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https://www.readbyqxmd.com/read/27915978/cell-compatibility-of-an-eposimal-vector-mediated-by-the-characteristic-motifs-of-matrix-attachment-regions
#1
Tian-Yun Wang, Li Wang, Yu-Xin Yang, Chun-Peng Zhao, Yan-Long Jia, Qin Li, Jun-He Zhang, Yi-You Peng, Miao Wang, Hong-Yan Xu, Xiao-Yin Wang
The characteristic sequence of β-interferon matrix attachment regions (MARs) can mediate transgene expression via episomal vectors in Chinese hamster ovary (CHO) cells. However, the host cells were from hamster ovaries, which are not suitable target cells for gene therapy. In this study, we aimed to evaluate the suitability of 12 different human cell lines as target cells for gene therapy. We transfected the cells with episomal vectors and obtained colonies stably expressing the vector products after G418 screening...
December 2, 2016: Current Gene Therapy
https://www.readbyqxmd.com/read/27852847/visualizing-the-essential-role-of-complete-virion-assembly-machinery-in-efficient-hepatitis-c-virus-cell-to-cell-transmission-by-viral-infection-activated-split-intein-mediated-reporter-system-visi
#2
Fanfan Zhao, Ting Zhao, Libin Deng, Dawei Lv, Xiaolong Zhang, Xiaoyu Pan, Jun Xu, Gang Long
: Hepatitis C virus (HCV) infects 2-3% of the world population and is a leading cause of liver diseases such as fibrosis, cirrhosis and hepatocellular carcinoma. Many aspects of HCV study, ranging from molecular virology, antiviral drug development and drug resistance profiling, were supported by straightforward assays of HCV replication and infection. Among these assays, HCV-dependent fluorescence relocalization (HDFR) system allows live-cell visualization of infection without modifying viral genome, but this strategy required careful recognition of fluorescence relocalization pattern for its high fluorescence background in cytoplasm...
November 16, 2016: Journal of Virology
https://www.readbyqxmd.com/read/27831485/analysis-of-membrane-proteins-localizing-to-the-inner-nuclear-envelope-in-living-cells
#3
Christine J Smoyer, Santharam S Katta, Jennifer M Gardner, Lynn Stoltz, Scott McCroskey, William D Bradford, Melainia McClain, Sarah E Smith, Brian D Slaughter, Jay R Unruh, Sue L Jaspersen
Understanding the protein composition of the inner nuclear membrane (INM) is fundamental to elucidating its role in normal nuclear function and in disease; however, few tools exist to examine the INM in living cells, and the INM-specific proteome remains poorly characterized. Here, we adapted split green fluorescent protein (split-GFP) to systematically localize known and predicted integral membrane proteins in Saccharomyces cerevisiae to the INM as opposed to the outer nuclear membrane. Our data suggest that components of the endoplasmic reticulum (ER) as well as other organelles are able to access the INM, particularly if they contain a small extraluminal domain...
November 9, 2016: Journal of Cell Biology
https://www.readbyqxmd.com/read/27742884/high-silicon-accumulation-in-the-shoot-is-required-for-down-regulating-the-expression-of-si-transporter-genes-in-rice
#4
Namiki Mitani-Ueno, Naoki Yamaji, Jian Feng Ma
Rice requires high silicon (Si) for its high and sustainable yield. The efficient uptake of Si in rice is mediated by two transporters OsLsi1 and OsLsi2, which function as influx and efflux transporters, respectively. Our previous studies showed that the mRNA expression levels of these transporter genes were down-regulated by Si. Herein we investigated the mechanism underlying regulation of OsLsi1 and OsLsi2 expression. There was a negative correlation between the expression level of OsLsi1 and OsLsi2 and shoot Si accumulation when the rice seedlings were exposed to different Si supply conditions...
