In zebrafish, the chorion is easily removed by Ringer's solution containing trypsin at pH 7.0 . The zebrafish has been established as the main vertebrate model system for whole organism screening applications. The zebrafish embryo is protected by the chorion, an acellular three-layered envelope with a thickness of 1.5-2.5 μm, composed of polypeptides. After chemical removal of the chorion (dechorionation), positively surface-charged SiO 2 NPs (25 nm) significantly reduced the survival rate of the fish compared to the control group. The National Toxicology Program's . Assembling and using the transplantation device. Removal of the chorion and renewal of exposure solutions increase toxicity of chemicals in a zebrafish embryo screening assay regardless of other protocol differences. (Research Article, Technical report) by "ALTEX: Alternatives to Animal Experimentation"; Zoology and wildlife conservation Science and technology, general Animal embryo Testing Animal models in research Animal research models Dispersion measures (Statistics) Methods Usage Embryonic development . This prompted us to further investigate the phenotype of the surviving atp6v1e1b -deficient larvae to assess whether dominant features in the clinical presentation of ARCL type 2C are present in mutant zebrafish. Automated Zebrafish Chorion Removal and Single Embryo Placement: . 17 66-74 We report the automated dechorionation of 1600 embryos at once at 4 h postfertilization (hpf) and placement of the dechorionated embryos into 96-well plates for exposure by 6 hpf. The chorion is the acellular envelope surrounding mature eggs of teleostean fish. Chorions can be removed easily with sharp forceps (Dumont No. Chorion. Muscular dystrophy is frequently caused by disruption of the dystrophin-glycoprotein complex (DGC), which links muscle cells to the extracellular matrix. Chorion Removal: Drain eggs add 5 ml of 0.5 mg/ml pronase for 3.5 min. Because the chorion is impermeable to water, it needs to be removed prior to metronidazole treatment and β-cell ablation in Tg(ins:NTR-mCherry) transgenic zebrafish embryos. It was found to be thinner than that of medaka Oryzias latipes and thicker than that of zebrafish Danio rerio. Zebrafish (D. rerio) has become a powerful and widely used model system for the analysis of vertebrate embryogenesis and organ development. One of the challenges of isolating RNA from zebrafish is the presence of a rigid and insoluble chorion. Structure and enzymatic removal of the chorion of embryos of the Nile tilapia Structure and enzymatic removal of the chorion of embryos of the Nile tilapia Morrison, C. M.; Pohajdak, B.; Henry, M.; Wright, J. R. 2003-12-01 00:00:00 Dechorionation or partial digestion of the egg chorion is necessary for introducing embryonic stem cells into blastulas for chimera production and for harvesting . STATUS: Four laboratories selected, study is ongoing. Removal of the chorion and renewal of exposure solutions increase toxicity of chemicals in a zebrafish embryo screening assay regardless of other protocol differences. PEG-b-PCL polymeric nano-micelle inhibits vascular angiogenesis by activating p53-dependent apoptosis in zebrafish Tian Zhou,1 Qinglei Dong,1 Yang Shen,2 Wei Wu,1 Haide Wu,1 Xianglin Luo,3 Xiaoling Liao,4 Guixue Wang1 1Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of . Zebrafish embryos at 48 h post-fertilisation (hpf) with (a) and after manual removal of the chorion (b) Full size image According to current European Union legislation for the protection of animals used for experimental and other scientific purposes, the use of embryonic stages of vertebrates is not regulated (Commission of the European . yet this is technically much more demanding and requires removal of the chorion prior to imaging. Furthermore, a . Dystroglycan, a central component of the DGC, serves as a laminin receptor via its extracellular α subunit, and interacts with dystrophin (and thus the actin cytoskeleton) through its integral membrane β subunit. Systematic Evaluation of the Application of Zebrafish In Toxicology: SEAZIT . J. • The homogenate was loaded into a 96 well clear-bottom black plate and scanned Step 10, Dehydration: 10-15 min + overnight. Although chorion removal at 24 vision camera and software could select a sufficiently iso- hpf from 50 to 100 zebrafish embryos at once has been lated embryo to map and direct the robot to retrieve. Figure 5: Zebrafish chorion spot analysis stained with Coomassie brilliant blue. Assembling the transplantation device. We hypothesized that E2 uptake would not be affected by chorion removal due to its small size, well under 3 kDa, and its lipophilicity as a steroid hormone. The embryonic zebrafish model has been used for this purpose in pharmaceutical development and in high-throughput screening programs at NTP and the EPA. 1 B). 