Transgenic fluorescent zebrafish lines that have revolutionized biomedical research.
Ontology highlight
ABSTRACT:
SUBMITTER: Choe CP
PROVIDER: S-EPMC8424172 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Amyotrophic lateral sclerosis (ALS) is a late-onset progressive neurodegenerative disorder that affects both upper and lower motor neurons, leading to muscle atrophy with spasticity and eventual death in 3-5 years after the disease onset. More than 50 mutations linked to ALS have been found in the gene TARDBP, encoding the protein TDP-43 that is the predominant component of neuronal inclusions in ALS. TDP-43 is an RNA binding protein with glycine-rich domains that binds to more than 6,000 RNAs in the human brain. However, ALS-related mutations do not appear to affect the function of these genes, indicating that a toxic gain-of-function may occur. We generated transgenic zebrafish lines expressing human TDP-43, either the wild-type form or the ALS-causative G348C mutation identified in a subset of ALS patients, with the transgene expression driven by an inducible heat shock promoter in order to bypass a potential early mortality. The expression of the mutant but not the wild-type human TDP-43 in zebrafish embryos induced a reduction of the locomotor activity in response to touch compared to controls and moderate axonopathy of the motor neurons of the spinal cord, with premature branching of the main axonal branch, recapitulating previous results obtained by mRNA injections. We used these lines to investigate transcriptomic changes due to the presence of mutant TDP-43 using RNA sequencing and have found 159 genes that are differentially expressed compared to control, with 67 genes up-regulated and 92 genes down-regulated. These transcriptomic changes are in line with recent transcriptomic data obtained in mouse models, indicating that these zebrafish transgenic lines are adequate to further study TDP-43-related ALS.
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Project description:Lung cancer cell lines have made a substantial contribution to lung cancer translational research and biomedical discovery. A systematic approach to initiating and characterizing cell lines from small cell and non-small cell lung carcinomas has led to the current collection of more than 200 lung cancer cell lines, a number that exceeds those for other common epithelial cancers combined. The ready availability and widespread dissemination of the lines to investigators worldwide have resulted in more than 9000 citations, including multiple examples of important biomedical discoveries. The high (but not perfect) genomic similarities between lung cancer cell lines and the lung tumor type from which they were derived provide evidence of the relevance of their use. However, major problems including misidentification or cell line contamination remain. Ongoing studies and new approaches are expected to reveal the full potential of the lung cancer cell line panel.
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Project description:Previously a standard toxicological test termed as DarT (Danio rerio Teratogenic assay) using wild type zebrafish embryos has been established and it is widely applied in toxicological and chemical screenings. As an increasing number of fluorescent transgenic zebrafish lines with specific fluorescent protein expression specifically expressed in different organs and tissues, we envision that the fluorescent markers may provide more sensitive endpoints for monitoring chemical induced phenotypical changes. Here we employed Tg(nkx2.2a:mEGFP) transgenic zebrafish which have GFP expression in the central nervous system to investigate its potential for screening neurotoxic chemicals. Five potential neurotoxins (acetaminophen, atenolol, atrazine, ethanol and lindane) and one neuroprotectant (mefenamic acid) were tested. We found that the GFP-labeled ventral axons from trunk motoneurons, which were easily observed in live fry and measured for quantification, were a highly sensitive to all of the five neurotoxins and the length of axons was significantly reduced in fry which looked normal based on DarT endpoints at low concentrations of neurotoxins. Compared to the most sensitive endpoints of DarT, ventral axon marker could improve the detection limit of these neurotoxins by about 10 fold. In contrast, there was no improvement for detection of the mefenamic acid compared to all DarT endpoints. Thus, ventral axon lengths provide a convenient and measureable marker specifically for neurotoxins. Our study may open a new avenue to use other fluorescent transgenic zebrafish embryos/fry to develop sensitive and specific toxicological tests for different categories of chemicals.
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Project description:Decoding the functional connectivity of the nervous system is facilitated by transgenic methods that express a genetically encoded reporter or effector in specific neurons; however, most transgenic lines show broad spatiotemporal and cell-type expression. Increased specificity can be achieved using intersectional genetic methods which restrict reporter expression to cells that co-express multiple drivers, such as Gal4 and Cre. To facilitate intersectional targeting in zebrafish, we have generated more than 50 new Cre lines, and co-registered brain expression images with the Zebrafish Brain Browser, a cellular resolution atlas of 264 transgenic lines. Lines labeling neurons of interest can be identified using a web-browser to perform a 3D spatial search (zbbrowser.com). This resource facilitates the design of intersectional genetic experiments and will advance a wide range of precision circuit-mapping studies.
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Project description:Selecting appropriate cell lines to represent a disease is crucial for the success of biomedical research, because the usage of less relevant cell lines could deliver misleading results. However, systematic guidance on cell line selection is unavailable. Here we developed a clinical Genomics-guided Prioritizing Strategy for Cancer Cell Lines (CCL-cGPS) and help to guide this process. Statistical analyses revealed CCL-cGPS selected cell lines were among the most appropriate models. Moreover, we observed a linear correlation between the drug response and CCL-cGPS score of cell lines for breast and thyroid cancers. Using RT4 cells selected by CCL-GPS, we identified mebendazole and digitoxin as candidate drugs against bladder cancer and validate their promising anticancer effect through in vitro and in vivo experiments. Additionally, a web tool was developed. In conclusion, CCL-cGPS bridges the gap between tumors and cell lines, presenting a helpful guide to select the most suitable cell line models.