October 13, 2016: Plant & Cell Physiology
https://www.readbyqxmd.com/read/27608276/split-gfp-sers-enhancers-in-plasmonic-nanocluster-probes
#5
Taerin Chung, Tugba Koker, Fabien Pinaud
The assembly of plasmonic metal nanoparticles into hot spot surface-enhanced Raman scattering (SERS) nanocluster probes is a powerful, yet challenging approach for ultrasensitive biosensing. Scaffolding strategies based on self-complementary peptides and proteins are of increasing interest for these assemblies, but the electronic and the photonic properties of such hybrid nanoclusters remain difficult to predict and optimize. Here, split-green fluorescence protein (sGFP) fragments are used as molecular glue and the GFP chromophore is used as a Raman reporter to assemble a variety of gold nanoparticle (AuNP) clusters and explore their plasmonic properties by numerical modeling...
September 8, 2016: Small
https://www.readbyqxmd.com/read/27604151/enhancing-endosomal-escape-for-intracellular-delivery-of-macromolecular-biologic-therapeutics
#6
Peter Lönn, Apollo D Kacsinta, Xian-Shu Cui, Alexander S Hamil, Manuel Kaulich, Khirud Gogoi, Steven F Dowdy
Bioactive macromolecular peptides and oligonucleotides have significant therapeutic potential. However, due to their size, they have no ability to enter the cytoplasm of cells. Peptide/Protein transduction domains (PTDs), also called cell-penetrating peptides (CPPs), can promote uptake of macromolecules via endocytosis. However, overcoming the rate-limiting step of endosomal escape into the cytoplasm remains a major challenge. Hydrophobic amino acid R groups are known to play a vital role in viral escape from endosomes...
2016: Scientific Reports
https://www.readbyqxmd.com/read/27574793/adipose-tissue-derived-microvascular-fragments-improve-vascularization-lymphangiogenesis-and-integration-of%C3%A2-dermal-skin-substitutes
#7
Florian S Frueh, Thomas Später, Nicole Lindenblatt, Maurizio Calcagni, Pietro Giovanoli, Claudia Scheuer, Michael D Menger, Matthias W Laschke
Full-thickness skin defects can be covered with dermal skin substitutes in combination with split-thickness skin grafts. However, slow vascularization of the matrices bears the risk of wound infection and extends the length of hospitalization. To overcome these problems, we describe a promising vascularization strategy. Green fluorescent protein(+) adipose tissue-derived microvascular fragments (ad-MVF) were isolated from epididymal fat pads of C57BL/6-Tg(CAG-EGFP)1Osb/J mice. ad-MVF were seeded on collagen-glycosaminoglycan matrices, which were implanted into full-thickness skin defects in the dorsal skinfold chamber of wild-type C57BL/6 mice...
August 26, 2016: Journal of Investigative Dermatology
https://www.readbyqxmd.com/read/27562135/automated-enrichment-transduction-and-expansion-of-clinical-scale-cd62l-t-cells-for-manufacturing-of-gtmps
#8
Christoph Priesner, Krasimira Aleksandrova, Ruth Esser, Nadine Mockel-Tenbrinck, Jana Leise, Katharina Drechsel, Michael Marburger, Andrea Quaiser, Lilia Goudeva, Lubomir Arseniev, Andrew Kaiser, Wolfgang Glienke, Ulrike Koehl
Multiple clinical studies demonstrated that adaptive immunotherapy using redirected T cells against advanced cancer has led to promising results with improved survival of the patients. The continuously increasing interest in those advanced Gene Therapy Medicinal Products (GTMPs) leads to a challenge on the manufacturing side regarding automation, process robustness and cell storage. Therefore, our study addresses the proof of principle in clinical-scale selection, stimulation, transduction and expansion of T cells using the automated closed CliniMACS® Prodigy system...
August 25, 2016: Human Gene Therapy
https://www.readbyqxmd.com/read/27422440/investigation-of-the-role-of-four-mitotic-septins-and-chitin-synthase-2-for-cytokinesis-in-kluyveromyces-lactis
#9
Dorthe Rippert, Jürgen J Heinisch
Septins are key components of the cell division machinery from yeast to humans. The model yeast Saccharomyces cerevisiae has five mitotic septins, Cdc3, Cdc10, Cdc11, Cdc12, and Shs1. Here we characterized the five orthologs from the genetically less-redundant milk yeast Kluyveromyces lactis. We found that except for KlSHS1 all septin genes are essential. Klshs1 deletions displayed temperature-sensitive growth and morphological defects. Heterologous complementation analyses revealed that all five K. lactis genes encode functional orthologs of their S...