17, Issue 1 Final concentration is 4% para-formaldehyde, 0.15 mM CaCl2, 4%sucrose in 0.1 M PO4 buffer. Systematic Evaluation of the Application of Zebrafish In Toxicology: SEAZIT . In zebrafish heart, the injury leads to a comparable fibrotic response: the intra-ventricular mechanical forces decrease during myocardial regeneration, leading to a down regulation of collagen synthesis and, eventually to the removal of the scar . CiteSeerX - Document Details (Isaac Councill, Lee Giles, Pradeep Teregowda): The potential of the developing zebrafish model for toxicology and drug discovery is limited by inefficient approaches to manipulating and chemically exposing zebrafish embryos—namely, manual placement of embryos into 96- or 384-well plates and exposure of embryos while still in the chorion, a barrier of poorly . Scale bars in G and H are . A few studies demonstrated increased sensitivity with chorion-on versus chorion-off embryos (Henn and Braunbeck, 2011; Kim and Tanguay, 2014), and even an automated chorion removal system has been developed (Mandrell et al., 2012) with the assumption that the presence of the chorion affects sensitivity to toxicological effects. Lab. stage zebrafish toxicity assay by leading to false positives. The zebrafish chorion has been reported to reduce the permeability of organic molecules (Brox et al., 2014) ; however, for heavy metals it could also represent a barrier since these could accumulate at the surface or inside the chorion (Henn and Braunbeck, 2011). Lab. Journal of Laboratory Automation. 2007). J Hamm . were observed on the chorion of ovulated eggs in stm−/− Scale bars in A-F are 1 mm. The chorion was thinned over a wide range of pH (7.0-11.0) and in concentrations of enzyme between 0.5-0.0125% within 21-36 hours. As a prerequisite to carry out proteomic experiments with early zebrafish embryos, we developed a . . To test if the chorion restricts movement and growth before hatching, we manually removed the chorion 3-6 days before the natural time of hatching (dechorionated) and compared movement, growth and oxygen consumption in dechorionated embryos and in embryos whose chorions remained intact until the time of hatching (chorionated). Environmental toxicology and chemistry. STATUS: Four laboratories selected, study is ongoing. Lab. Automated zebrafish chorion removal and single embryo placement: optimizing throughput of zebrafish developmental toxicity screens. 16,105-111 (2011).Crossref, Medline, Google Scholar; 16 Mandrell D, Truong L, Jephson C et al. J. 2009). To test whether the chorion influences E2 uptake, we exposed embryos at 6 and 24 hpf, with chorions intact and following manual chorion removal, to 5 nM [3 H]E2 for either 1 or 24 h. At 6 . Warga and Kimmel (1990) briefly described stages of the blastula and gastrula. While genetic methods are readily available in zebrafish, protocols for two dimensional (2D) gel electrophoresis and proteomics have yet to be developed. The macromolecular composition of the zebrafish (Danio rerio) egg chorion, organised as a three-layered structure, has been analysed.SDS-PAGE analysis, under reducing conditions, of isolated and purified chorions revealed a reproducible pattern of four major polypeptides (116, 97, 50 and 43kDa) and several minor . Herein we report rapid and cost-effective automated removal of the chorion from 2000 embryos at once at the As a prerequisite to carry out proteomic experiments with early zebrafish embryos, we developed a . This can be achieved either with forceps or by enzymatic removal using Pronase. J Lab Autom, (1):66-74 2012 MED: 22357610 1-4).The ZFE as a vertebrate model species combines advantages such as rapid development and transparency of embryos and is considered as an alternative . Perfluorooctanesulfonic acid (PFOS) is a perfluorinated compound used in many industrial and consumer products. Oxygen consumption allows measuring the metabolic activity of organisms. We have removed the . 17, 66-74. pmid:22357610 . Perfluorooctanesulfonic Acid-Induced Toxicity on Zebrafish Embryos in the Presence or Absence of the Chorion. Introduction To investigate the role of a gene during development, overexpression or misexpression of your gene of interest is a fast assay. . Zebrafish Course, Mullins, August, 2008 Microinjection Techniques: Injecting through the chorion I. Step 1. ref. While technical solutions to the orientation problem exist, dissemination is often . 