Interlaboratory validation of the ToxTracker assay: An in vitro reporter assay for mechanistic genotoxicity assessment.

ToxTracker is a mammalian cell reporter assay that predicts the genotoxic properties of compounds with high accuracy. By evaluating induction of various reporter genes that play a key role in relevant cellular pathways, it provides insight into chemical mode-of-action (MoA), thereby supporting discrimination of direct-acting genotoxicants and cytotoxic chemicals. A comprehensive interlaboratory validation trial was conducted, in which the principles outlined in OECD Guidance Document 34 were followed, with the primary objectives of establishing transferability and reproducibility of the assay and confirming the ability of ToxTracker to correctly classify genotoxic and non-genotoxic compounds. Reproducibility of the assay to predict genotoxic MoA was confirmed across participating laboratories and data were evaluated in terms of concordance with in vivo genotoxicity outcomes. Seven laboratories tested a total of 64 genotoxic and non-genotoxic chemicals that together cover a broad chemical space. The within-laboratory reproducibility (WLR) was up to 98% (73%-98% across participants) and the overall between-laboratory reproducibility (BLR) was 83%. This trial confirmed the accuracy of ToxTracker to predict in vivo genotoxicants with a sensitivity of 84.4% and a specificity of 91.2%. We concluded that ToxTracker is a robust in vitro assay for the accurate prediction of in vivo genotoxicity. Considering ToxTracker's robust standalone accuracy and that it can provide important information on the MoA of chemicals, it is seen as a valuable addition to the regulatory in vitro genotoxicity battery that may even have the potential to replace certain currently used in vitro battery assays.

A novel G3BP1-GFP reporter human lung cell system enabling real-time monitoring of stress granule dynamics for in vitro lung toxicity assessment.

Under various cellular stress conditions, including exposure to toxic chemicals, RNA-binding proteins (RBPs), including Ras GTPase-activating protein-binding protein 1 (G3BP1), aggregate and form stress granule complexes, which serve as hallmarks of cellular stress. The existing methods for analyzing stress granule assembly have limitations in the rapid detection of dynamic cellular stress and ignore the effects of constitutively overexpressed RBP on cellular stress and stress-related processes. Therefore, to overcome these limitations, we established a G3BP1-GFP reporter in a human lung epithelial cell line using CRISPR/Cas9-based knock-in as an alternative system for stress granule analysis. We showed that the G3BP1-GFP reporter system responds to stress conditions and forms a stress granule complex similar to that of native G3BP1. Furthermore, we validated the stress granule response of an established cell line under exposure to various household chemicals. Overall, this novel G3BP1-GFP reporter human lung cell system is capable of monitoring stress granule dynamics in real time and can be used for assessing the lung toxicity of various substances in vitro.

Molecular Imaging with Aquaporin-Based Reporter Genes: Quantitative Considerations from Monte Carlo Diffusion Simulations.

Aquaporins provide a unique approach for imaging genetic activity in deep tissues by increasing the rate of cellular water diffusion, which generates a magnetic resonance contrast. However, distinguishing aquaporin signals from the tissue background is challenging because water diffusion is influenced by structural factors, such as cell size and packing density. Here, we developed a Monte Carlo model to analyze how cell radius and intracellular volume fraction quantitatively affect aquaporin signals. We demonstrated that a differential imaging approach based on subtracting signals at two diffusion times can improve specificity by unambiguously isolating aquaporin signals from the tissue background. We further used Monte Carlo simulations to analyze the connection between diffusivity and the percentage of cells engineered to express aquaporin and established a mapping that accurately determined the volume fraction of aquaporin-expressing cells in mixed populations. The quantitative framework developed in this study will enable a broad range of applications in biomedical synthetic biology, requiring the use of aquaporins to noninvasively monitor the location and function of genetically engineered devices in live animals.

Establishment of an In Bacterio Assay for the Assessment of Carbon Storage Regulator A (CsrA) Inhibitors.

