Biology subjects

Wilson, J. A.

Publications and source records attributed to Wilson, J. A..

7 recordsLinked to original sources

bioRxiv2026

An optimized protocol for single-brain isolation of sex-defined pure mouse astrocyte cultures

BackgroundPrimary astrocyte cultures derived from neonatal rodent cortices provide a controlled system for investigating astrocyte-specific mechanisms. However, mixed glial preparations frequently contain contaminating microglia and oligodendrocyte progenitor cells, and most existing protocols require pooling tissue from multiple mouse pups to obtain sufficient astrocyte yields. This approach is impractical as it obscures sex and genotype, limits investigations of sex dependent astrocyte phenotypes, and precludes studies in certain transgenic models. To address this gap, our protocol achieves a high astrocyte yield from a single neonatal mouse brain, enabling sex- and genotype-specific cultures without the need for pooling. Mechanical removal of oligodendrocyte progenitors combined with pharmacological depletion of microglia using a Colony Stimulating Factor 1 Receptor (CSF1R) inhibitor produces highly enriched astrocytes suitable for functional assays, including those focused on sex-specific biology. MethodsCortical tissue was isolated from a single mouse pup is mechanically dissociated in astrocyte media. Cell suspensions are transferred to poly-D-lysine-coated flasks in astrocyte media. After 10-15 days in culture, OPCs are mechanically removed by horizontal shaking and microglia are selectively depleted by incubating cultures with CSF1R inhibitor PLX5622 for 24, 48, 72 and 96 hours. After PLX treatment, media is replaced and enriched astrocytes were maintained or passaged for experimentation. The sex of the pups is determined by PCR performed on DNA extracted from tail biopsies. ResultsImmunocytochemical analysis for astrocyte and microglia markers (GFAP and Iba1, respectively) showed that 24 hours of PLX5622 treatment did not fully eliminate microglia from mixed glial cultures. Extending treatment to 48 hours effectively depleted microglia while minimizing cytotoxicity and astrocyte loss and produced a pure, high-yield, sex-specific primary astrocyte culture. PCR reliably enabled the sex identification of pups used in culture using DNA extracted from tail biopsies. DiscussionThis protocol provides an efficient and reproducible method for generating high-purity, sex-specific primary astrocyte cultures from a single mouse brain. It improves consistency and purity while eliminating the need to pool tissue, preserving sex and genotype and enabling studies in transgenic mouse lines of both sexes.

pharmacology and toxicology↗

bioRxiv2025

Genome-wide Screening Identifies Unique Host-Directed Drugs and Pro-viral Signalling Pathways for SARS-CoV-2

SARS-CoV-2 is a positive-sense RNA virus and was responsible for the devastating COVID-19 pandemic. Although the current disease burden is less severe, there are limited treatment options, significant gaps in knowledge, and a looming threat of the emergence of variants and future pandemics. To address these challenges, we performed genome-wide CRISPR knockout screens in a novel human lung cell line NCI-H23ACE2, as well as in HEK293TACE2 cells, with SARS-CoV-2 Wuhan virus, with the aim of identifying host-dependency factors that could predict effective antivirals. We identified four host-directed drugs, donepezil, dH-ergocristine, trametinib and sorafenib, that could potentially be repurposed to treat coronavirus infections. Three of the drugs inhibited SARS-CoV-2, HCoV-229E, and HCoV-OC43, suggesting they could be used as pan-coronavirus antivirals. We also confirmed that SARS-CoV-2 relies on the NRAS/Raf/MEK/ERK signaling pathway for its replication. Our study highlights the robustness and efficiency of a bilateral approach of gene silencing and antiviral screening to identify host-dependency factors and effective antivirals.