17 66-74 Steps 11-13, Embryo transfer and day 1 of ISH: 2.5 h . Step 3. Autom. The chorion is then held in a region . An earlier staging series for zebrafish, although less complete than the present one, fairly accurately por- trays the first third (or 1st day) of embryonic develop- ment, and includes useful sets of photographs (Hisaoka and Battle, 1958; Hisaoka and Firlit, 1960). Step 2. Journal of laboratory automation 17 (1), 66-74 , 2012 Project Summary There has been a tremendously increasing interest in the use of small animals such as zebrafish in drug discovery A number of recent studies have proposed the use of zebrafish in drug discovery workflows where they could be used to test drug candidates prior to being tested on expensive mammalian models One significant advantage . Add dH2O to a final volume of 25.0 ml. Only As(V) resulted in significant developmental abnormalities with LOEL of 500 μM. It includes information on the importance of acellular chorion removal, detailed chorion removal procedures, and a complete protocol for the toxicity assessment of nanomaterials using dechorionated zebrafish embryos. Autom. Carefully grab the chorion of the embryo with a pair of fine-tip tweezers, without disrupting the embryo itself. The endpoints are related to developmental effects occurring in dechorionated zebrafish embryos for 120 hours post fertilization and . It includes information on the importance of acellular chorion removal, detailed chorion removal procedures, and a complete protocol for the toxicity assessment of nanomaterials using dechorionated zebrafish embryos. Chorions can be removed easily using two forceps (it is critical that the tips are sharp and that their ends can touch). similar developmental stage (gastrulation-stage) as in in vivo EFD studies Test concentration: - Wide range of concentrations tested (typically up to 1000uM) tailored to Mandrell D, Truong L, Jephson C, Sarker M R, Moore A, Lang C, Simonich M T and Tanguay R L 2012 Automated zebrafish chorion removal and single embryo placement: optimizing throughput of zebrafish developmental toxicity screens J. In zebrafish, the chorion is easily removed by Ringer's solution containing trypsin at pH 7.0 . The chorion is the membrane that exists around the developing embryo. Trypsin and urea solution are also effective for chorion removal in goldfish [ 19 ] and common carp Cyprinus carpio [ 20 ]. Enzymatic degradation of the chorion is possible, but will most likely affect the transcriptome. Rinse 3x more with 200 ml washes of 8x water. Autom. 1 1 The role of chorion on toxicity of silver nanoparticles in the embryonic zebrafish assay 2 Original Article 3 Ki-Tae Kim1),*, Robert L. Tanguay2) 4 5 1) Department of Environmental Engineering, Seoul National University of Science and 6 Technology, Seoul 139-743, Korea 7 2) Department of Environmental and Molecular Toxicology, Oregon State University, USA Zebrafish (D. rerio) has become a powerful and widely used model system for the analysis of vertebrate embryogenesis and organ development. Lab. - Chorion-off embryos (pronase treatment & manually dissected) Onset of compound administration: - 5-8 hr post fertilization (hpf) embryos i.e. The chorion of Nile tilapia possesses multiple lamellae as found in most teleost species that have been studied. On the other hand, chorion removal involves complex processes with non-environmental relevance, resulting in inhibition of the hatching process, low survival rate, and sensitivity [18-20]. During natural hatching the chorion was digested from the inner surface, and tail movements helped to break the remaining . Steps 7-9, Chemical removal, chorion removal and fixation of embryos: 1 h + overnight. We show that the removal of the embryonic chorion is beneficial for oxygen consumption rates (OCR) and . Introduction The zebrafish (Danio rerio) embryo (ZFE) toxicity test is a versatile in vitro method to assess the hazard of chemicals and has been applied in several studies for the toxicological testing of nanoparticles (NPs) (e.g. Therefore, we chose for a sample preparation method involving mechanical disruption of the chorion under frozen conditions. It is more realistic to think of the chorion as a sieve but not a wall (Schreiber et al. These results indicate that zebrafish chorion acts as a physical barrier against SiO 2 1. Obtain a zebrafish embryo with a chorion, as pictured above. The process removed ≥95% of the embryos from their chorions with 2% embryo mortality by 24 hpf, and 2% of the embryos malformed at 120 hpf. At 32 hpf, more n-PS was adsorbed on the chorion and almost covered the entire surface of the embryonic chorion ( Fig. When raised in Embryo Medium at 28.5°C, zebrafish develop normally outside their chorions. Warga and Kimmel (1990) briefly described stages of the blastula and gastrula. It was found to be thinner than that of medaka Oryzias latipes and thicker than that of zebrafish Danio rerio. Once reported, initiating embryo exposure so late in development the source dish was depleted to less than 300 embryos and is likely to be of . These results indicate that zebrafish chorion acts as a physical barrier against SiO 2 NPs, and removing the chorions in ZFET might be necessary for evaluation of . Upon manual removal of the chorion, the mutant zebrafish survived until 8 dpf albeit with a