scholarly journals Comparative assessment of fluorescent proteins forin vivoimaging in an animal model system

2016 ◽  
Author(s):  
Jennifer K Heppert ◽  
Daniel J Dickinson ◽  
Ariel M Pani ◽  
Christopher D Higgins ◽  
Annette Steward ◽  
...  
Keyword(s):  
Animal Model ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Model System ◽  
C Elegans ◽  
Red Fluorescent Proteins ◽  
Fluorescent Protein Tags ◽  
Protein Tags

Fluorescent protein tags are fundamental tools used to visualize gene products and analyze their dynamicsin vivo. Recent advances in genome editing have enabled precise insertion of fluorescent protein tags into the genomes of diverse organisms. These advances expand the potential ofin vivoimaging experiments, and they facilitate experimentation with new, bright, photostable fluorescent proteins. Most quantitative comparisons of the brightness and photostability of different fluorescent proteins have been madein vitro, removed from biological variables that govern their performance in cells or organisms. To address the gap we quantitatively assessed fluorescent protein propertiesin vivoin an animal model system. We generated transgenicC. elegansstrains expressing green, yellow, or red fluorescent proteins in embryos, and we imaged embryos expressing different fluorescent proteins under the same conditions for direct comparison. We found that mNeonGreen was not brightin vivoas predicted based onin vitrodata, but that mNeonGreen is a better tag than GFP for specific kinds of experiments, and we report on optimal red fluorescent proteins. These results identify ideal fluorescent proteins for imagingin vivoinC. elegansembryos, and they suggest good candidate fluorescent proteins to test in other animal model systems.

scholarly journals Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system

2016 ◽  
Vol 27 (22) ◽  
pp. 3385-3394 ◽  
Author(s):  
Jennifer K. Heppert ◽  
Daniel J. Dickinson ◽  
Ariel M. Pani ◽  
Christopher D. Higgins ◽  
Annette Steward ◽  
...  
Keyword(s):  
Animal Model ◽  
In Vivo Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Model System ◽  
Red Fluorescent Proteins ◽  
Fluorescent Protein Tags ◽  
Protein Tags

Fluorescent protein tags are fundamental tools used to visualize gene products and analyze their dynamics in vivo. Recent advances in genome editing have expedited the precise insertion of fluorescent protein tags into the genomes of diverse organisms. These advances expand the potential of in vivo imaging experiments and facilitate experimentation with new, bright, photostable fluorescent proteins. Most quantitative comparisons of the brightness and photostability of different fluorescent proteins have been made in vitro, removed from biological variables that govern their performance in cells or organisms. To address the gap, we quantitatively assessed fluorescent protein properties in vivo in an animal model system. We generated transgenic Caenorhabditis elegans strains expressing green, yellow, or red fluorescent proteins in embryos and imaged embryos expressing different fluorescent proteins under the same conditions for direct comparison. We found that mNeonGreen was not as bright in vivo as predicted based on in vitro data but is a better tag than GFP for specific kinds of experiments, and we report on optimal red fluorescent proteins. These results identify ideal fluorescent proteins for imaging in vivo in C. elegans embryos and suggest good candidate fluorescent proteins to test in other animal model systems for in vivo imaging experiments.

scholarly journals Supramolecular Encapsulation of Small-Ultra Red Fluorescent Proteins in Virus-Like Nanoparticles for Non-Invasive In Vivo Imaging Agents

2020 ◽  
Author(s):  
Fabian C. Herbert ◽  
Olivia Brohlin ◽  
Tyler Galbraith ◽  
Candace Benjamin ◽  
Cesar A. Reyes ◽  
...  
Keyword(s):  
In Vivo Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Ex Vivo ◽  
Imaging Agents ◽  
Non Invasive ◽  
Red Fluorescent Proteins ◽  
Ex Vivo Imaging

<div> <div> <div> <p>Icosahedral virus-like particles (VLPs) derived from bacteriophages Qβ and PP7 encapsulating small-ultra red fluorescent protein (smURFP) were produced using a versatile supramolecualr capsid dissassemble-reassemble approach. The generated fluorescent VLPs display identical structural properties to their non-fluorescent analogs. Encapsulated smURFP shows indistinguishable photochemical properties to its unencapsulated counterpart, exhibits outstanding stability towards pH, and produces bright in vitro images following phagocytosis by macrophages. In vivo imaging allows biodistribution to be imaged at different time points. Ex vivo imaging of intravenously administered encapsulated smURFP reveleas localization in the liver and </p> </div> </div> <div> <div> <p>kidneys after 2 h blood circulation and substantial elimination constructs as non-invasive in vivo imaging agents. </p> </div> </div> </div>

scholarly journals Supramolecular Encapsulation of Small-Ultra Red Fluorescent Proteins in Virus-Like Nanoparticles for Non-Invasive In Vivo Imaging Agents

