Biology subjects

Tsou, T.

Publications and source records attributed to Tsou, T..

6 recordsLinked to original sources

bioRxiv2026

Improved vector toolkit for genome writing in mammalian cells

Efficient genome writing in mammalian cells requires robust methods for integrating large DNA payloads. The previously described method mammalian Switching Antibiotic resistance markers Progressively for Integration (mSwAP-In) enables iterative, biallelic genome rewriting in mammalian stem cells with DNA payloads exceeding 100 kb. However, the lack of standardized vectors and certain technical constraints have limited its broader adoption. Here we present an improved plasmid toolkit designed to streamline the implementation of mSwAP-In. The toolkit includes two core vectors. pLP-TK (pCTC174) is a landing-pad plasmid compatible with Golden Gate assembly of genomic homology arms and supports both mSwAP-In and the recombinase-mediated cassette exchange method Big-IN. mSwAP-In MC2v2 (pKBA135) is a versatile Big DNA assembly and delivery vector that supports Gibson-based assembly and incorporates positive, negative, and fluorescent selection markers, as well as a backbone counterselection cassette to minimize unwanted plasmid integration. The vector architecture also enables propagation in yeast and bacterial hosts, inducible plasmid copy-number amplification in standard E. coli strains, and CRISPR/Cas9-mediated payload release through preinstalled guide RNA target sites. We further characterize the FCU1/5-FC counterselection system in mouse embryonic stem cells and define conditions that minimize its bystander toxicity. Finally, we provide a set of Cas9-gRNA expression plasmids optimized for common mSwAP-In applications. Together, these reagents constitute a standardized and experimentally validated toolkit that simplifies large-scale genome writing using mSwAP-In.

synthetic biology↗

bioRxiv2025

Designing signaling environments to modulate neural progenitor cell differentiation with regulatory network models

During development, combinatorial signals modulate progenitor differentiation to build populations of diverse cell types. Classical deterministic fate-control circuits focus on stable commitment but do not explain how a diverse cell population is generated and tuned by signals. Here, using single-cell RNA-seq profiling of neural progenitor cells (NPCs) upon combinatorial signal inputs, we identify a candidate circuit-level mechanism for controlling cell type compositions through a noise-gated toggle switch. With 40 signaling conditions, we show that combinatorial signal inputs modulate the neuronal-glial population proportions following a simple log-linear model. Regulatory network models built by D-SPIN quantitatively capture cell state distribution shifts induced by signal combinations, and indicate a circuit model for the probabilistic fate specification by a toggle switch with controlled transcriptional heterogeneity. Noise levels drive the system from highly plastic to committed states, allowing the relative level of signal inputs to shift population structures. The model further predicts an early bipotent state expressing lineage-specifying factors of both fates, which we verify in our single-cell profiling experiment and a mouse embryonic visual cortex development dataset. Our work identifies a design principle of noise-driven additive regulation in the logarithmic cell-fate probability space, providing a new strategy for population-level stem cell control.

systems biology↗

bioRxiv2024

Control of odor sensation by light and cryptochrome in the Drosophila antenna

Olfaction is a sense employed by insects to differentiate safe from harmful food options, evaluate potential mates, and identify oviposition sites. Here, we found that the fruit fly, Drosophila melanogaster, responds differently to a set of repulsive odors depending on ambient light conditions. Ultraviolet (UV) or blue light reduces the behavioral aversion and electrophysiological responses of olfactory receptor neurons (ORNs) to certain repellent odors, such as benzaldehyde. We found that cryptochrome (cry) is strongly expressed in the antennal support cells that lie adjacent to ORNs, and mutation of cry eliminates the light-dependent reduction in aversion. Thus, these data demonstrate that support cells in an olfactory organ serve a sensory function as light receptor cells. It has been shown that light activation of Cry creates reactive oxygen species (ROS), and ROS activates the TRPA1 channel. We demonstrate that the TRPA1-C isoform is expressed and required in ORNs for benzaldehyde repulsion, and that TRPA1-C is activated in vitro by benzaldehyde. Overexpression of dual oxygenase, which generates hydrogen peroxide, reduced the aversion under dark conditions. Our data support the model that light-dependent creation of hydrogen peroxide persistently activates TRPA1-C. Consequently, the channel is no longer effectively activated by benzaldehyde. Since flies sleep much more at night, and begin feeding at dawn, we propose that the light-induced reduction in aversion to certain odors provides a mechanism to lower the barrier to feeding following the transition from night to day.

