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Mathias J.R.,Luminomics, Inc. | Zhang Z.,Georgia Regents University | Saxena M.T.,Luminomics, Inc. | Mumm J.S.,Georgia Regents University | Mumm J.S.,Wilmer Eye Institute
Zebrafish | Year: 2014

Transgenic expression of bacterial nitroreductase (NTR) facilitates chemically-inducible targeted cell ablation. In zebrafish, the NTR system enables studies of cell function and cellular regeneration. Metronidazole (MTZ) has become the most commonly used prodrug substrate for eliciting cell loss in NTR-expressing transgenic zebrafish due to the cell-specific nature of its cytotoxic derivatives. Unfortunately, MTZ treatments required for effective cell ablation border toxic effects, and, thus, likely incur undesirable nonspecific effects. Here, we tested whether a triple mutant variant of NTR, previously shown to display improved activity in bacterial assays, can solve this issue by promoting cell ablation in zebrafish using reduced prodrug treatment regimens. We generated several complementary transgenic zebrafish lines expressing either wild-type or mutant NTR (mutNTR) in specific neural cell types, and assayed prodrug-induced cell ablation kinetics using confocal time series imaging and plate reader-based quantification of fluorescent reporters expressed in targeted cell types. The results show that cell ablation can be achieved in mutNTR expressing transgenic lines with markedly shortened prodrug exposure times and/or at lower prodrug concentrations. The mutNTR variant characterized here can circumvent problematic nonspecific/toxic effects arising from low prodrug conversion efficiency, thus increasing the effectiveness and versatility of this selective cell ablation methodology. © Copyright 2014, Mary Ann Liebert, Inc. 2014.


Xie X.,University of Georgia | Mathias J.R.,Luminomics, Inc. | Smith M.-A.,Luminomics, Inc. | Walker S.L.,University of Georgia | And 8 more authors.
BMC Biology | Year: 2012

Background: We have investigated a simple strategy for enhancing transgene expression specificity by leveraging genetic silencer elements. The approach serves to restrict transgene expression to a tissue of interest - the nervous system in the example provided here - thereby promoting specific/exclusive targeting of discrete cellular subtypes. Recent innovations are bringing us closer to understanding how the brain is organized, how neural circuits function, and how neurons can be regenerated. Fluorescent proteins enable mapping of the 'connectome', optogenetic tools allow excitable cells to be short-circuited or hyperactivated, and targeted ablation of neuronal subtypes facilitates investigations of circuit function and neuronal regeneration. Optimally, such toolsets need to be expressed solely within the cell types of interest as off-site expression makes establishing causal relationships difficult. To address this, we have exploited a gene 'silencing' system that promotes neuronal specificity by repressing expression in non-neural tissues. This methodology solves non-specific background issues that plague large-scale enhancer trap efforts and may provide a means of leveraging promoters/enhancers that otherwise express too broadly to be of value for in vivo manipulations.Results: We show that a conserved neuron-restrictive silencer element (NRSE) can function to restrict transgene expression to the nervous system. The neuron-restrictive silencing factor/repressor element 1 silencing transcription factor (NRSF/REST) transcriptional repressor binds NRSE/repressor element 1 (RE1) sites and silences gene expression in non-neuronal cells. Inserting NRSE sites into transgenes strongly biased expression to neural tissues. NRSE sequences were effective in restricting expression of bipartite Gal4-based 'driver' transgenes within the context of an enhancer trap and when associated with a defined promoter and enhancer. However, NRSE sequences did not serve to restrict expression of an upstream activating sequence (UAS)-based reporter/effector transgene when associated solely with the UAS element. Morpholino knockdown assays showed that NRSF/REST expression is required for NRSE-based transgene silencing.Conclusions: Our findings demonstrate that the addition of NRSE sequences to transgenes can provide useful new tools for functional studies of the nervous system. However, the general approach may be more broadly applicable; tissue-specific silencer elements are operable in tissues other than the nervous system, suggesting this approach can be similarly applied to other paradigms. Thus, creating synthetic associations between endogenous regulatory elements and tissue-specific silencers may facilitate targeting of cellular subtypes for which defined promoters/enhancers are lacking. © 2012 Xie et al; licensee BioMed Central Ltd.


Mathias J.R.,Luminomics, Inc. | Saxena M.T.,Luminomics, Inc. | Mumm J.S.,University of Georgia
Future Medicinal Chemistry | Year: 2012

Due to several inherent advantages, zebrafish are being utilized in increasingly sophisticated screens to assess the physiological effects of chemical compounds directly in living vertebrate organisms. Diverse screening platforms showcase these advantages. Morphological assays encompassing basic qualitative observations to automated imaging, manipulation, and data-processing systems provide whole organism to subcellular levels of detail. Behavioral screens extend chemical screening to the level of complex systems. In addition, zebrafish-based disease models provide a means of identifying new potential therapeutic strategies. Automated systems for handling/sorting, high-resolution imaging and quantitative data collection have significantly increased throughput in recent years. These advances will make it easier to capture multiple streams of information from a given sample and facilitate integration of zebrafish at the earliest stages of the drug-discovery process, providing potential solutions to current drug-development bottlenecks. Here we outline advances that have been made within the growing field of zebrafish chemical screening. © 2012 Future Science Ltd.


