scholarly journals Construction of Two mCherry Plasmids (pXG-mCherry) for Transgenic Leishmania: Valuable Tools for Future Molecular Analysis

2017 ◽  
Vol 2017 ◽  
pp. 1-11 ◽  
Author(s):  
Andrés Vacas ◽  
Conor Sugden ◽  
Óscar Velasco-Rodriguez ◽  
Miriam Algarabel-Olona ◽  
José Peña-Guerrero ◽  
...  
Keyword(s):  
Drug Screening ◽  
Fluorescent Protein ◽  
Red Fluorescent Protein ◽  
Fluorescent Reporter ◽  
The World ◽  
Colorimetric Assays ◽  
Reporter Proteins ◽  
Indicator Dyes ◽  
Multiple Cloning Sites

Leishmania is the causative agent of leishmaniasis, a neglected tropical disease that affects more than 12 million people around the world. Current treatments are toxic and poorly effective due to the acquisition of resistance within Leishmania populations. Thus, the pursuit for new antileishmanial drugs is a priority. The available methods for drug screening based on colorimetric assays using vital dyes are time-consuming. Currently, the use of fluorescent reporter proteins is replacing the use of viability indicator dyes. We have constructed two plasmids expressing the red fluorescent protein mCherry with multiple cloning sites (MCS), adequate for N- and C-terminal fusion protein constructs. Our results also show that the improved pXG-mCherry plasmid can be employed for drug screening in vitro. The use of the red fluorescent protein, mCherry, is an easier tool for numerous assays, not only to test pharmacological compounds, but also to determine the subcellular localization of proteins.

scholarly journals An alternative in vitro drug screening test using Leishmania amazonensis transfected with red fluorescent protein

2013 ◽  
Vol 75 (3) ◽  
pp. 282-291 ◽  
Author(s):  
Marcele N. Rocha ◽  
Célia M. Corrêa ◽  
Maria N. Melo ◽  
Stephen M. Beverley ◽  
Olindo Assis Martins-Filho ◽  
...  
Keyword(s):  
Drug Screening ◽  
Screening Test ◽  
Fluorescent Protein ◽  
Leishmania Amazonensis ◽  
Red Fluorescent Protein ◽  
In Vitro Drug Screening

scholarly journals The fluorescent protein stability assay: an efficient method for monitoring intracellular protein stability

BioTechniques ◽  
2021 ◽  
Author(s):  
Armelle Roisin ◽  
Samuel Buchsbaum ◽  
Vincent Mocquet ◽  
Pierre Jalinot
Keyword(s):  
Protein Stability ◽  
Efficient Method ◽  
Fluorescent Protein ◽  
Specific Protein ◽  
Intracellular Protein ◽  
Fluorescent Reporter ◽  
Cellular Environment ◽  
Intracellular Proteins ◽  
Reporter Proteins ◽  
The Stability

The stability of intracellular proteins is highly variable, from a few minutes to several hours, and can be tightly regulated to respond to external and internal cellular environment changes. Several techniques can be used to study the stability of a specific protein, including pulse-chase labeling and blocking of translation. Another approach that has gained interest in recent years is fusing a protein of interest to a fluorescent reporter. In this report, the authors present a new version of this approach aimed at optimizing expression and comparison of the two reporter proteins. The authors show that the system works efficiently in various cells and can be useful for studying changes in protein stability and assessing the effects of drugs.

Non-invasive bioluminescence imaging of caspase-3 activity in Breast Cancer

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e14557-e14557
Author(s):  
C. C. Olsen ◽  
F. Li ◽  
Z. He ◽  
W. Li ◽  
C. Li
Keyword(s):  
Breast Cancer ◽  
Drug Exposure ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Fold Increase ◽  
Reporter System ◽  
Reporter Protein ◽  
Reporter Proteins