Polymicrobial infections involving various combinations of microorganisms, such as Escherichia, Pseudomonas, or Yersinia, can lead to acute and chronic diseases in for example the gastrointestinal and respiratory tracts. Our aim is to modulate microbial communities by targeting the posttranscriptional regulator system called carbon storage regulator A (CsrA) (or also repressor of secondary metabolites (RsmA)). In previous studies, we identified easily accessible CsrA binding scaffolds and macrocyclic CsrA binding peptides through biophysical screening and phage display technology. However, due to the lack of an appropriate in bacterio assay to evaluate the cellular effects of these inhibitor hits, the focus of the present study is to establish an in bacterio assay capable of probing and quantifying the impact on CsrA-regulated cellular mechanisms. We have successfully developed an assay based on a luciferase reporter gene assay, which in combination with a qPCR expression gene assay, allows for the monitoring of expression levels of different downstream targets of CsrA. The chaperone protein CesT was used as a suitable positive control for the assay, and in time-dependent experiments, we observed a CesT-mediated increase in bioluminescence over time. By this means, the cellular on-target effects of non-bactericidal/non-bacteriostatic virulence modulating compounds targeting CsrA/RsmA can be evaluated.

Proteomics coupled with AhR-reporter gene bioassay for human and environmental safety assessment of sewage sludge and hydrochar.

Today application of sewage sludge (SL) and hydrochar (HC) in agriculture is a common practice for soil conditioning and crop fertilization, however safety concerns for human and environmental health due to the presence of toxic compounds have recently been expressed. Our aim was to test the suitability of proteomics coupled with bioanalytical tools for unravelling mixture effects of these applications in human and environmental safety assessment. We conducted proteomic and bioinformatic analysis of cell cultures used in the DR-CALUX® bioassay to identify proteins differentially abundant after exposure to SL and the corresponding HC, rather than only using the Bioanalytical Toxicity Equivalents (BEQs) obtained by DR-CALUX®. DR-CALUX® cells exposed to SL or HC showed a differential pattern of protein abundance depending on the type of SL and HC extract. The modified proteins are involved in antioxidant pathways, unfolded protein response and DNA damage that have close correlations with the effects of dioxin on biological systems and with onset of cancer and neurological disorders. Other cell response evidence suggested enrichment of heavy metals in the extracts. The present combined approach represents an advance in the application of bioanalytical tools for safety assessment of complex mixtures such as SL and HC. It proved successful in screening proteins, the abundance of which is determined by SL and HC and by the biological activity of legacy toxic compounds, including organohalogens.

High sensitivity and low-cost flavin luciferase (FLUXVc)-based reporter gene for mammalian cell expression.

Luciferase-based gene reporters generating bioluminescence signals are important tools for biomedical research. Amongst the luciferases, flavin-dependent enzymes use the most economical chemicals. However, their applications in mammalian cells are limited due to their low signals compared to other systems. Here, we constructed Flavin Luciferase from Vibrio campbellii (Vc) for Mammalian Cell Expression (FLUXVc) by engineering luciferase from V. campbellii (the most thermostable bacterial luciferase reported to date) and optimizing its expression and reporter assays in mammalian cells which can improve the bioluminescence light output by >400-fold as compared to the nonengineered version. We found that the FLUXVc reporter gene can be overexpressed in various cell lines and showed outstanding signal-to-background in HepG2 cells, significantly higher than that of firefly luciferase (Fluc). The combined use of FLUXVc/Fluc as target/control vectors gave the most stable signals, better than the standard set of Fluc(target)/Rluc(control). We also demonstrated that FLUXVc can be used for testing inhibitors of the NF-κB signaling pathway. Collectively, our results provide an optimized method for using the more economical flavin-dependent luciferase in mammalian cells.

Comprehensive assessment of NR ligand polypharmacology by a multiplex reporter NR assay.

Nuclear receptors (NR) are ligand-modulated transcription factors that regulate multiple cell functions and thus represent excellent drug targets. However, due to a considerable NR structural homology, NR ligands often interact with multiple receptors. Here, we describe a multiplex reporter assay (the FACTORIAL NR) that enables parallel assessment of NR ligand activity across all 48 human NRs. The assay comprises one-hybrid GAL4-NR reporter modules transiently transfected into test cells. To evaluate the reporter activity, we assessed their RNA transcripts. We used a homogeneous RNA detection approach that afforded equal detection efficacy and permitted the multiplex detection in a single-well format. For validation, we examined a panel of selective NR ligands and polypharmacological agonists and antagonists of the progestin, estrogen, PPAR, ERR, and ROR receptors. The assay produced highly reproducible NR activity profiles (r > 0.96) permitting quantitative assessment of individual NR responses. The inferred EC50 values agreed with the published data. The assay showed excellent quality ( = 0.73) and low variability ( = 7.2%). Furthermore, the assay permitted distinguishing direct and non-direct NR responses to ligands. Therefore, the FACTORIAL NR enables comprehensive evaluation of NR ligand polypharmacology.