Walker S.L.,University of Georgia | Ariga J.,University of Georgia | Mathias J.R.,Luminomics, Inc. | Coothankandaswamy V.,University of Georgia | And 10 more authors.
PLoS ONE | Year: 2012

Reporter-based assays underlie many high-throughput screening (HTS) platforms, but most are limited to in vitro applications. Here, we report a simple whole-organism HTS method for quantifying changes in reporter intensity in individual zebrafish over time termed, Automated Reporter Quantification in vivo (ARQiv). ARQiv differs from current "high-content" (e.g., confocal imaging-based) whole-organism screening technologies by providing a purely quantitative data acquisition approach that affords marked improvements in throughput. ARQiv uses a fluorescence microplate reader with specific detection functionalities necessary for robust quantification of reporter signals in vivo. This approach is: 1) Rapid; achieving true HTS capacities (i.e., >50,000 units per day), 2) Reproducible; attaining HTS-compatible assay quality (i.e., Z'-factors of ≥0.5), and 3) Flexible; amenable to nearly any reporter-based assay in zebrafish embryos, larvae, or juveniles. ARQiv is used here to quantify changes in: 1) Cell number; loss and regeneration of two different fluorescently tagged cell types (pancreatic beta cells and rod photoreceptors), 2) Cell signaling; relative activity of a transgenic Notch-signaling reporter, and 3) Cell metabolism; accumulation of reactive oxygen species. In summary, ARQiv is a versatile and readily accessible approach facilitating evaluation of genetic and/or chemical manipulations in living zebrafish that complements current "high-content" whole-organism screening methods by providing a first-tier in vivo HTS drug discovery platform. © 2012 Walker et al.


Teng Y.,University of Georgia | Xie X.,University of Georgia | Walker S.,University of Georgia | Saxena M.,Luminomics, Inc. | And 3 more authors.
PLoS ONE | Year: 2011

Mutations in the LGI1 gene predispose to a hereditary epilepsy syndrome and is the first gene associated with this disease which does not encode an ion channel protein. In zebrafish, there are two paralogs of the LGI1 gene, lgi1a and lgi1b. Knockdown of lgi1a results in a seizure-like hyperactivity phenotype with associated developmental abnormalities characterized by cellular loss in the eyes and brain. We have now generated knockdown morphants for the lgi1b gene which also show developmental abnormalities but do not show a seizure-like behavior. Instead, the most striking phenotype involves significant enlargement of the ventricles (hydrocephalus). As shown for the lgi1a morphants, however, lgi1b morphants are also sensitized to PTZ-induced hyperactivity. The different phenotypes between the two lgi1 morphants support a subfunctionalization model for the two paralogs. © 2011 Teng et al.


PubMed | Luminomics, Inc.
Type: Journal Article | Journal: Future medicinal chemistry | Year: 2012

Due to several inherent advantages, zebrafish are being utilized in increasingly sophisticated screens to assess the physiological effects of chemical compounds directly in living vertebrate organisms. Diverse screening platforms showcase these advantages. Morphological assays encompassing basic qualitative observations to automated imaging, manipulation, and data-processing systems provide whole organism to subcellular levels of detail. Behavioral screens extend chemical screening to the level of complex systems. In addition, zebrafish-based disease models provide a means of identifying new potential therapeutic strategies. Automated systems for handling/sorting, high-resolution imaging and quantitative data collection have significantly increased throughput in recent years. These advances will make it easier to capture multiple streams of information from a given sample and facilitate integration of zebrafish at the earliest stages of the drug-discovery process, providing potential solutions to current drug-development bottlenecks. Here we outline advances that have been made within the growing field of zebrafish chemical screening.


Grant
Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 225.00K | Year: 2014

DESCRIPTION (provided by applicant): Our goal is to create a compact, affordable, fully automated, screening system for large-scale drug 2 testing in living small animal disease models (e.g., worms, flies, and fish). Modern drug discovery is 3 driven by high-throughput screening (HTS) systems that have the capacity to evaluate large chemical 4 compound 'libraries'. The majority of hits identified by in vitro HTS assays fail at animal testing stages 5 after significant investments in time and costs have already been made. This increase drug 6 development costs and presents a critical barrier to the discovery of novel therapies. A solution to this 7 problem is to integrate small animal disease models into HTS platforms at the earliest stage of the 8 discovery process. Powerful 'high-content' in vivo screening systems use automated imaging 9 processes to collect phenotypic data following drug exposures. However, these methods cannot attain 10 true HTS capacities and therefore cannot fully address current


