple binding and washing steps. Here, we describe an experimental and analytical framework for the development of simple and modular "mix-and-read" enzymatic complementation assays based on split luciferase that enable sensitive detection and quantification of analytes in solution. In this assay, two engineered protein binders targeting non-overlapping epitopes on the target analyte were each fused to non-active fragments of luciferase to create biosensor probes. Binding proteins to two model targets, lysozyme and Sso6904, were isolated from a combinatorial library of Sso7d mutants using yeast surface display. In the presence of the analyte, probes were brought into close proximity, reconstituting enzymatic activity of luciferase and enabling detection of low picomolar concentrations of the analyte by chemiluminescence. Subsequently, we constructed an equilibrium binding model that relates binding affinities of the binding proteins for the target, assay parameters such as the concentrations of probes used, and assay performance (limit of detection and concentration range over which the target can be quantified). Overall, our experimental and analytical framework provide the foundation for the development of split luciferase assays for detection and quantification of various targets.

bioengineering↗

bioRxiv2020

Discovery of membrane-permeating cyclic peptides via mRNA display

Small synthetic peptides capable of crossing biological membranes represent valuable tools in cell biology and drug delivery. While several cell-penetrating peptides (CPPs) of natural or synthetic origin have been reported, no peptide is currently known to cross both cytoplasmic and outer embryonic membranes. Here we describe a method to engineer membrane-permeating cyclic peptides (MPPs) with broad permeation activity by screening mRNA display libraries of cyclic peptides against embryos at different developmental stages. The proposed method was demonstrated by identifying peptides capable of permeating Drosophila melanogaster (fruit fly) embryos and mammalian cells. The selected peptide cyclo[Glut-MRKRHASRRE-K*] showed a strong permeation activity of embryos exposed to minimal permeabilization pretreatment, as well as human embryonic stem cells and a murine fibroblast cell line. Notably, in both embryos and mammalian cells, the cyclic peptide outperformed its linear counterpart and the control MPPs. Confocal microscopy and single cell flow cytometry analysis were utilized to assess the degree of permeation both qualitatively and quantitatively. These MPPs have potential application in studying and non-disruptively controlling intracellular or intraembryonic processes.

biochemistry↗

bioRxiv2019

Spatiotemporal control of gene expression boundaries using a feedforward loop

A feed forward loop (FFL) is commonly observed in several biological networks. The FFL network motif has been mostly been studied with respect to variation of the input signal in time, with only a few studies of FFL activity in a spatially distributed system such as morphogen-mediated tissue patterning. However, most morphogen gradients also evolve in time. We studied the spatiotemporal behavior of a coherent FFL in two contexts: (1) a generic, oscillating morphogen gradient and (2) the dorsal-ventral patterning of the early Drosophila embryo by a gradient of the NF-{kappa}B homolog Dorsal with its early target Twist. In both models, we found features in the dynamics of the intermediate node - phase difference and noise filtering - that were largely independent of the parameterization of the models, and thus were functions of the structure of the FFL itself. In the Dorsal gradient model, we also found that the dynamics of Dorsal require maternal pioneering factor Zelda for proper target gene expression.

developmental biology↗

bioRxiv2019

Shuttling of Dorsal by Cactus: mechanism and implications

In a developing animal, morphogen gradients act to pattern tissues into distinct domains of cell types. However, despite their prevalence in development, morphogen gradient formation is a matter of debate. In our recent publication, we showed that the Dorsal/NF-{kappa}B morphogen gradient, which patterns the DV axis of the early Drosophila embryo, is partially established by a mechanism of facilitated diffusion. This mechanism, also known as \"shuttling,\" occurs when a binding partner of the morphogen facilitates the diffusion of the morphogen, allowing it to accumulate at a given site. In this case, the inhibitor Cactus/I{kappa}B facilitates the diffusion of Dorsal/NF-{kappa}B. In the fly embryo, we used computation and experiment to not only show that shuttling occurs in the embryo, but also that it enables the viability of embryos that inherit only one copy of dorsal maternally. Here we further discuss our evidence behind the shuttling mechanism, the previous literature data explained by the mechanism, and how it may also be critical for robustness of development. Finally, we describe an interaction between Dorsal and BMP signaling that is likely affected by shuttling.