e14557 Background: Apoptosis is a major form of tumor cells death during cytotoxic therapy. Understanding the kinetics of apoptosis would greatly facilitate development of more effective therapeutic approaches. In order to monitor apoptosis activities in vivo, we developed a novel bioluminescence-based reporter gene to detect caspase 3 activities, which are elevated at the execution phase of apoptosis. Methods: A caspase-3 reporter system was constructed by combining two different reporter proteins; green fluorescent protein (GFP) and firefly luciferase (FL) linked through multiple polyubiquitin domains with a caspase-3 recognition site. Under normal circumstances, the reporter proteins are rapidly degraded by the proteasome system.. During apoptosis, activated caspse 3 cleaves off the multi-ubiquitin domain from the reporter protein. This enable the GFP and luciferase fusion reporter to be stabilized and achieve a significant gain in GFP protein and luciferase activities, which in turn could be monitored both in vitro and in vivo. 4T1 cells transduced with CMV-luc or Caspase-3 reporter xenografts were treated with both chemotherapy and radiation therapy and monitored for apoptosis activity. Results: In vitro experiments demonstrated increased luciferase with increasing radiation dose reflective of apoptosis with background levels nearly undetectable. Taxol was associated with a time-dependent increase from 24 to 72hrs after drug exposure, indicating that apoptosis is a gradual, heterogeneous process. EGFP signal increased from 1.85% in controls to 80.6% in cells treated with 1uM Taxol. Xenografts showed nearly undetectable luciferase background with Cytoxan therapy resulting in a 90-fold increase, 10 Gy a 24 fold increase and fractionated RT (5Gy x3) with a 46-fold increase. Conclusions: We developed a novel in vivo caspase reporter based on the ubiquitous proteosome system of protein degradation and bioluminsecence imaging. This allowed us to assess activation of apoptosis in response to chemoradiation therapy in tissue culture and breast cancer xenografts over the course of 2–3 weeks, which has not been possible with other technologies. No significant financial relationships to disclose.

scholarly journals Illuminating the Formation of Lumens

2007 ◽  
Vol 15 (3) ◽  
pp. 3-5
Author(s):  
Stephen W. Carmichael
Keyword(s):  
Animal Model ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Solid Core ◽  
Red Fluorescent Protein ◽  
Neuronal Process ◽  
Two Photon ◽  
Green Fluorescent

How do lumens form? Two mechanisms that come readily to mind are a wrapping model, similar to the wrapping of the myelin sheath around a neuronal process, and a solid core of cells followed by apoptosis of the central cells. Another obvious mechanism that was suggested over 100 years ago is the fusion of intracellular vacuoles. Whereas several recent studies have supported this latter mechanism, it has not yet been proven. Now, the appropriate animal model (zebrafish), the modern techniques (transgenic chimeras), dyes (green fluorescent protein and monomeric red fluorescent protein) that can be linked to proteins to label vacuoles, and two-photon imaging in real time finally have provided the strongest support yet. In an article by Makoto Kamei, Brian Saunders, Kayla Bayless, Louis Dye, George Davis, and Brant Weinstein the assembly of endothelial tubes from intracellular vacuoles was observed in vitro and in vivo.

scholarly journals A Fluorescent Split Aptamer for Visualizing RNA-RNA Assembly In Vivo

2017 ◽  
Author(s):  
Khalid K. Alam ◽  
Kwaku D. Tawiah ◽  
Matthew F. Lichte ◽  
David Porciani ◽  
Donald H. Burke
Keyword(s):  
Self Assembly ◽  
Living Cells ◽  
Rna Aptamers ◽  
Genetic Circuits ◽  
Fluorescent Reporter ◽  
Indirect Measures ◽  
Reporter Proteins ◽  
Circuit Function