2020 ◽  
Author(s):  
Fabian C. Herbert ◽  
Olivia Brohlin ◽  
Tyler Galbraith ◽  
Candace Benjamin ◽  
Cesar A. Reyes ◽  
...  
Keyword(s):  
In Vivo Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Ex Vivo ◽  
Imaging Agents ◽  
Non Invasive ◽  
Red Fluorescent Proteins ◽  
Ex Vivo Imaging

<div> <div> <div> <p>Icosahedral virus-like particles (VLPs) derived from bacteriophages Qβ and PP7 encapsulating small-ultra red fluorescent protein (smURFP) were produced using a versatile supramolecualr capsid dissassemble-reassemble approach. The generated fluorescent VLPs display identical structural properties to their non-fluorescent analogs. Encapsulated smURFP shows indistinguishable photochemical properties to its unencapsulated counterpart, exhibits outstanding stability towards pH, and produces bright in vitro images following phagocytosis by macrophages. In vivo imaging allows biodistribution to be imaged at different time points. Ex vivo imaging of intravenously administered encapsulated smURFP reveleas localization in the liver and </p> </div> </div> <div> <div> <p>kidneys after 2 h blood circulation and substantial elimination constructs as non-invasive in vivo imaging agents. </p> </div> </div> </div>

scholarly journals Split-wrmScarlet and split-sfGFP: Tools for faster, easier fluorescent l abeling of endogenous proteins in Caenorhabditis elegans

Genetics ◽  
2021 ◽  
Author(s):  
Jérôme Goudeau ◽  
Catherine S Sharp ◽  
Jonathan Paw ◽  
Laura Savy ◽  
Manuel D Leonetti ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Labeling ◽  
Large Fragment ◽  
C Elegans ◽  
High Affinity ◽  
Red Fluorescent Proteins ◽  
Invaluable Tool ◽  
Endogenous Proteins ◽  
Fluorescent Tags

Abstract We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous C. elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663

scholarly journals Properties of Fluorescent Far-Red Anti-TNF Nanobodies

Antibodies ◽  
2018 ◽  
Vol 7 (4) ◽  
pp. 43 ◽  
Author(s):  
Ekaterina Gorshkova ◽  
Grigory Efimov ◽  
Ksenia Ermakova ◽  
Ekaterina Vasilenko ◽  
Diana Yuzhakova ◽  
...  
Keyword(s):  
Autoimmune Diseases ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Humanized Mice ◽  
Whole Body ◽  
Whole Body Imaging ◽  
Theranostic Agent ◽  
Variable Heavy

Upregulation of the expression of tumor necrosis factor (TNF-α, TNF) has a significant role in the development of autoimmune diseases. The fluorescent antibodies binding TNF may be used for personalized therapy of TNF-dependent diseases as a tool to predict the response to anti-TNF treatment. We generated recombinant fluorescent proteins consisting of the anti-TNF module based on the variable heavy chain (VHH) of camelid antibodies fused with the far-red fluorescent protein Katushka (Kat). Two types of anti-TNF VHH were developed: one (BTN-Kat) that was bound both human or mouse TNF, but did not neutralize their activity, and a second (ITN-Kat) that was binding and neutralizing human TNF. BTN-Kat does not interfere with TNF biological functions and can be used for whole-body imaging. ITN-Kat can be evaluated in humanized mice or in cells isolated from humanized mice. It is able to block human TNF (hTNF) activities both in vitro and in vivo and may be considered as a prototype of a theranostic agent for autoimmune diseases.

scholarly journals C-terminal eYFP fusion impairs Escherichia coli MinE function

Open Biology ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 200010
Author(s):  
Navaneethan Palanisamy ◽  
Mehmet Ali Öztürk ◽  
Emir Bora Akmeriç ◽  
Barbara Di Ventura
Keyword(s):  
Escherichia Coli ◽  
In Silico ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Time Lapse ◽  
Enhanced Yellow Fluorescent Protein ◽  
Min Proteins

The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo , in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.

Cellular Uptake of Carbon Nanotubes Conjugated to Semiconductor Quantum Dots for Breast Cancer Imaging and Treatment Applications

2010 ◽  
Author(s):  
Kristen A. Zimmermann ◽  
Jianfei Zhang ◽  
Harry Dorn ◽  
Christopher Rylander ◽  
Marissa Nichole Rylander
Keyword(s):  
Carbon Nanotubes ◽  
Quantum Dots ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Properties ◽  
Breast Cancer Imaging ◽  
Fluorescent Markers ◽  
Green Fluorescent

Carbon nanotubes (CNTs) are attractive materials for early detection, treatment, and imaging of cancer malignancies; however, they are limited by their inability to be monitored in vitro and in vivo [1]. Unlabeled CNTs are difficult to distinguish using elemental analysis because they are composed entirely of carbon, which is also characteristic of cellular membranes. Although some single walled nanotubes (SWNT) have been found to exhibit fluorescent properties, not all particles in a single batch fluoresce [2]. Additionally, these emissions may be too weak to be detected using conventional imaging modalities [3]. Incorporating fluorescent markers, such as fluorescent proteins or quantum dots, allows the non-fluorescent particles to be visualized. Previously, fluorophores, such as green fluorescent protein (GFP) or red fluorescent protein (RFP), have been used to visualize and track cells or other particles in biological environments, but their low quantum yield and tendency to photobleach generate limitations for their use in such applications.