severe systemic phenotype. However, there are few systematic examinations of the utility of chorion removal in the literature. Grab the other end of the chorion with a second pair of fine-tip tweezers. Autom. Here, the development of a bio-derived material suitable for stem cell culture and improvement of differentiation efficiency to specific cell lineages with no stimulating agents by using a chorion obtained from a fertilized zebrafish egg through the removal of the yolk and embryonic cell mass from the egg is reported. The potential of the developing zebrafish model for toxicology and drug discovery was limited by inefficient approaches such as, 1) plating zebrafish into multi-well plates; 2) chorion removal; 3) chemical exposures; 4) imaging; 5) adult behavioral assessments; 6) and data analysis. Automated zebrafish chorion removal and single embryo placement: optimizing throughput of zebrafish developmental toxicity screens. The earliest time of removal should be after completion of The study is designed to determine the effect of chorion removal . The chorion of Nile tilapia possesses multiple lamellae as found in most teleost species that have been studied. Removal of the chorion prior to As(III) and As(V) exposure was associated with increased incidence of mortality and developmental abnormality suggesting that the chorion may normally attenuate mass uptake of these metals by the embryo. • Zebrafish were exposed to 0 and 500 ppm Rhodamine B labeled CNC. After chemical removal of the chorion (dechorionation), positively surface-charged SiO 2 NPs (25 nm) significantly reduced the survival rate of the fish compared to the control group. When zebrafish embryo was exposed to fluorescent n-PS, the particles accumulated soon on the surface of the embryonic chorion at 10 hpf ( Fig. Contraction and generation of mechanical forces are very important for cardiac development and . During natural hatching the chorion was digested from the inner surface, and tail movements helped to break the remaining . Read more related scholarly scientific articles and abstracts. Free Online Library: Characterizing Sources of Variability in Zebrafish Embryo Screening Protocols. Micro CT scan images from Contrary no signal of binding of anti-Stm antibody anterior (A and B), left lateral (C and D), posterior (E and F) and dorsal (G and H) sides of adult WT zebrafish and the stm−/− mutant are indicated. Dilute eggs with 200 ml of 8x water. The data consists of 19 toxicological endpoints on unique 1018 compounds measured in relatively low concentration ranges. Automated Zebrafish Chorion Removal and Single Embryo Placement: Optimizing Throughput of Zebrafish Developmental Toxicity Screens journal, February 2012 Mandrell, David; Truong, Lisa; Jephson, Caleb Journal of Laboratory Automation, Vol. Removal of the chorion membrane sensitizes zebrafish embryos to MSN toxicity Next we removed the protective chorion membrane around the embryo to allow better penetration of all the differently . Although the embryos frequently escaped from the thinned . The National Toxicology Program's . The IGG identified two specific parameters that can potentially influence study outcomes for chemical screening using zebrafish embryos: the presence versus absence of the chorion and the use of the static versus static-renewal exposure procedure. Trypsin and urea solution are also effective for chorion removal in goldfish [ 19 ] and common carp Cyprinus carpio [ 20 ]. Pores in the chorion are about 0.5 μm in diameter, and are about 2 μm apart (Rawson et al. J. • The chorion of the fish was either left on or manually removed before exposure. [PMC free article] [Google Scholar] Morin C, de Souza Silva MA, Muller CP, Hardigan P, Spieler RE. Step 4. This document specifies a method for rapidly assessing nanomaterial toxicity (fish early life stage, 0 HPF to 120 HPF). Here, we adopted the multi-well plate-based respirometry of the extracellular flux analyzer (Seahorse XF96) to investigate the effect of temperature on the bioenergetics of zebrafish embryos (Danio rerio) in situ. A zebrafish embryo's chorion is intact until 2 or 3 days postfertilization (dpf). This document specifies a method for rapidly assessing nanomaterial toxicity (fish early life stage, 0 HPF to 120 HPF). by J Erik Mylroie, Mitchell S Wilbanks, Ashley N Kimble, Kimberly T To, Catherine S Cox, Sheila J McLeod, Kurt A Gust, David W Moore, Edward J Perkins, Natàlia Garcia-Reyero. • The fish were homogenized on days 1-5 after thorough rinsing and manual chorion removal. 5) by gently making a tear in . Mandrell D, Truong L, Jephson C, Sarker MR, Moore A, Lang C, et al. The CompTox Chemistry Dashboard (ToxCast) contains one of the largest public databases on Zebrafish (Danio rerio) developmental toxicity. 