developmental biology↗

bioRxiv2019

Robustness of the Dorsal morphogen gradient with respect to morphogen dosage

In multicellular organisms, the timing and placement of gene expression in a developing tissue assigns the fate of each cell in the embryo in order for a uniform field of cells to differentiate into a reproducible pattern of organs and tissues. This positional information is often achieved through the action of spatial gradients of morphogens. Spatial patterns of gene expression are paradoxically robust to variations in morphogen dosage, given that, by definition, gene expression must be sensitive to morphogen concentration. In this work we investigate the robustness of the Dorsal/NF-{kappa}B signaling module with respect to perturbations to the dosage of maternally-expressed dorsal mRNA. The Dorsal morphogen gradient patterns the dorsal-ventral axis of the early Drosophila embryo, and we found that an empirical description of the Dorsal gradient is highly sensitive to maternal dorsal dosage. In contrast, we found experimentally that gene expression patterns are highly robust. Although the components of this signaling module have been characterized in detail, how their function is integrated to produce robust gene expression patterns to variations in the dorsal maternal dosage is still unclear. Therefore, we analyzed a mechanistic model of the Dorsal signaling module and found that Cactus, a cytoplasmic inhibitor for Dorsal, must be present in the nucleus for the system to be robust. Furthermore, active Toll, the receptor that dissociates Cactus from Dorsal, must be saturated. Finally, the vast majority of robust descriptions of the system require facilitated diffusion of Dorsal by Cactus. Each of these three recently-discovered mechanisms of the Dorsal module are critical for robustness. Our work highlights the need for quantitative understanding of biophysical mechanisms of morphogen gradients in order to understand emergent phenotypes, such as robustness.\n\nAuthor SummaryThe early stages of development of an embryo are crucial for laying the foundation of the body plan. The blueprint of this plan is encoded in long-range spatial protein gradients called morphogens. This positional information is then interpreted by nuclei that begin to differentiate by expressing different genes. In fruit fly embryos, the Dorsal morphogen forms a gradient along the dorsal-ventral axis, with a maximum at the ventral midline. This gradient, and the resulting gene expression patterns are extraordinarily robust to variations in developmental conditions, even during early stages of development. Since positional information is interpreted in terms of concentration of the morphogen, one would expect that doubling or halving dosage would result in disastrous consequences for the embryo. However, we observed that development remains robust. We quantified the effect of dosage by experimentally measuring the boundaries of 2 genes, - sna and sog, expressed along the DV axis and found that variation in the boundaries of these genes was minimal, across embryos with different dosages of Dl. We then used a mathematical model to discern components of the Dl system responsible for buffering the effects of dosage and found three specific mechanisms - deconvolution, Toll saturation and shuttling

developmental biology↗

bioRxiv2019

Tunable self-cleaving ribozymes for modulating gene expression in eukaryotic systems

Advancements in the field of synthetic biology have been possible due to the development of genetic tools that are able to regulate gene expression. However, the current toolbox of gene regulatory tools for eukaryotic systems have been outpaced by those developed for simple, single-celled systems. Here, we engineered a set of gene regulatory tools by combining self-cleaving ribozymes with various upstream competing sequences that were designed to disrupt ribozyme self-cleavage. As a proof-of-concept, we were able to modulate GFP expression in mammalian cells, and then showed the feasibility of these tools in Drosophila embryos. For each system, the fold-reduction of gene expression was influenced by the location of the self-cleaving ribozyme/upstream competing sequence (i.e. 5 untranslated region (UTR) vs. 3UTR) and the competing sequence used. Together, this work provides a set of genetic tools that can be used to tune gene expression across various eukaryotic systems.

bioengineering↗