scholarly journals Transgenic fluorescent zebrafish lines that have revolutionized biomedical research

2021 ◽  
Vol 37 (1) ◽  
Author(s):  
Chong Pyo Choe ◽  
Seok-Yong Choi ◽  
Yun Kee ◽  
Min Jung Kim ◽  
Seok-Hyung Kim ◽  
...  
Keyword(s):  
Biomedical Research ◽  
Digestive System ◽  
Fluorescent Proteins ◽  
Model Organism ◽  
Urogenital System ◽  
Research Fields ◽  
Intracellular Organelle ◽  
Vertebrate Model ◽  
Intracellular Organelles

AbstractSince its debut in the biomedical research fields in 1981, zebrafish have been used as a vertebrate model organism in more than 40,000 biomedical research studies. Especially useful are zebrafish lines expressing fluorescent proteins in a molecule, intracellular organelle, cell or tissue specific manner because they allow the visualization and tracking of molecules, intracellular organelles, cells or tissues of interest in real time and in vivo. In this review, we summarize representative transgenic fluorescent zebrafish lines that have revolutionized biomedical research on signal transduction, the craniofacial skeletal system, the hematopoietic system, the nervous system, the urogenital system, the digestive system and intracellular organelles.

A Novel TMTP1-Modified Theranostic Nanoplatform for Targeted In Vivo NIR-II Fluorescence Imaging-Guided Chemotherapy of Cervical Cancer

2022 ◽  
Author(s):  
Nuernisha Alifu ◽  
Rong Ma ◽  
Lijun Zhu ◽  
Zhong Du ◽  
Shuang Chen ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Fluorescence Imaging ◽  
Spatial Resolution ◽  
Biomedical Research ◽  
Near Infrared ◽  
High Sensitivity ◽  
Research Fields ◽  
Excellent Spatial Resolution ◽  
Fluorescence Bioimaging

Near-infrared II (NIR-II, 900-1700 nm) fluorescence bioimaging with advantages of good biosafety, excellent spatial resolution, high sensitivity, and contrast has attracted great attentions in biomedical research fields. However, most of...

scholarly journals Efficient Neuroprotective Rescue of Sacsin-Related Disease Phenotypes in Zebrafish

2021 ◽  
Vol 22 (16) ◽  
pp. 8401
Author(s):  
Valentina Naef ◽  
Maria Marchese ◽  
Asahi Ogi ◽  
Gianluca Fichi ◽  
Daniele Galatolo ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Molecular Mechanisms ◽  
Motor Impairment ◽  
Model Organism ◽  
Cell Degeneration ◽  
Vertebrate Model ◽  
Therapeutic Compounds

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a multisystem hereditary ataxia associated with mutations in SACS, which encodes sacsin, a protein of still only partially understood function. Although mouse models of ARSACS mimic largely the disease progression seen in humans, their use in the validation of effective therapies has not yet been proposed. Recently, the teleost Danio rerio has attracted increasing attention as a vertebrate model that allows rapid and economical screening, of candidate molecules, and thus combines the advantages of whole-organism phenotypic assays and in vitro high-throughput screening assays. Through CRISPR/Cas9-based mutagenesis, we generated and characterized a zebrafish sacs-null mutant line that replicates the main features of ARSACS. The sacs-null fish showed motor impairment, hindbrain atrophy, mitochondrial dysfunction, and reactive oxygen species accumulation. As proof of principle for using these mutant fish in high-throughput screening studies, we showed that both acetyl-DL-leucine and tauroursodeoxycholic acid improved locomotor and biochemical phenotypes in sacs−/− larvae treated with these neuroprotective agents, by mediating significant rescue of the molecular functions altered by sacsin loss. Taken together, the evidence here reported shows the zebrafish to be a valuable model organism for the identification of novel molecular mechanisms and for efficient and rapid in vivo optimization and screening of potential therapeutic compounds. These findings may pave the way for new interventions targeting the earliest phases of Purkinje cell degeneration in ARSACS.

scholarly journals In vivo characterisation of fluorescent proteins in budding yeast

2018 ◽  
Author(s):  
Dennis Botman ◽  
Daan Hugo de Groot ◽  
Phillipp Schmidt ◽  
Joachim Goedhart ◽  
Bas Teusink
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescent Proteins ◽  
Codon Optimization ◽  
Budding Yeast ◽  
Model Organism ◽  
Ph Sensitivity ◽  
Future Studies ◽  
Signal Readout

AbstractFluorescent proteins (FPs) are widely used in many organisms, but are commonly characterised in vitro. However, the in vitro properties may poorly reflect in vivo performance. Therefore, we characterised 27 FPs in vivo using Saccharomyces cerevisiae as model organism. We linked the FPs via a T2A peptide to a control FP, producing equimolar expression of the 2 FPs from 1 plasmid. Using this strategy, we characterised the FPs for brightness, photostability, photochromicity and pH-sensitivity, achieving a comprehensive in vivo characterisation. Many FPs showed different in vivo properties compared to existing in vitro data. Additionally, various FPs were photochromic, which affects readouts due to complex bleaching kinetics. Finally, we codon optimized the best performing FPs for optimal expression in yeast, and found that codon-optimization alters FP characteristics. These FPs improve experimental signal readout, opening new experimental possibilities. Our results may guide future studies in yeast that employ fluorescent proteins.

scholarly journals Tracking Diacylglycerol and Phosphatidic Acid Pools in Budding Yeast