2000; Lee et al. It has been linked to a broad range of adverse effects in several species, including zebrafish (Danio rerio).The zebrafish embryo is a widely used vertebrate model to elucidate potential adverse effects of chemicals because it is amenable to medium and high throughput. An earlier staging series for zebrafish, although less complete than the present one, fairly accurately por- trays the first third (or 1st day) of embryonic develop- ment, and includes useful sets of photographs (Hisaoka and Battle, 1958; Hisaoka and Firlit, 1960). Removal of the chorion increased sensitivity to OPEs: we found higher incidence of mortality and malformation in dechorionated embryos. Connect the Luer tip 25 µL gas-tight syringe and a micropipette holder with Luer lock fitting to assemble the transplantation device (Figure 1A).NOTE: Wetting the Luer tip with a thin film of water can help to enhance the connection by holding the parts together via water adhesion and cohesion. J Hamm . While genetic methods are readily available in zebrafish, protocols for two dimensional (2D) gel electrophoresis and proteomics have yet to be developed. However, deficits in the following key areas hinder the broader adoption of the zebrafish model for toxicological screening: . When we consider the obvious potential for false negatives due to the aforementioned barrier effect, chorion removal is critical to improve the predictivity of the assay. Lab. Automated zebrafish chorion removal and single embryo placement: Optimizing throughput of zebrafish developmental toxicity screens. 2012; 17:66-74. 8x water = 12 ml stock salts per liter dH2O Although removal of the embryo chorion is efficient at epiboly or later stages (Truong et al., Reference Truong, Harper and Tanguay 2011) even with automated dechorionation systems (Mandrall et al., Reference Mandrall, Truong, Jephson, Sarker, Moore, Lang, Simonich and Tanguay 2012), unfortunately at the mid blastula transition (MBT) stage (3 . However, the lack of consistent positioning of zebrafish embryos within wells of microtiter plates remains an obstacle for the comparative analysis of images acquired in automated screening assays. 1 A). R Core Team R: A Language and Environment for Statistical Computing. Automated Zebrafish Chorion Removal and Single Embryo Placement. Mandrell D, Truong L, Jephson C, Sarker MR, Moore A, Lang C, Simonich MT, Tanguay RL. It includes information on the importance of acellular chorion removal, detailed chorion removal procedures, and a complete protocol for the toxicity assessment of nanomaterials using dechorionated zebrafish embryos. In a behavioral assay, the locomotive activity of zebrafish larvae was consistently inhibited by OPEs except tris (1-chloropropyl) phosphate regardless of chorion presence. When raised in E3 medium at 28.5 °C, zebrafish embryos develop normally outside of their chorions. Some observations of zebrafish development can be made directly through the chorion.i.Chorion;, however for most procedures it is better to remove the chorion. Image-based fluidic sorting system for automated Zebrafish egg sorting into multiwell plates. The chorion can significantly confound the early life stage zebrafish toxicity assay by leading to false positives. Using one forcep to hold the chorion, make a tear in the chorion with the other forcep. You can inject mRNA or DNA of the wild-type gene or a With the use of NIH image J software, the images were obtained from chorion pieces, previously stained with Coomassie brilliant blue, as described in the Materials and Methods section, and was subjected to filter band passed to give uniform spot areas from 0.5 to 5 . Mandrell D, Truong L, Jephson C, Sarker M R, Moore A, Lang C, Simonich M T and Tanguay R L 2012 Automated zebrafish chorion removal and single embryo placement: optimizing throughput of zebrafish developmental toxicity screens J. Automated zebrafish chorion removal and single embryo placement: optimizing throughput of zebrafish developmental toxicity screens. This document specifies a method for rapidly assessing nanomaterial toxicity (fish early life stage, 0 HPF to 120 HPF). When we consider the obvious potential for false negatives due to the aforementioned barrier effect, chorion removal is critical to improve the predictivity of the assay. The chorions of 4 h post fertilization (hpf) zebrafish embryos were enzymatically removed using a custom automated dechorionator [100]. Automated zebrafish chorion removal and single embryo placement: optimizing throughput of zebrafish developmental toxicity screens D Mandrell, L Truong, C Jephson, MR Sarker, A Moore, C Lang, . The potential of the developing zebrafish model for toxicology and drug discovery is limited by inefficient approaches to manipulating and chemically exposing zebrafish embryos—namely, manual placement of embryos into 96- or 384-well plates and exposure of embryos while .

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