microbiology↗

bioRxiv2023

Ultra-high throughput mapping of genetic design space

Massively parallel genetic screens have been used to map sequence-to-function relationships for a variety of genetic elements. However, because these approaches only interrogate short sequences, it remains challenging to perform high throughput (HT) assays on constructs containing combinations of multiple sequence elements arranged across multi-kb length scales. Overcoming this barrier could accelerate synthetic biology; by screening diverse gene circuit designs and learning "composition-to-function" mappings that reveal genetic part composability rules and enable rapid identification of behavior-optimized variants. Here, we introduce CLASSIC, a genetic screening platform that combines long- and short-read next-generation sequencing (NGS) modalities to quantitatively assess pools of constructs of arbitrary length containing diverse part compositions. We show that CLAS-SIC can measure expression profiles of >105 gene circuit designs (from 5-20 kb) in a single experiment in human cells. The resulting datasets can be used to train ML models that accurately predict circuit behavior across expansive circuit design landscapes, revealing part composability rules that govern circuit performance. Our work shows that by expanding the throughput of each design-build-test-learn (DBTL) cycle, CLASSIC enhances the pace and scale of synthetic biology and establishes an experimental basis for data-driven design of complex genetic systems.

synthetic biology↗

bioRxiv2023

Generation of a SARS-CoV-2 reverse genetics system and novel human lung cell lines that exhibit high virus-induced cytopathology

The global COVID-19 pandemic continues with an increasing number of cases worldwide and the emergence of new SARS-CoV-2 variants. In our study, we have developed novel tools with applications for screening antivirals, identifying virus-host dependencies, and characterizing viral variants. Using reverse genetics, we rescued SARS-CoV-2 Wuhan1 (D614G variant) wild type (WTFL) and reporter virus (NLucFL) using molecular BAC clones. The replication kinetics, plaque morphology and titers were comparable between rescued molecular clones and a clinical isolate (VIDO-01 strain), thus providing confidence that the rescued viruses can be used as effective replication tools. Furthermore, the reporter SARS-CoV-2 NLucFL virus exhibited robust luciferase values over the time course of infection and was used to develop a rapid antiviral assay using remdesivir as proof-of-principle. In addition, as a tool to study lung-relevant virus-host interactions, we established novel human lung cell lines that support SARS-CoV-2 infection with high virus-induced cytopathology. Six lung cell lines (NCI-H23, A549, NCI-H1703, NCI-H520, NCI-H226, and HCC827) and HEK293T cells, were transduced to stably express ACE2 and tested for their ability to support virus infection. A549ACE2 B1 and HEK293TACE2 A2 cell lines exhibited more than 70% virus-induced cell death and a novel lung cell line NCI-H23ACE2 A3 showed about [~]99% cell death post-infection. These cell lines are ideal for assays relying on live-dead selection and are currently being used in CRISPR knockout and activation screens in our lab. ImportanceWe used a reverse genetics system to generate a wild type as well as a nanoluciferase-expressing reporter clone of SARS-CoV-2. The reporter virus allows for rapid transient replication assays and high throughput screens by detection of virus replication using luciferase assays. In addition, the reverse genetic system can be used to generate mutant viruses to study phenotypes of variant mutations. Additionally, unique human lung cell lines supporting SARS-CoV-2 replication will aid in studying the virus in a lung-relevant environment and based on high cytopathology induced in some cell lines, will be useful for screens that rely on virus-induced cell death for selection. Our study aims to enhance and contribute to the current replication tools available to study SARS-CoV-2 by providing rapid methods, virus clones and novel lung cell lines.

molecular biology↗

bioRxiv2021

miR-122 affects both the initiation and maintenance of Hepatitis C Virus infections

A liver-specific microRNA, miR-122, anneals to the HCV genomic 5 terminus and is essential for virus replication in cell culture. However, bicistronic HCV replicons and full length RNAs with specific mutations in the 5 UTR can replicate, albeit to low levels, without miR-122. In this study, we have identified that HCV RNAs lacking the structural gene region or having EMCV IRES-regulated translation had reduced requirements for miR-122. In addition, we found that a smaller proportion of cells supported miR-122-independent replication when compared a population of cells supporting miR-122-dependent replication, while viral protein levels per positive cell were similar. Further, the proportion of cells supporting miR-122-independent replication increased with the amount of viral RNA delivered, suggesting that establishment of miR-122-independent replication in a cell is affected by amount of viral RNA delivered. HCV RNAs replicating independent of miR-122 were not affected by supplementation with miR-122, suggesting that miR-122 is not essential for maintenance of a miR-122-independent HCV infection. However, miR-122 supplementation had a small positive impact on miR-122-dependent replication suggesting a minor role in enhancing ongoing virus RNA accumulation. We suggest that miR-122 functions primarily to initiate an HCV infection but has a minor influence on its maintenance, and we present a model in which miR-122 is required for replication complex formation at the beginning of an infection, and also supports new replication complex formation during ongoing infection and after infected cell division. IMPORTANCEThe mechanism by which miR-122 promotes the HCV life cycle is not well understood, and a role in directly promoting genome amplification is still debated. In this study, we have shown that miR-122 increases the rate of viral RNA accumulation and promotes the establishment of an HCV infection in a greater number of cells than in the absence of miR-122. However, we also confirm a minor role in promoting ongoing virus replication and propose a role in the initiation of new replication complexes throughout a virus infection. This study has implications for the use of anti-miR-122 as potential HCV therapy.