neuroscience↗

bioRxiv2023

D-SPIN constructs gene regulatory network models from multiplexed scRNA-seq data revealing organizing principles of cellular perturbation response

Gene regulatory networks within cells modulate the expression of the genome in response to signals and changing environmental conditions. Reconstructions of gene regulatory networks can reveal the information processing and control principles used by cells to maintain homeostasis and execute cell-state transitions. Here, we introduce a computational framework, D-SPIN, that generates quantitative models of gene regulatory networks from single-cell mRNA-seq datasets collected across thousands of distinct perturbation conditions. D-SPIN constructs probabilistic models of regulatory interactions between genes or gene-expression programs to fit the cell state distributions under different perturbations. Using large Perturb-seq and drug-response datasets, we demonstrate that D-SPIN models reveal key regulators of cell fate decisions and the coordination of distant cellular pathways in response to gene knockdown perturbations. D-SPIN also dissects gene-level drug response mechanisms in heterogeneous cell populations, elucidating how combinations of immunomodulatory drugs acting on distinct regulators induce novel cell states through additive recruitment of gene expression programs. The D-SPIN model further predicts cell state distributions under drug dosage combinations beyond the training data. D-SPIN provides a computational framework for constructing interpretable models of gene regulatory networks to reveal principles of cellular information processing and physiological control.

bioinformatics↗

bioRxiv2019

Designing signaling environments to steer transcriptional diversity in neural progenitor cell populations

Stem and progenitor populations within developing embryos are diverse, composed of different subpopulations of precursor cells with varying developmental potential. How these different subpopulations are coordinately regulated by their signaling environments is not well understood. In this paper we develop a framework for controlling progenitor population structure in cell culture using high-throughput single cell mRNA-seq and computational analysis. We find that the natural transcriptional diversity of neural stem cell populations from the developing mouse brain collapses during in vitro culture. Cell populations are depleted of committed neuroblast progenitors and become dominated by a single pre-astrocytic cell population. By analyzing the response of neural stem cell populations to forty combinatorial signaling conditions, we demonstrate that signaling environments can restructure cell populations by modulating the relative abundance of pre-astrocytic and pre-neuronal subpopulations according to a simple log-linear model. Our work demonstrates that single-cell RNA-seq can be applied to learn how to modulate the diversity of stem cell populations, providing a new strategy for population-level stem cell control. HighlightsO_LINatural progenitor diversity in the brain collapses during in vitro culture to a single progenitor type C_LIO_LILoss of progenitor diversity alters fate potential of cells during differentiation C_LIO_LILarge scale single-cell signaling screen identifies signals that reshape population structure towards neuronal cell types C_LIO_LISignals regulate population structure according to a simple log-linear model C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/890087v3_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@e5286corg.highwire.dtl.DTLVardef@c0e69borg.highwire.dtl.DTLVardef@640b20org.highwire.dtl.DTLVardef@16779e_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2016

Transcriptomic Description of an Endogenous Female State in C. elegans

Understanding genome and gene function in a whole organism requires us to fully understand the life cycle and the physiology of the organism in question. Although it is traditionally though of as a hermaphrodite, C. elegans XX animals become (endogenous) females after 3 days of egg-laying. The molecular physiology of this state has not been studied as intensely as other parts of the life cycle, in spite of documented changes in behavior and metabolism that occur at this stage. To study the female state of C. elegans, we designed an experiment to measure the transcriptome of 1st day adult females, endogenous, 6th day adult females, as well as mutant feminized worms that never go through a hermaphrodite stage at these time points. Using this experimental design, we were able to measure the effects of biological aging from the transition into the female state. We find that spermless young adult animals partially phenocopy 6 day old wild-type animals that have depleted their sperm after egg-laying, and that spermless animals also exhibit fewer transcriptomic changes associated with aging throughout these 6 days. Our results indicate that sperm loss is responsible for some of the naturally occuring transcriptomic changes that occur during the life cycle of these animals. These changes involve a variety of factors, and they are enriched in transcription factors canonically associated with neuronal development and differentiation. Our data provide a high-quality picture of the changes that happen in global gene expression throughout the period of early aging in the worm.

genetics↗