A novel two-factor monosynaptic TRIO tracing method for assessment of circuit integration of hESC-derived dopamine transplants.

Transplantation in Parkinson's disease using human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons is a promising future treatment option. However, many of the mechanisms that govern their differentiation, maturation, and integration into the host circuitry remain elusive. Here, we engrafted hESCs differentiated toward a ventral midbrain DA phenotype into the midbrain of a preclinical rodent model of Parkinson's disease. We then injected a novel DA-neurotropic retrograde MNM008 adeno-associated virus vector capsid, into specific DA target regions to generate starter cells based on their axonal projections. Using monosynaptic rabies-based tracing, we demonstrated for the first time that grafted hESC-derived DA neurons receive distinctly different afferent inputs depending on their projections. The similarities to the host DA system suggest a previously unknown directed circuit integration. By evaluating the differential host-to-graft connectivity based on projection patterns, this novel approach offers a tool to answer outstanding questions regarding the integration of grafted hESC-derived DA neurons.

An ultra-low-cost electroporator with microneedle electrodes (ePatch) for SARS-CoV-2 vaccination.

Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other pathogens with pandemic potential requires safe, protective, inexpensive, and easily accessible vaccines that can be developed and manufactured rapidly at a large scale. DNA vaccines can achieve these criteria, but induction of strong immune responses has often required bulky, expensive electroporation devices. Here, we report an ultra-low-cost (<1 USD), handheld (<50 g) electroporation system utilizing a microneedle electrode array ("ePatch") for DNA vaccination against SARS-CoV-2. The low cost and small size are achieved by combining a thumb-operated piezoelectric pulser derived from a common household stove lighter that emits microsecond, bipolar, oscillatory electric pulses and a microneedle electrode array that targets delivery of high electric field strength pulses to the skin's epidermis. Antibody responses against SARS-CoV-2 induced by this electroporation system in mice were strong and enabled at least 10-fold dose sparing compared to conventional intramuscular or intradermal injection of the DNA vaccine. Vaccination was well tolerated with mild, transient effects on the skin. This ePatch system is easily portable, without any battery or other power source supply, offering an attractive, inexpensive approach for rapid and accessible DNA vaccination to combat COVID-19, as well as other epidemics.

Toxicity assessment of urban marine sediments from Western Norway using a battery of stress-activated receptors and cell-based bioassays from fish.

A luciferase reporter gene-based bioassay battery consisting of stress-activated receptors from fish, complemented with traditional fish cell-based bioassays, were used to assess the toxicity of marine sediment samples from the Byfjorden area around the city of Bergen (Norway). The reporter assays covered a wide range of cellular signalling and metabolic pathways, representing different molecular initiating events in the adverse outcome pathway framework. Cytotoxicity, generation of reactive oxygen-species, and induction of 7-ethoxyresorufin-O-deethylase activity were analysed using fish liver and gill cell lines. Chemical analyses of the sediment extracts revealed complex contamination profiles, especially at the innermost stations, which contained a wide array of persistent organic pollutants, polycyclic aromatic hydrocarbons, and metals. Sediment extracts from these sites were more potent in activating the stress-activated receptors than the other extracts, reflecting their toxicant profiles. Importantly, receptor- and cell-based bioassays complemented the chemical analyses and provided important data for future environmental risk assessments of urban marine sediments.

Assessment of heterogeneity in collective endothelial cell behavior with multicolor clonal cell tracking to predict arteriovenous remodeling.

Arteries and veins form in a stepwise process that combines vasculogenesis and sprouting angiogenesis. Despite extensive data on the mechanisms governing blood vessel assembly at the single-cell level, little is known about how collective cell migration contributes to the organization of the balanced distribution between arteries and veins. Here, we use an endothelial-specific zebrafish reporter, arteriobow, to label small cohorts of arterial cells and trace their progeny from early vasculogenesis throughout arteriovenous remodeling. We reveal that the genesis of arteries and veins relies on the coordination of 10 types of collective cell dynamics. Within these behavioral categories, we identify a heterogeneity of collective cell motion specific to either arterial or venous remodeling. Using pharmacological blockade, we further show that cell-intrinsic Notch signaling and cell-extrinsic blood flow act as regulators in maintaining the heterogeneity of collective endothelial cell behavior, which, in turn, instructs the future territory of arteriovenous remodeling.