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 217.53K | Year: 2010

DESCRIPTION (provided by applicant): 1 Our ultimate goal is to discover how cells targeted for destruction during degenerative disease 2 can be regenerated from adult stem cells. The specific goal of this proposal is to create 3 transgenic zebrafish that can be used to model the regeneration of motor neuron (MN) cells, the 4 cell type lost in all motor neuron diseases (MND). Zebrafish have a remarkable capacity for 5 cellular regeneration that extends even to the nervous system, including MN cells in adult 6 zebrafish. Zebrafish are also an established model system for large-scale forward genetic 7 screens, whereby the genome is randomly mutated to identify genes which are required for a 8 specific biological process. To combine these attributes, we have developed simple screening 9 methods around an inducible cellular ablation platform that can be used to identify regeneration- 10 deficient mutants, in this case, genes required for MN regeneration. Specifically, transgenic 11 methods will be used to target the expression of a pro-drug converting enzyme, nitroreductase 12 (NTR), to MN subpopulations. NTR functions to convert water soluble pro-drugs into cellular 13 toxins, thereby ablating the MN specifically expressing the enzyme. A fusion between NTR and 14 a fluorescent reporter (NTR-FP) allows the presence or absence of targeted cells to be easily 15 monitored over time in living zebrafish. In this system FP loss would indicate MN degeneration 16 while subsequent gains in FP signal provide evidence of MN regeneration. By using high- 17 throughput plate readers for quantitative detection of fluorescent reporters in living zebrafish, a 18 large-scale genetic screen could be performed (Phase II) to identify multiple genes required for 19 MN regeneration. Thus, the identification of molecular factors which promote MN regeneration 20 in a vertebrate model system such as zebrafish should provide a means to explore the 21 possibility of regenerative therapies for human MND. PUBLIC HEALTH RELEVANCE: Motor neurons are the cell type lost in a number of debilitating degenerative diseases, including Lou Gehrig's disease. The goal of this proposal is to discover genes required for motor neuron regeneration by identifying mutations that disrupt motor neuron regeneration in a small model organism, the zebrafish - a species with a regenerative capacity that extends even to the nervous system. The motor neuron disease models produced and genetic insights gained during these studies will facilitate efforts to discover drugs that promote motor neuron regeneration, thus suggesting possible avenues of regenerative therapies for human motor neuron diseases.


Luminomics, Inc. | Entity website


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 177.07K | Year: 2011

DESCRIPTION (provided by applicant): Zebrafish are an important animal model system for both basic science research and preclinical disease modeling. Complementary to mammalian models, the zebrafish system facilitates methods that are not practical (e.g.,large-scale forward genetic screens), not possible (in vivo imaging of embryonic development), or not cost-effective (high-throughput chemical screens for drug discovery) in mice or rats. Until recently, however, methods for targeted genetic manipulations(e.g., knockout) have eluded the zebrafish field. Targeted genetic modifications stand as the single most desired methodology of the rapidly growing zebrafish market. The advent of zinc finger nuclease (ZFN)-based genome modification has brought gene knockout, and potentially knock-in, strategies to zebrafish researchers. Yet, the process of identifying appropriate ZFN pairs for a given gene target is not trivial, resulting in only a small number of labs that have successfully applied this approach, to date. Here we propose to test whether customized ZFN pairs from Sigma's Advanced Genetic Engineering (SAGE) group improve the efficiency of creating targeted genetic modifications in zebrafish. If proven effective, we will partner with SAGE - which holds anexclusive license from Sangamo Biosciences for creating ZFN-based animal models - to create a catalog of knockout and knock-in zebrafish. Proprietary techniques that SAGE has applied to other species (e.g. rodents) will be employed both in terms of identifying optimal ZFN targets/pairs and in terms of facilitating ZFN-based modifications. Aim 1: Create three knockout disease models in zebrafish using customized ZFN pairs from SAGE targeting 1three genetic loci: 1) sapje, 2) pink1, 3) kif1b, corresponding to knockout models for Muscular Dystrophy, Parkinson's Disease, and Multiple Sclerosis, respectively. Aim 2: Test knock-in efficiency using customized ZFN pairs from SAGE. Although ZFN-based knockout methods have been validated in zebrafish, ZFN-basedknock-in success has not been demonstrated. For this pilot study, proprietary information from SAGE, our own insights regarding transgenesis, and data from groups that have created ZFN-based knock-ins in other systems, we be employed to introduce a fluorescent reporter into the krox20 locus. Successful demonstration of ZFN-induced knock-in would pave the way for the creation of a catalog highly versatile research models. Success of this Phase I proposal will be followed by scale-up initiatives in Phase II and partnering with SAGE in Phase III (see letter of support) to bring 2 both Knockout zebrafish (KOZTM) and Knock-in zebrafish (KIZTM) models to market. We anticipate that development of a strong disease model catalog, coupled with an extremely strongIP position, will promote lucrative relationships with pharmaceutical partners in our efforts to bring insights afforded by the zebrafish system to bear on human disease. PUBLIC HEALTH RELEVANCE: The ability to manipulate genes in a targeted manner revolutionized the fields of molecular genetics and disease modeling but, until recently, was only applicable in the mouse (e.g., gene knockouts). The advent of zinc finger nuclease (ZFN) technology facilitates targeted gene manipulation in any species in which the genome has been sequenced. Accordingly, Luminomics proposes to create ZFN-induced gene knockout and knock-in models in zebrafish, the fastest growing vertebrate model species, as an off-the-shelf product line of broad appeal to both academic and commercial sectors of the zebrafish research community.

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