scholarly journals Fully Automated Pipetting Sorting System for Different Morphological Phenotypes of Zebrafish Embryos

2017 ◽  
Vol 23 (2) ◽  
pp. 128-133 ◽  
Author(s):  
Helmut Breitwieser ◽  
Thomas Dickmeis ◽  
Marcel Vogt ◽  
Marco Ferg ◽  
Christian Pylatiuk
Keyword(s):  
Template Matching ◽  
Zebrafish Embryo ◽  
Petri Dish ◽  
Microtiter Plate ◽  
Model Organisms ◽  
Zebrafish Embryos ◽  
Wild Type ◽  
Patterning Defect ◽  
Glucocorticoid Deficiency ◽  
Sorting System

Systems biology methods, such as transcriptomics and metabolomics, require large numbers of small model organisms, such as zebrafish embryos. Manual separation of mutant embryos from wild-type embryos is a tedious and time-consuming task that is prone to errors, especially if there are variable phenotypes of a mutant. Here we describe a zebrafish embryo sorting system with two cameras and image processing based on template-matching algorithms. In order to evaluate the system, zebrafish rx3 mutants that lack eyes due to a patterning defect in brain development were separated from their wild-type siblings. These mutants show glucocorticoid deficiency due to pituitary defects and serve as a model for human secondary adrenal insufficiencies. We show that the variable phenotypes of the mutant embryos can be safely distinguished from phenotypic wild-type zebrafish embryos and sorted from one petri dish into another petri dish or into a 96-well microtiter plate. On average, classification of a zebrafish embryo takes approximately 1 s, with a sensitivity and specificity of 87% to 95%, respectively. Other morphological phenotypes may be classified and sorted using similar techniques.

scholarly journals Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

2005 ◽  
Vol 71 (7) ◽  
pp. 3468-3474 ◽  
Author(s):  
Gyeong Tae Eom ◽  
Jae Kwang Song ◽  
Jung Hoon Ahn ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Abc Transporter ◽  
Microtiter Plate ◽  
Single Amino Acid ◽  
Wild Type ◽  
E Coli ◽  
Single Amino Acid Change ◽  
Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

scholarly journals The Relationship between Dissolution Behavior and the Toxicity of Silver Nanoparticles on Zebrafish Embryos in Different Ionic Environments

Nanomaterials ◽  
2018 ◽  
Vol 8 (9) ◽  
pp. 652 ◽  
Author(s):  
Wang Lee ◽  
Eungwang Kim ◽  
Hyun-Ju Cho ◽  
Taejoon Kang ◽  
Bongsoo Kim ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Polyethylene Glycol ◽  
Zebrafish Embryo ◽  
Antibacterial Properties ◽  
Dissolution Behavior ◽  
Aquatic Environments ◽  
Zebrafish Embryos ◽  
Negative Effects ◽  
The Relationship ◽  
Water Filters

A silver nanoparticle is one of the representative engineered nanomaterials with excellent optical, electrical, antibacterial properties. Silver nanoparticles are being increasingly used for medical products, water filters, and cosmetics, etc. However, silver nanoparticles are known to cause adverse effects on the ecosystem and human health. To utilize silver nanoparticles with minimized negative effects, it is important to understand the behavior of silver nanoparticles released to the environment. In this study, we compared toxicity behaviors of citrate-stabilized silver nanoparticles with polyethylene glycol coated silver nanoparticles in two different ionic environments, which are aquatic environments for developing zebrafish embryo. Depending on the composition of the ionic environment, citrate-stabilized silver nanoparticles and polyethylene glycol coated silver nanoparticles exhibited different behaviors in dissolution, aggregation, or precipitation, which governed the toxicity of silver nanoparticles on zebrafish embryos.

scholarly journals Genome-Scale Screening and Combinatorial Optimization of Gene Overexpression Targets to Improve Cadmium Tolerance in Saccharomyces cerevisiae