AbstractRNA-RNA assembly governs key biological processes and is a powerful tool for engineering synthetic genetic circuits. Characterizing RNA assembly in living cells often involves monitoring fluorescent reporter proteins, which are at best indirect measures of underlying RNA-RNA hybridization events and are subject to additional temporal and load constraints associated with translation and activation of reporter proteins. In contrast, RNA aptamers that sequester small molecule dyes and activate their fluorescence are increasingly utilized in genetically-encoded strategies to report on RNA-level events. Split-aptamer systems have been rationally designed to generate signal upon hybridization of two or more discrete RNA transcripts, but none directly function when expressed in vivo. We reasoned that the improved physiological properties of the Broccoli aptamer enable construction of a split-aptamer system that could function in living cells. Here we present the Split-Broccoli system, in which self-assembly is nucleated by a thermostable, three-way junction RNA architecture and fluorescence activation requires both strands. Functional assembly of the system approximately follows second order kinetics in vitro and improves when cotranscribed, rather than when assembled from purified components. Split-Broccoli fluorescence is digital in vivo and retains functional modularity when fused to RNAs that regulate circuit function through RNA-RNA hybridization, as demonstrated with an RNA Toehold switch. Split-Broccoli represents the first functional split-aptamer system to operate in vivo. It offers a genetically-encoded and nondestructive platform to monitor and exploit RNA-RNA hybridization, whether as an all-RNA, stand-alone AND gate or as a tool for monitoring assembly of RNA-RNA hybrids.

scholarly journals A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein

2017 ◽  
Author(s):  
Yi Shen ◽  
Hod Dana ◽  
Ahmed S. Abdelfattah ◽  
Ronak Patel ◽  
Jamien Shea ◽  
...  
Keyword(s):  
Calcium Ion ◽  
Fluorescent Protein ◽  
Brain Slices ◽  
Sea Anemone ◽  
Red Fluorescent Protein ◽  
Combined Use ◽  
Development Structure ◽  
Organotypic Brain Slices

AbstractGenetically-encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring Ca2+ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs enable multicolor visualization with blue or cyan-excitable fluorophores and combined use with blue or cyan-excitable optogenetic actuators. Here we report the development, structure, and validation of a new red fluorescent Ca2+ indicator, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We characterized the performance of K-GECO1 in cultured HeLa cells, dissociated neurons, stem cell derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo.

scholarly journals Structure Basis for Inhibition of SARS-CoV-2 by the Feline Drug GC376

2020 ◽  
Author(s):  
Xiaodong Luan ◽  
Weijuan Shang ◽  
Yifei Wang ◽  
Wanchao Yin ◽  
Yi Jiang ◽  
...  
Keyword(s):  
Drug Screening ◽  
Antiviral Drug ◽  
High Specificity ◽  
Catalytic Site ◽  
Viral Translation ◽  
Important Protein ◽  
Potential Target ◽  
The World

AbstractThe pandemic of SARS-CoV-2 coronavirus disease-2019 (COVID-19) caused by SARS-COV-2 continues to ravage many countries in the world. Mpro is an indispensable protein for viral translation in SARS-CoV-2 and a potential target in high-specificity anti-SARS-CoV-2 drug screening. In this study, to explore potential drugs for treating COVID-19, we elucidated the structure of SARS-CoV-2 Mpro and explored the interaction between Mpro and GC376, an antiviral drug used to treat a range of coronaviruses in Feline via inhibiting Mpro. The availability and safety of GC376 were proved by biochemical and cell experiments in vitro. We determined the structure of an important protein, Mpro, in SARS-CoV-2, and revealed the interaction of GC376 with the viral substrate and inhibition of the catalytic site of SARS-CoV-2 Mpro.

FluoroCalins: engineered lipocalins with novel binding functions fused to a fluorescent protein for applications in biomolecular imaging and detection

2019 ◽  
Vol 32 (6) ◽  
pp. 289-296 ◽  
Author(s):  
Evelyn Eggenstein ◽  
Antonia Richter ◽  
Arne Skerra
Keyword(s):  
Fluorescent Protein ◽  
Signal To Noise Ratio ◽  
Growth Factor Receptor ◽  
High Specificity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Reporter Protein ◽  
Fluorescent Reporter ◽  
Promising Alternative ◽  
Biomolecular Imaging