Characterization of unsubstituted and methylated polycyclic aromatic hydrocarbons in settled dust: Combination of instrumental analysis and in vitro reporter gene assays and implications for cancer risk assessment.

Concentrations of 34 unsubstituted and methylated polycyclic aromatic hydrocarbons (PAHs and Me-PAHs) and AhR-mediated activities in settled dust samples were determined by a combination of gas chromatography-mass spectrometry and an in vitro reporter gene assay (PAH-CALUX). The levels of Σ34PAHs and bioassay-derived benzo[a]pyrene equivalents (CALUX BaP-EQs) were significantly higher in workplace dust from informal end-of-life vehicle dismantling workshops than in common house dust and road dust. In all the samples, the theoretical BaP-EQs of PAHs (calculated using PAH-CALUX relative potencies) accounted for 28 ± 19% of the CALUX BaP-EQs, suggesting significant contribution of aryl hydrocarbon receptor (AhR) agonists and/or mixture effects. Interestingly, the bioassay-derived BaP-EQs in these samples were significantly correlated with not only unsubstituted PAHs with known carcinogenic potencies but also many Me-PAHs, which should be included in future monitoring and toxicity tests. The bioassay responses of many sample extracts were substantially reduced but not suppressed with sulfuric acid treatment, indicating contribution of persistent AhR agonists. Cancer risk assessment based on the CALUX BaP-EQs has revealed unacceptable level of risk in many cases. The application of bioassay-derived BaP-EQs may reduce underestimation in environmental management and risk evaluation regarding PAHs and their derivatives (notably Me-PAHs), suggesting a consideration of using in vitro toxic activity instead of conventional chemical-specific approach in such assessment practices.

Assessment of AXL and mTOR genes expression in medullary thyroid carcinoma (MTC) cell line in relation with over expression of miR-144 and miR-34a.

OBJECTIVES: The aim of the present study was to investigate the expression of AXL and mTOR genes and their targeting microRNAs (miRNAs) including miR-34a and miR-144 in Medullary Thyroid Carcinoma (MTC) cell line, TT, and determine the effect of these two miRNAs on their target genes to introduce new molecular markers or therapeutics. METHODS: The expression of miR-34a, miR-144, and their targets genes including AXL and mTOR was evaluated by quantitative Real-time PCR. Luciferase assay was performed to confirm the interaction between miRNAs and their target mRNAs. The expression level of AXL and mTOR was evaluated before and after miRNAs induction in TT cell line compared with Cos7 as control cells. RESULTS: The expression of AXL and mTOR were up-regulated significantly, while miR-34a and miR-144 were down-regulated in TT cell line compared to Cos7. After transduction, the overexpression of miR-34a and 144 caused down-regulation of both genes. Luciferase assay results showed that the mTOR is targeted by miR-34a and miR-144 and the intensity of luciferase decreased in the presence of miRNAs. CONCLUSIONS: Based on the results of the present study and since AXL and mTOR genes play a critical role in variety of human cancers, suppression of these genes by their targeting miRNAs, especially miR-34a and miR-144, can be propose as a new strategy for MTC management. However, more studies are needed to approve the hypothesis.

Assessment of chemical mixtures using biomarkers of combined biological activity: A screening study in human placentas.

Humans are simultaneously exposed to complex mixtures of chemicals with limited knowledge on potential health effects, therefore improved tools for assessing these mixtures are needed. As part of the Human Biomonitoring for Europe (HBM4EU) Project, we aimed to examine the combined biological activity of chemical mixtures extracted from human placentas using one in vivo and four in vitro bioassays, also known as biomarkers of combined effect. Relevant endocrine activities (proliferative and/or reporter gene assays) and four endpoints were tested: the estrogen receptor (ER), androgen receptor (AR), and aryl hydrocarbon receptor (AhR) activities, as well as thyroid hormone (TH) signaling. Correlations among bioassays and their functional shapes were evaluated. Results showed that all placental extracts agonized or antagonized at least three of the abovementioned endpoints. Most placentas induced ER-mediated transactivation and ER-dependent cell proliferation, together with a strong inhibition of TH signaling and the AR transactivity; while the induction of the AhR was found in only one placental extract. The effects in the two estrogenic bioassays were positively and significantly correlated and the AR-antagonism activity showed a positive borderline-significant correlation with both estrogenic bioassay activities. However, the in vivo anti-thyroid activities of placental extracts were not correlated with any of the tested in vitro assays. Findings highlight the importance of comprehensively mapping the biological effects of "real-world" chemical mixtures present in human samples, through a battery of in vitro and in vivo bioassays. This approach should be a complementary tool for epidemiological studies to further elucidate the combined biological fingerprint triggered by chemical mixtures.