2021 ◽  
Vol 12 ◽  
Author(s):  
Yongcan Chen ◽  
Jun Liang ◽  
Zhicong Chen ◽  
Bo Wang ◽  
Tong Si
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Engineering ◽  
Heavy Metal Contamination ◽  
Cadmium Toxicity ◽  
Biomass Accumulation ◽  
Model Organisms ◽  
Cadmium Tolerance ◽  
Wild Type ◽  
Cadmium Resistance ◽  
Genome Scale

Heavy metal contamination is an environmental issue on a global scale. Particularly, cadmium poses substantial threats to crop and human health. Saccharomyces cerevisiae is one of the model organisms to study cadmium toxicity and was recently engineered as a cadmium hyperaccumulator. Therefore, it is desirable to overcome the cadmium sensitivity of S. cerevisiae via genetic engineering for bioremediation applications. Here we performed genome-scale overexpression screening for gene targets conferring cadmium resistance in CEN.PK2-1c, an industrial S. cerevisiae strain. Seven targets were identified, including CAD1 and CUP1 that are known to improve cadmium tolerance, as well as CRS5, NRG1, PPH21, BMH1, and QCR6 that are less studied. In the wild-type strain, cadmium exposure activated gene transcription of CAD1, CRS5, CUP1, and NRG1 and repressed PPH21, as revealed by real-time quantitative PCR analyses. Furthermore, yeast strains that contained two overexpression mutations out of the seven gene targets were constructed. Synergistic improvement in cadmium tolerance was observed with episomal co-expression of CRS5 and CUP1. In the presence of 200 μM cadmium, the most resistant strain overexpressing both CAD1 and NRG1 exhibited a 3.6-fold improvement in biomass accumulation relative to wild type. This work provided a new approach to discover and optimize genetic engineering targets for increasing cadmium resistance in yeast.

scholarly journals A red herring in zebrafish genetics: allele-specific gene expression can underlie altered transcript abundance in zebrafish mutants

2021 ◽  
Author(s):  
Richard J White ◽  
Eirinn Mackay ◽  
Stephen W Wilson ◽  
Elisabeth M Busch-Nentwich
Keyword(s):  
Differentially Expressed Genes ◽  
Differentially Expressed Gene ◽  
Transcript Abundance ◽  
Differentially Expressed ◽  
Model Organisms ◽  
Specific Gene ◽  
Wild Type ◽  
Specific Expression ◽  
Genomic Location ◽  
Allele Specific

In model organisms, RNA sequencing is frequently used to assess the effect of genetic mutations on cellular and developmental processes. Typically, animals heterozygous for a mutation are crossed to produce offspring with different genotypes. Resultant embryos are grouped by genotype to compare homozygous mutant embryos to heterozygous and wild-type siblings. Genes that are differentially expressed between the groups are assumed to reveal insights into the pathways affected by the mutation. Here we show that in zebrafish, differentially expressed genes are often overrepresented on the same chromosome as the mutation due to different levels of expression of alleles from different genetic backgrounds. Using an incross of haplotype-resolved wild-type fish, we found evidence of widespread allele-specific expression, which appears as differential expression when comparing embryos homozygous for a region of the genome to their siblings. When analysing mutant transcriptomes, this means that differentially expressed genes on the same chromosome as a mutation of interest may not be caused by that mutation. Typically, the genomic location of a differentially expressed gene is not considered when interpreting its importance with respect to the phenotype. This could lead to pathways being erroneously implicated or overlooked due to the noise of spurious differentially expressed genes on the same chromosome as the mutation. These observations have implications for the interpretation of RNA-seq experiments involving outbred animals and non-inbred model organisms.

scholarly journals Microfluidic chambers using fluid walls for cell biology

2018 ◽  
Vol 115 (26) ◽  
pp. E5926-E5933 ◽  
Author(s):  
Cristian Soitu ◽  
Alexander Feuerborn ◽  
Ann Na Tan ◽  
Henry Walker ◽  
Pat A. Walsh ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Soft Lithography ◽  
Petri Dish ◽  
Microtiter Plate ◽  
The Media ◽  
Downstream Analysis ◽  
Microfluidic Chambers ◽  
Made In