scholarly journals Visualization of Periplasmic and Cytoplasmic Proteins with a Self-Labeling Protein Tag

2016 ◽  
Vol 198 (7) ◽  
pp. 1035-1043 ◽  
Author(s):  
Na Ke ◽  
Dirk Landgraf ◽  
Johan Paulsson ◽  
Mehmet Berkmen
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Functional Protein ◽  
Content Type ◽  
Additional Tool

ABSTRACTThe use of fluorescent and luminescent proteins in visualizing proteins has become a powerful tool in understanding molecular and cellular processes within living organisms. This success has resulted in an ever-increasing demand for new and more versatile protein-labeling tools that permit light-based detection of proteins within living cells. In this report, we present data supporting the use of the self-labeling HaloTag protein as a light-emitting reporter for protein fusions within the model prokaryoteEscherichia coli. We show that functional protein fusions of the HaloTag can be detected bothin vivoandin vitrowhen expressed within the cytoplasmic or periplasmic compartments ofE. coli. The capacity to visually detect proteins localized in various prokaryotic compartments expands today's molecular biologist toolbox and paves the path to new applications.IMPORTANCEVisualizing proteins microscopically within living cells is important for understanding both the biology of cells and the role of proteins within living cells. Currently, the most common tool is green fluorescent protein (GFP). However, fluorescent proteins such as GFP have many limitations; therefore, the field of molecular biology is always in need of new tools to visualize proteins. In this paper, we demonstrate, for the first time, the use of HaloTag to visualize proteins in two different compartments within the model prokaryoteEscherichia coli. The use of HaloTag as an additional tool to visualize proteins within prokaryotes increases our capacity to ask about and understand the role of proteins within living cells.

scholarly journals Robust genome editing with short single-stranded and long, partially single-stranded DNA donors in C. elegans

2018 ◽  
Author(s):  
Gregoriy A. Dokshin ◽  
Krishna S. Ghanta ◽  
Katherine M. Piscopo ◽  
Craig C. Mello
Keyword(s):  
Green Fluorescent Protein ◽  
Cost Effectiveness ◽  
Genome Editing ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
C Elegans ◽  
Multiple Loci ◽  
Green Fluorescent ◽  
Fluorescent Protein Tags ◽  
Protein Tags

AbstractCRISPR-based genome editing using ribonucleoprotein (RNP) complexes and synthetic single stranded oligodeoxynucleotide (ssODN) donors can be highly effective. However, reproducibility can vary, and precise, targeted integration of longer constructs – such as green fluorescent protein (GFP) tags remains challenging in many systems. Here we describe a streamlined and optimized editing protocol for the nematode C. elegans. We demonstrate its efficacy, flexibility, and cost-effectiveness by affinity-tagging all twelve of the Worm-specific Argonaute (WAGO) proteins in C. elegans using ssODN donors. In addition, we describe a novel PCR-based partially single-stranded “hybrid” donor design that yields high efficiency editing with large (kilobase-scale) constructs. We use these hybrid donors to introduce fluorescent protein tags into multiple loci achieving editing efficiencies that approach those previously obtained only with much shorter ssODN donors. The principals and strategies described here are likely to translate to other systems and should allow researchers to reproducibly and efficiently obtain both long and short precision genome edits.

scholarly journals Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells

2017 ◽  
Author(s):  
Stephen D. Carter ◽  
Shrawan K. Mageswaran ◽  
Zachary J. Farino ◽  
João I. Mamede ◽  
Catherine M. Oikonomou ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Organic Dyes ◽  
Fluorescent Proteins ◽  
Secretory Granules ◽  
Light And Electron Microscopy ◽  
Low Contrast ◽  
Fluorescent Protein Tags ◽  
Protein Tags

AbstractCryogenic correlated light and electron microscopy (cryo-CLEM) is a valuable tool for studying biological processes in situ. In cryo-CLEM, a target protein of interest is tagged with a fluorophore and the location of the corresponding fluorescent signal is used to identify the structure in low-contrast but feature-rich cryo-EM images. To date, cryo-CLEM studies of mammalian cells have relied on very bright organic dyes or fluorescent protein tags concentrated in virus particles. Here we describe a method to expand the application of cryo-CLEM to cells harboring genetically-encoded fluorescent proteins. We discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80K). Compared to fluorescent protein tags, these sources of autofluorescence exhibit a broader spectrum of fluorescence, which we exploited to develop a simple, robust approach to discriminate between the two. We validate this method in INS-1 E cells using a mitochondrial marker, and apply it to study the ultrastructural variability of secretory granules in a near-native state within intact INS-1E pancreatic cells by high-resolution 3D electron cryotomography.

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