A practical method for multimodal registration and assessment of whole-brain disease burden using PET, MRI, and optical imaging.

Many neurological diseases present with substantial genetic and phenotypic heterogeneity, making assessment of these diseases challenging. This has led to ineffective treatments, significant morbidity, and high mortality rates for patients with neurological diseases, including brain cancers and neurodegenerative disorders. Improved understanding of this heterogeneity is necessary if more effective treatments are to be developed. We describe a new method to measure phenotypic heterogeneity across the whole rodent brain at multiple spatial scales. The method involves co-registration and localized comparison of in vivo radiologic images (e.g. MRI, PET) with ex vivo optical reporter images (e.g. labeled cells, molecular targets, microvasculature) of optically cleared tissue slices. Ex vivo fluorescent images of optically cleared pathology slices are acquired with a preclinical in vivo optical imaging system across the entire rodent brain in under five minutes, making this methodology practical and feasible for most preclinical imaging labs. The methodology is applied in various examples demonstrating how it might be used to cross-validate and compare in vivo radiologic imaging with ex vivo optical imaging techniques for assessing hypoxia, microvasculature, and tumor growth.

Silicon Photomultipliers as a Low-Cost Fluorescence Detector for Capillary Electrophoresis.

Capillary electrophoresis (CE) is a highly efficient separation method capable of handling small sample volumes (∼pL) and low (∼yoctomole) detection limits and, as such, is ideal for applications that require high sensitivity, such as single-cell analysis (Chen et al. Anal. Chem. 1996, 68 (4), 690-696; Cohen et al. Annu. Rev. Anal. Chem. 2008, 1 (1), 165-190; Vickerman et al. ACS Chem. Biol. 2018, 13 (7), 1741-1751). Low-cost CE instrumentation is quickly expanding, but low-cost, open-source fluorescence detectors with ultrasensitive detection limits are lacking (Vickerman et al. ACS Chem. Biol. 2018, 13 (7), 1741-1751; Fang et al. Electrophoresis 2016, 37 (17-18), 2376-2383; Casto et al. Anal. Chem. 2019, 40 (1), 65-78). Silicon photomultipliers (SiPM) are inexpensive, low-footprint detectors with the potential to fill the role as a detector when cost, size, and customization are important. In this work, we demonstrate the use of a SiPM in CE with zeptomolar detection limits and a dynamic range spanning 5 orders of magnitude, comparable to photomultiplier detectors. The performance of these detectors was measured using a continuous wave excitation laser in an epifluorescence detection configuration. We characterize the performance of the SiPM as a highly sensitive detector by measuring enzyme activity in single cells. This simple, small footprint, and low-cost (<$130) light detection circuit will be beneficial for open-source, portable, and budget-friendly instrumentation requiring high sensitivity.

Simultaneous quantitative assessment of two distinct cell lineages with a nuclear-localized dual genetic reporter.

Genetic lineage tracing has been widely used for studying in vivo cell fate plasticity during embryogenesis, tissue homeostasis, and disease development. Recent applications with multiple site-specific recombinases have been used in complex and sophisticated genetic fate mapping studies. However, the previous multicolor reporters for dual recombinases had limitations of precise in situ quantification of cell number, which is mainly due to the intermingling of cells in condensed tissues. Here, we generated a dual recombinase-mediated nuclear-localized GFP and tdTomato reporter line, which enables clear, simultaneous quantification of two distinct cell lineages in vivo. Combining this dual genetic reporter with Tbx18-Cre and Cdh5-Dre lines, which genetically trace epicardial and endothelial cells, respectively, we obtained high-resolution images for the anatomic distribution of the descendants of these two distinct cell lineages in the valve mesenchyme during development, remodeling, and maturation stages. This new dual genetic reporter is expected to facilitate fate tracing of two cell lineages and their objective quantification in vivo.