Many proofs of concept have demonstrated the potential of microfluidics in cell biology. However, the technology remains inaccessible to many biologists, as it often requires complex manufacturing facilities (such as soft lithography) and uses materials foreign to cell biology (such as polydimethylsiloxane). Here, we present a method for creating microfluidic environments by simply reshaping fluids on a substrate. For applications in cell biology, we use cell media on a virgin Petri dish overlaid with an immiscible fluorocarbon. A hydrophobic/fluorophilic stylus then reshapes the media into any pattern by creating liquid walls of fluorocarbon. Microfluidic arrangements suitable for cell culture are made in minutes using materials familiar to biologists. The versatility of the method is demonstrated by creating analogs of a common platform in cell biology, the microtiter plate. Using this vehicle, we demonstrate many manipulations required for cell culture and downstream analysis, including feeding, replating, cloning, cryopreservation, lysis plus RT-PCR, transfection plus genome editing, and fixation plus immunolabeling (when fluid walls are reconfigured during use). We also show that mammalian cells grow and respond to stimuli normally, and worm eggs develop into adults. This simple approach provides biologists with an entrée into microfluidics.

oko meduzy mutations affect neuronal patterning in the zebrafish retina and reveal cell-cell interactions of the retinal neuroepithelial sheet

Development ◽  
1999 ◽  
Vol 126 (6) ◽  
pp. 1235-1246 ◽  
Author(s):  
J. Malicki ◽  
W. Driever
Keyword(s):  
Critical Role ◽  
Cell Types ◽  
Cell Interactions ◽  
Retinal Cell ◽  
Wild Type ◽  
Vertebrate Retina ◽  
Neuroepithelial Cells ◽  
Patterning Defect ◽  
Cell Cell

Mutations of the oko meduzy (ome) locus cause drastic neuronal patterning defect in the zebrafish retina. The precise, stratified appearance of the wild-type retina is absent in the mutants. Despite the lack of lamination, at least seven retinal cell types differentiate in oko meduzy. The ome phenotype is already expressed in the retinal neuroepithelium affecting morphology of the neuroepithelial cells. Our experiments indicate that previously unknown cell-cell interactions are involved in development of the retinal neuroepithelial sheet. In genetically mosaic animals, cell-cell interactions are sufficient to rescue the phenotype of oko meduzy retinal neuroepithelial cells. These cell-cell interactions may play a critical role in the patterning events that lead to differentiation of distinct neuronal laminae in the vertebrate retina.

Patterning of angiogenesis in the zebrafish embryo

Development ◽  
2002 ◽  
Vol 129 (4) ◽  
pp. 973-982 ◽  
Author(s):  
Sarah Childs ◽  
Jau-Nian Chen ◽  
Deborah M. Garrity ◽  
Mark C. Fishman
Keyword(s):  
Zebrafish Embryo ◽  
Regular Pattern ◽  
Wild Type ◽  
Cell Fates ◽  
Mutagenesis Screen ◽  
The Third ◽  
Shaped Cell ◽  
Vessel Growth ◽  
Lateral Posterior ◽  
Lineage Analysis