2015 ◽  
Vol 8s1 ◽  
pp. LPI.S31781 ◽  
Author(s):  
Suriakarthiga Ganesan ◽  
Brittney N. Shabits ◽  
Vanina Zaremberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Phosphatidic Acid ◽  
Physical Properties ◽  
Negative Curvature ◽  
Fluorescent Proteins ◽  
Budding Yeast ◽  
Model Organism ◽  
Current Evidence ◽  
C1 Domain

Phosphatidic acid (PA) and diacylglycerol (DAG) are key signaling molecules and important precursors for the biosynthesis of all glycerolipids found in eukaryotes. Research conducted in the model organism Saccharomyces cerevisiae has been at the forefront of the identification of the enzymes involved in the metabolism and transport of PA and DAG. Both these lipids can alter the local physical properties of membranes by introducing negative curvature, but the anionic nature of the phosphomonoester headgroup in PA sets it apart from DAG. As a result, the mechanisms underlying PA and DAG interaction with other lipids and proteins are notoriously different. This is apparent from the analysis of the protein domains responsible for recognition and binding to each of these lipids. We review the current evidence obtained using the PA-binding proteins and domains fused to fluorescent proteins for in vivo tracking of PA pools in yeast. In addition, we present original results for visualization of DAG pools in yeast using the C1 domain from mammalian PKCδ. An emerging first cellular map of the distribution of PA and DAG pools in actively growing yeast is discussed.

scholarly journals K-means clustering of zebrafish embryos images acquired with AOTF-based hyperspectral microscope

2021 ◽  
Vol 2127 (1) ◽  
pp. 012062
Author(s):  
A B Burlakov ◽  
S V Shirokov ◽  
C C Huang ◽  
D D Khokhlov
Keyword(s):  
Clustering Algorithm ◽  
Hyperspectral Image ◽  
Model Organism ◽  
Tunable Filter ◽  
Zebrafish Embryos ◽  
Non Invasive ◽  
Research Fields ◽  
Automated Observation ◽  
Convenient Model

Abstract Model organism studies are widely implemented in biomedical research fields. Zebrafish is a common and convenient model organism. To provide in vivo investigation of living zebrafish the non-invasive imaging methods are implemented. Hyperspectral imaging utilizing acousto-optic tunable filters is a perspective modality for zebrafish embryos and larvae automated observation. In this paper, the hyperspectral microscope based on the acousto-optical tunable filter is described. Using the hyperspectral image arrays obtained with the described setup, the K-means clustering algorithm is tested. The results obtained for different number of clusters are presented and discussed.

scholarly journals From seeing to believing: labelling strategies for in vivo cell-tracking experiments

2013 ◽  
Vol 3 (3) ◽  
pp. 20130001 ◽  
Author(s):  
Fränze Progatzky ◽  
Margaret J. Dallman ◽  
Cristina Lo Celso
Keyword(s):  
High Resolution ◽  
Cell Tracking ◽  
Genetic Manipulation ◽  
Fluorescent Proteins ◽  
Ex Vivo ◽  
Cell Types ◽  
Model Organisms ◽  
Technological Advances ◽  
Vertebrate Model

Intravital microscopy has become increasingly popular over the past few decades because it provides high-resolution and real-time information about complex biological processes. Technological advances that allow deeper penetration in live tissues, such as the development of confocal and two-photon microscopy, together with the generation of ever-new fluorophores that facilitate bright labelling of cells and tissue components have made imaging of vertebrate model organisms efficient and highly informative. Genetic manipulation leading to expression of fluorescent proteins is undoubtedly the labelling method of choice and has been used to visualize several cell types in vivo . This approach, however, can be technically challenging and time consuming. Over the years, several dyes have been developed to allow rapid, effective and bright ex vivo labelling of cells for subsequent transplantation and imaging. Here, we review and discuss the advantages and limitations of a number of strategies commonly used to label and track cells at high resolution in vivo in mouse and zebrafish, using fluorescence microscopy. While the quest for the perfect label is far from achieved, current reagents are valuable tools enabling the progress of biological discovery, so long as they are selected and used appropriately.

Three-Dimensional Subcellular Organization of Hepatocytes in Intact Liver: Simultaneous Visualization of Cytoskeletal and Membrane Markers by Confocal Microscopy

1996 ◽  
Vol 54 ◽  
pp. 288-289
Author(s):  
Greg V. Martin ◽  
Ann L. Hubbard
Keyword(s):  
Three Dimensional ◽  
Integral Membrane Proteins ◽  
Polarized Secretion ◽  
Microscope Images ◽  
Computer Aided ◽  
Intracellular Organelles ◽  
Cytoskeletal Elements ◽  
Simultaneous Visualization

The microtubule (MT) cytoskeleton is necessary for many of the polarized functions of hepatocytes. Among the functions dependent on the MT-based cytoskeleton are polarized secretion of proteins, delivery of endocytosed material to lysosomes, and transcytosis of integral plasma membrane (PM) proteins. Although microtubules have been shown to be crucial to the establishment and maintenance of functional and structural polarization in the hepatocyte, little is known about the architecture of the hepatocyte MT cytoskeleton in vivo, particularly with regard to its relationship to PM domains and membranous organelles. Using an in situ extraction technique that preserves both microtubules and cellular membranes, we have developed a protocol for immunofluorescent co-localization of cytoskeletal elements and integral membrane proteins within 20 µm cryosections of fixed rat liver. Computer-aided 3D reconstruction of multi-spectral confocal microscope images was used to visualize the spatial relationships among the MT cytoskeleton, PM domains and intracellular organelles.

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

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