In Vivo Assessment of Size-Selective Glomerular Sieving in Transplanted Human Induced Pluripotent Stem Cell-Derived Kidney Organoids.

BACKGROUND: The utility of kidney organoids in regenerative medicine will rely on the functionality of the glomerular and tubular structures in these tissues. Recent studies have demonstrated the vascularization and subsequent maturation of human pluripotent stem cell-derived kidney organoids after renal subcapsular transplantation. This raises the question of whether the glomeruli also become functional upon transplantation. METHODS: We transplanted kidney organoids under the renal capsule of the left kidney in immunodeficient mice followed by the implantation of a titanium imaging window on top of the kidney organoid. To assess glomerular function in the transplanted human pluripotent stem cell-derived kidney tissue 1, 2, and 3 weeks after transplantation, we applied high-resolution intravital multiphoton imaging through the imaging window during intravenous infusion of fluorescently labeled low and high molecular mass dextran molecules or albumin. RESULTS: After vascularization, glomerular structures in the organoid displayed dextran and albumin size selectivity across their glomerular filtration barrier. We also observed evidence of proximal tubular dextran reuptake. CONCLUSIONS: Our results demonstrate that human pluripotent stem cell-derived glomeruli can develop an appropriate barrier function and discriminate between molecules of varying size. These characteristics together with tubular presence of low molecular mass dextran provide clear evidence of functional filtration. This approach to visualizing glomerular filtration function will be instrumental for translation of organoid technology for clinical applications as well as for disease modeling.

The mito-QC Reporter for Quantitative Mitophagy Assessment in Primary Retinal Ganglion Cells and Experimental Glaucoma Models.

Mitochondrial damage plays a prominent role in glaucoma. The only way cells can degrade whole mitochondria is via autophagy, in a process called mitophagy. Thus, studying mitophagy in the context of glaucoma is essential to understand the disease. Up to date limited tools are available for analyzing mitophagy in vivo. We have taken advantage of the mito-QC reporter, a recently generated mouse model that allows an accurate mitophagy assessment to fill this gap. We used primary RGCs and retinal explants derived from mito-QC mice to quantify mitophagy activation in vitro and ex vivo. We also analyzed mitophagy in retinal ganglion cells (RGCs), in vivo, using different mitophagy inducers, as well as after optic nerve crush (ONC) in mice, a commonly used surgical procedure to model glaucoma. Using mito-QC reporter we quantified mitophagy induced by several known inducers in primary RGCs in vitro, ex vivo and in vivo. We also found that RGCs were rescued from some glaucoma relevant stress factors by incubation with the iron chelator deferiprone (DFP). Thus, the mito-QC reporter-based model is a valuable tool for accurately analyzing mitophagy in the context of glaucoma.

Assessment of faithful interleukin-3 production by novel bicistronic interleukin-3 reporter mice.

Interleukin-3 (IL-3) is an important hematopoietic growth factor and immunregulatory cytokine. Although activated T helper cells represent a main source of IL-3, other cell types have been reported to express this cytokine. However, precise identification and quantification of the cells that produce IL-3 in vivo have not been performed. Therefore, we used a CRISPR/Cas approach to engineer mice containing a bicistronic mRNA linking a readily identifiable reporter, enhanced green fluorescent protein (ZsGreen1), to IL-3 expression. To characterize these novel reporter mice, we first examined ZsGreen1 expression by CD4 T cells subsets primed and activated in vitro. We found that activated Th1 cells expressed ∼4-fold higher levels of ZsGreen1 as compared to Th0 and Th2 cells. Endogenous IL-3 expression remained intact although reporter Th1 cells secreted ∼33 % less IL-3 than similarly activated wild-type cells. To characterize the ability of reporter mice to accurately mark IL-3-producing cells in vivo, we infected mice with Nippostrongylus brasiliensis. Low but significant numbers of ZsGreen1+ CD4 T cells were detected in the mesenteric lymph nodes and lung following both primary and secondary infection. No difference in basophil and intestinal mast cell numbers were observed between infected reporter and wild-type mice indicating that reporter mice secreted IL-3 levels in vivo that results in IL-3-driven biological activities which are indistinguishable from those observed in corresponding wild-type mice. These IL-3 reporter mice will be a valuable resource to investigate IL-3-dependent immune responses in vivo.