microbiology↗

bioRxiv2021

Enhanced virus translation enables miR-122-independent Hepatitis C Virus propagation

The 5UTR of the Hepatitis C Virus genome forms RNA structures that regulate virus replication and translation. The region contains a viral internal ribosomal entry site and a 5 terminal region. Binding of the liver specific miRNA, miR-122, to two conserved binding sites in the 5 terminal region regulates viral replication, translation, and genome stability, and is essential for efficient virus replication, but its precise mechanism of its action is still under debate. A current hypothesis is that miR-122 binding stimulates viral translation by facilitating the viral 5 UTR to form the translationally active HCV IRES RNA structure. While miR-122 is essential for detectable virus replication in cell culture, several viral variants with 5 UTR mutations exhibit low level replication in the absence of miR-122. We show that HCV mutants capable of replicating independently of miR-122 also replicate independently of other microRNAs generated by the canonical miRNA synthesis pathway. Further, we also show that the mutant genomes display an enhanced translation phenotype that correlates with their ability to replicate independently of miR-122. Finally, we provide evidence that translation regulation is the major role for miR-122, and show that miR-122-independent HCV replication can be rescued to miR-122-dependent levels by the combined impacts of 5 UTR mutations that stimulate translation, and by stabilizing the viral genome by knockdown of host exonucleases and phosphatases that degrade the genome. Thus, we provide a model suggesting that translation stimulation and genome stabilization are the primary roles for miR-122 in the virus life cycle. IMPORTANCEThe unusual role of miR-122 in promoting HCV propagation is incompletely understood but is essential for an HCV infection. To better understand its role, we have analyzed HCV mutants capable of replicating independently of miR-122. Our data show that the ability of viruses to replicate independently of miR-122 correlates with enhanced virus translation, but that genome stabilization is required to restore efficient HCV replication. This suggests that viruses must gain both abilities to escape the need for miR-122 and impacts the possibility that HCV can evolve to replicate outside of the liver.

microbiology↗

bioRxiv2020

Location specific small RNA annealing to the HCV 5' UTR promotes Hepatitis C Virus replication by favoring IRES formation and stimulating virus translation

Hepatitis C virus (HCV) genome replication requires annealing of a liver specific small-RNA, miR-122 to 2 sites on 5 untranslated region (UTR). Annealing has been reported to a) stabilize the genome, b) promote translation, and c) induce the canonical HCV 5 UTR Internal Ribosome Entry Site (IRES) structure. In this report we identify the relative impact of small RNA annealing on the three functions ascribed to miR-122 and generate a mechanistic model for miR-122 promotion of HCV. First, we identified that perfectly complementary small RNAs that anneal to different locations on the HCV 5 UTR stimulate replication with varying efficiencies and mapped the region on the HCV genome to which small RNA annealing promotes virus replication. Second, by using a panel of small RNAs that promote with varying efficiencies we link HCV replication induction with translation stimulation and 5 UTR RNA structure modifications. However, replication promotion was not linked to genome stabilization since all small RNAs tested could stabilize the viral genome regardless of their ability to promote replication. Thus, we propose that miR-122 annealing promotes HCV replication primarily by activating the HCV IRES and stimulating translation, and that miR-122-induced HCV genome stabilization is insufficient alone but enhances virus replication. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/008417v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@178a857org.highwire.dtl.DTLVardef@741083org.highwire.dtl.DTLVardef@1e84743org.highwire.dtl.DTLVardef@9eadd2_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

microbiology↗