Little is known about how vascular patterns are generated in the embryo. The vasculature of the zebrafish trunk has an extremely regular pattern. One intersegmental vessel (ISV) sprouts from the aorta, runs between each pair of somites, and connects to the dorsal longitudinal anastomotic vessel (DLAV). We now define the cellular origins, migratory paths and cell fates that generate these metameric vessels of the trunk. Additionally, by a genetic screen we define one gene, out of bounds (obd), that constrains this angiogenic growth to a specific path. We have performed lineage analysis, using laser activation of a caged dye and mosaic construction to determine the origin of cells that constitute the ISV. Individual angioblasts destined for the ISVs arise from the lateral posterior mesoderm (LPM), and migrate to the dorsal aorta, from where they migrate between somites to their final position in the ISVs and dorsal longitudinal anastomotic vessel (DLAV). Cells of each ISV leave the aorta only between the ventral regions of two adjacent somites, and migrate dorsally to assume one of three ISV cell fates. Most dorsal is a T-shaped cell, based in the DLAV and branching ventrally; the second constitutes a connecting cell; and the third an inverted T-shaped cell, based in the aorta and branching dorsally. The ISV remains between somites during its ventral course, but changes to run mid-somite dorsally. This suggests that the pattern of ISV growth ventrally and dorsally is guided by different cues. We have also performed an ENU mutagenesis screen of 750 mutagenized genomes and identified one mutation, obd that disrupts this pattern. In obd mutant embryos, ISVs sprout precociously at abnormal sites and migrate anomalously in the vicinity of ventral somite. The dorsal extent of the ISV is less perturbed. Precocious sprouting can be inhibited in a VEGF morphant, but the anomalous site of origin of obd ISVs remains. In mosaic embryos, obd somite causes adjacent wild-type endothelial cells to assume the anomalous ISV pattern of obd embryos. Thus, the launching position of the new sprout and its initial trajectory are directed by inhibitory signals from ventral somites. Zebrafish ISVs are a tractable system for defining the origins and fates of vessels, and for dissecting elements that govern patterns of vessel growth.

scholarly journals Ultraspecific live imaging of the dynamics of zebrafish neutrophil granules by a histopermeable fluorogenic benzochalcone probe

2019 ◽  
Vol 10 (12) ◽  
pp. 3654-3670 ◽  
Author(s):  
Emma Colucci-Guyon ◽  
Ariane S. Batista ◽  
Suellen D. S. Oliveira ◽  
Magali Blaud ◽  
Ismael C. Bellettini ◽  
...  
Keyword(s):  
Live Imaging ◽  
Zebrafish Embryos ◽  
Wild Type

A fluorogenic benzochalcone specifically labels live neutrophil granules in whole wild-type, GFP- or RFP-expressing zebrafish embryos and larvae.

scholarly journals Standardized mounting method of (zebrafish) embryos using a 3D-printed stamp for high-content, semi-automated confocal imaging

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
David Simon Kleinhans ◽  
Virginie Lecaudey
Keyword(s):  
Coordinate System ◽  
Light Exposure ◽  
Confocal Imaging ◽  
Model Organisms ◽  
Limiting Factor ◽  
Phenotypic Traits ◽  
Embryo Morphology ◽  
Zebrafish Embryos ◽  
High Content Imaging ◽  
3D Printed

Abstract Background Developmental biology relies to a large extent on the observation and comparison of phenotypic traits through time using high resolution microscopes. In this context, transparent model organisms such as the zebrafish Danio rerio in which developing tissues and organs can be easily observed and imaged using fluorescent proteins have become very popular. One limiting factor however is the acquisition of a sufficient amount of data, in standardized and reproducible conditions, to allow robust quantitative analysis. One way to improve this is by developing mounting methods to increase the number of embryos that can be imaged simultaneously in near-to-identical orientation. Results Here we present an improved mounting method allowing semi-automated and high-content imaging of zebrafish embryos. It is based on a 3D-printed stamp which is used to create a 2D coordinate system of multiple μ-wells in an agarose cast. Each μ-well models a negative of the average zebrafish embryo morphology between 22 and 96 h-post-fertilization. Due to this standardized and reproducible arrangement, it is possible to define a custom well plate in the respective imaging software that allows for a semi-automated imaging process. Furthermore, the improvement in Z-orientation significantly reduces post-processing and improves comparability of volumetric data while reducing light exposure and thus photo-bleaching and photo-toxicity, and improving signal-to-noise ratio (SNR). Conclusions We present here a new method that allows to standardize and improve mounting and imaging of embryos. The 3D-printed stamp creates a 2D coordinate system of μ-wells in an agarose cast thus standardizing specimen mounting and allowing high-content imaging of up to 44 live or mounted zebrafish embryos simultaneously in a semi-automated, well-plate like manner on inverted confocal microscopes. In summary, image data quality and acquisition efficiency (amount of data per time) are significantly improved. The latter might also be crucial when using the services of a microscopy facility.

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