Abstract FluoroCalins represent novel bifunctional protein reagents derived from engineered lipocalins fused to a fluorescent reporter protein, here the enhanced green fluorescent protein (eGFP). We demonstrate the construction, facile bacterial production and broad applicability of FluoroCalins using two Anticalin® molecules directed against the tumor vasculature-associated extra domain B of fibronectin (ED-B) and the vascular endothelial growth factor receptor 3, a marker of tumor and lymphangiogenesis. FluoroCalins were prepared with two different spacers: (i) a short Ser3Ala linker and (ii) a long hydrophilic and conformationally unstructured PASylation® polypeptide comprising 200 Pro, Ala and Ser residues. These FluoroCalins were applied for direct target quantification in enzyme-linked immunosorbent assay as well as target detection by flow cytometry and fluorescence microscopy of live and fixed cells, respectively, demonstrating high specificity and signal-to-noise ratio. Hence, FluoroCalins offer a promising alternative to antibody-based reagents for state of the art fluorescent in vitro detection and biomolecular imaging.

20 GENERATION AND CHARACTERIZATION OF TRANSGENIC-CLONED PIGS EXPRESSING THE FAR-RED FLUORESCENT PROTEIN MONOMERIC PLUM

2014 ◽  
Vol 26 (1) ◽  
pp. 124
Author(s):  
M. Kobayashi ◽  
M. Watanabe ◽  
H. Matsunari ◽  
K. Nakano ◽  
T. Kanai ◽  
...  
Keyword(s):  
Mixed Culture ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Red Fluorescent Protein ◽  
Cell Stage ◽  
Fetal Fibroblasts ◽  
Morula Stage ◽  
Clear Identification ◽  
Green Fluorescent

Transgenic (Tg) pigs expressing a fluorescent protein are extremely useful for research into transplantation and regenerative medicine. This study aimed to create Tg pigs expressing monomeric Plum (mPlum), a far-red fluorescent protein with a longer wavelength than enhanced green fluorescent protein (EGFP) and humanized Kusabira Orange (huKO), the two fluorescent proteins that have been used previously for Tg pig production. A linearized CAG-mPlum transgene construct was transferred into porcine fetal fibroblasts (PFF) by electroporation. mPlum fluorescence-positive cells were collected using a cell sorter and used as nuclear donors (mPlum-PFF) for somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were obtained from porcine cumulus–oocyte complexes cultured in NCSU23-based medium and were used to obtain recipient oocytes for SCNT after enucleation. Then, SCNT was performed as reported previously (Matsunari et al., 2008). The reconstructed embryos were cultured for 7 days in porcine zygote medium-5 (PZM-5). mPlum fluorescence expression was screened during the early development of the embryos. After 5 or 6 days of culture, the SCNT embryos were surgically transferred to the uterus of a recipient gilt. We first obtained fetuses on Day 36 or 37 of gestation by Caesarean section and the PFF were retrieved from their skin. Fluorescence expression was analysed using fluorescence microscope, and the number of transgene copies in each fetus was determined by Southern blot analysis. We also analysed whether unique spectral properties of mPlum are suitable for multicolor imaging using confocal microscope and flow cytometer. The identification of mPlum-expressing PFF under the mixed culture of PFF expressing EGFP and huKO was examined. The 2 cell lines of PFF expressing EGFP and huKO were previously generated in our laboratory. Rates of normal cleavage and blastocyst formation occurred in the SCNT embryos generated with mPlum-PFF (mPlum embryos) were equivalent to those of SCNT embryos derived from nontransgenic PFF (34/42, 81.0%; 33/42, 78.6% v. 37/40, 92.5%; 30/40, 75.0%). Total cell numbers in mPlum and control blastocysts did not differ significantly (88.3 ± 6.0 v. 99.9 ± 8.8). Fluorescence expression in the mPlum embryos began at the 8-cell stage and became brighter from the morula stage. The gilt into which 103 mPlum embryos were transferred produced 3 fetuses. These fetuses expressed mPlum fluorescence systemically and had 1 to 5 copies of the transgene. Multicolor fluorescence imaging and flow cytometric analyses of a mixed culture of mPlum PFF and PFF expressing EGFP and huKO showed that clear identification and isolation of cells displaying each of the 3 fluorescence signals was possible. These observations demonstrate that the transfer of CAG-mPlum did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of Tg cloned pigs that systemically expressed mPlum. This work was supported by JSPS KAKENHI Grant Number 25293279.

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