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Project description:The Rab family of small GTPases function as molecular switches regulating membrane and protein trafficking. Individual Rab isoforms define and are required for specific endosomal compartments. To facilitate in vivo investigation of specific Rab proteins, and endosome biology in general, we have generated transgenic zebrafish lines to mark and manipulate Rab proteins. We also developed software to track and quantify endosome dynamics within time-lapse movies. The established transgenic lines ubiquitously express EGFP fusions of Rab5c (early endosomes), Rab11a (recycling endosomes), and Rab7 (late endosomes) to study localization and dynamics during development. Additionally, we generated UAS-based transgenic lines expressing constitutive active (CA) and dominant-negative (DN) versions for each of these Rab proteins. Predicted localization and functional consequences for each line were verified through a variety of assays, including lipophilic dye uptake and Crumbs2a localization. In summary, we have established a toolset for in vivo analyses of endosome dynamics and functions.
| S-EPMC3197968 | biostudies-literature
Project description:Transgenic methods enable the selective manipulation of neurons for functional mapping of neuronal circuits. Using confocal microscopy, we have imaged the cellular-level expression of 109 transgenic lines in live 6 day post fertilization larvae, including 80 Gal4 enhancer trap lines, 9 Cre enhancer trap lines and 20 transgenic lines that express fluorescent proteins in defined gene-specific patterns. Image stacks were acquired at single micron resolution, together with a broadly expressed neural marker, which we used to align enhancer trap reporter patterns into a common 3-dimensional reference space. To facilitate use of this resource, we have written software that enables searching for transgenic lines that label cells within a selectable 3-dimensional region of interest (ROI) or neuroanatomical area. This software also enables the intersectional expression of transgenes to be predicted, a feature which we validated by detecting cells with co-expression of Cre and Gal4. Many of the imaged enhancer trap lines show intrinsic brain-specific expression. However, to increase the utility of lines that also drive expression in non-neuronal tissue we have designed a novel UAS reporter, that suppresses expression in heart, muscle, and skin through the incorporation of microRNA binding sites in a synthetic 3' untranslated region. Finally, we mapped the site of transgene integration, thus providing molecular identification of the expression pattern for most lines. Cumulatively, this library of enhancer trap lines provides genetic access to 70% of the larval brain and is therefore a powerful and broadly accessible tool for the dissection of neural circuits in larval zebrafish.
| S-EPMC4656851 | biostudies-literature
Project description:IntroductionThe gene encoding isocitrate dehydrogenase 1 (IDH1) is frequently mutated in several tumor types including gliomas. The most prevalent mutation in gliomas is a missense mutation leading to a substitution of arginine with histidine at the residue 132 (R132H). Wild type IDH1 catalyzes oxidative decarboxylation of isocitrate to ?-ketoglutarate (?-KG) whereas mutant IDH1 converts ?-KG into D2-hydroxyglutarate (D2HG). Unfortunately, there are few in vivo model systems for IDH-mutated tumors to study the effects of IDH1 mutations in tumor development. We have therefore created transgenic zebrafish lines that express various IDH1 mutants.Materials and methodsIDH1 mutations (IDH1R132H, IDH1R132C and loss-of-function mutation IDH1G70D), IDH1wildtype or eGFP were cloned into constructs with several brain-specific promoters (Nestin, Gfap or Gata2). These constructs were injected into fertilized zebrafish eggs at the one-cell stage.ResultsIn total more than ten transgenic zebrafish lines expressing various brain-specific IDH1 mutations were created. A significant increase in the level of D2HG was observed in all transgenic lines expressing IDH1R132C or IDH1R132H, but not in any of the lines expressing IDH1wildtype, IDH1G70D or eGFP. No differences in 5-hydroxymethyl cytosine and mature collagen IV levels were observed between wildtype and mutant IDH1 transgenic fish. To our surprise, we failed to identify any strong phenotype, despite increased levels of the oncometabolite D2HG. No tumors were observed, even when backcrossing with tp53-mutant fish which suggests that additional transforming events are required for tumor formation. Elevated D2HG levels could be lowered by treatment of the transgenic zebrafish with an inhibitor of mutant IDH1 activity.ConclusionsWe have generated a transgenic zebrafish model system for mutations in IDH1 that can be used for functional analysis and drug screening. Our model systems help understand the biology of IDH1 mutations and its role in tumor formation.
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Project description:Herein, we report on near-infrared (NIR) fluorescent nanoparticles generated from an emergent class of materials we refer to as a Group of Uniform Materials Based on Organic Salts (GUMBOS). GUMBOS are largely frozen ionic liquids, although the concept is more general and is also easily applied to solid ionic materials with melting points in excess of 100 degrees C. Nanoparticles based on GUMBOS (nanoGUMBOS) derived from a NIR fluorophore are prepared using a reprecipitation method and evaluated for in vivo fluorescence imaging. Due to their uniformity, single-step preparation, and composite nature, nanoGUMBOS help to resolve issues with dye leakage problems innate to alternate cellular stains and unlock a myriad of applications for these materials, highlighting exciting possibilities for multifunctional nanoGUMBOS.
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Project description:In this protocol, we describe steps that utilize the optical clarity of the zebrafish larvae and the stereotyped motor neuron axon structure in the trunk to measure spontaneous or evoked motor neuron axon activity. This activity is detected with transgenic fluorescent indicators introduced into the larvae by zygotic injection. Fluorescent indicator intensity changes in the small neuromuscular junctions are quantified to measure the presynaptic calcium activity and consequent synaptic vesicle release. For complete details on the use and execution of this protocol, please refer to Mandal et al. (2020).
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