September 2016: Fungal Genetics and Biology: FG & B
https://www.readbyqxmd.com/read/27390781/illuminating-the-sites-of-enterovirus-replication-in-living-cells-by-using-a-split-gfp-tagged-viral-protein
#10
H M van der Schaar, C E Melia, J A C van Bruggen, J R P M Strating, M E D van Geenen, A J Koster, M Bárcena, F J M van Kuppeveld
Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles [ROs]) with a unique protein and lipid composition on which they multiply their viral genome. Suitable tools for live-cell imaging of enterovirus ROs are currently unavailable, as recombinant enteroviruses that carry genes that encode RO-anchored viral proteins tagged with fluorescent reporters have not been reported thus far. To overcome this limitation, we used a split green fluorescent protein (split-GFP) system, comprising a large fragment [strands 1 to 10; GFP(S1-10)] and a small fragment [strand 11; GFP(S11)] of only 16 residues...
July 2016: MSphere
https://www.readbyqxmd.com/read/27385335/detection-of-protein-protein-interactions-at-the-septin-collar-in-saccharomyces-cerevisiae-using-a-tripartite-split-gfp-system
#11
Gregory C Finnigan, Angela Duvalyan, Elizabeth N Liao, Aspram Sargsyan, Jeremy Thorner
Various methods can provide a readout of the physical interaction between two biomolecules. A recently described tripartite split-GFP system has the potential to report by direct visualization via a fluorescence signal the intimate association of minimally tagged proteins expressed at their endogenous level in their native cellular milieu and can capture transient or weak interactions. Here we document the utility of this tripartite split-GFP system to assess in living cells protein-protein interactions in a dynamic cytoskeletal structure-the septin collar at the yeast bud neck...
September 1, 2016: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/27351653/development-of-a-novel-murine-model-for-treatment-of-infected-mesh-scenarios
#12
Arnab Majumder, Clayton C Petro, Lijia Liu, Mojtaba Fayezizadeh, Yuri W Novitsky
BACKGROUND: Indications regarding hernia repair after removal of previously infected prostheses remain unclear. Patients may receive staged primary repair or single-stage reconstructions, neither of which may be ideal. Although animal models have simulated contamination by direct inoculation of implants with bacteria, there remains a paucity of literature, which simulates a field following mesh infection and removal. We aimed to develop a murine model to mimic this complex scenario to allow for further testing of various implants...
June 28, 2016: Surgical Endoscopy
https://www.readbyqxmd.com/read/27282591/improving-analytical-methods-for-protein-protein-interaction-through-implementation-of-chemically-inducible-dimerization
#13
Tonni Grube Andersen, Sebastian J Nintemann, Magdalena Marek, Barbara A Halkier, Alexander Schulz, Meike Burow
When investigating interactions between two proteins with complementary reporter tags in yeast two-hybrid or split GFP assays, it remains troublesome to discriminate true- from false-negative results and challenging to compare the level of interaction across experiments. This leads to decreased sensitivity and renders analysis of weak or transient interactions difficult to perform. In this work, we describe the development of reporters that can be chemically induced to dimerize independently of the investigated interactions and thus alleviate these issues...
2016: Scientific Reports
https://www.readbyqxmd.com/read/27274053/a-scalable-strategy-for-high-throughput-gfp-tagging-of-endogenous-human-proteins
#14
Manuel D Leonetti, Sayaka Sekine, Daichi Kamiyama, Jonathan S Weissman, Bo Huang
A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9 nuclease/single-guide RNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless, and specific tagging of endogenous human genes with GFP...
June 21, 2016: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/27268028/combinatorial-pairwise-assembly-efficiently-generates-high-affinity-binders-and-enables-a-mix-and-read-detection-scheme
#15
Kevin B Carlin, Carlos A Cruz-Teran, Jay Prakash Kumar, Catherina Gomes, Balaji M Rao
We show that a combinatorial library constructed by random pairwise assembly of low affinity binders can efficiently generate binders with increased affinity. Such a library based on the Sso7d scaffold, from a pool of low affinity binders subjected to random mutagenesis, contained putative high affinity clones for a model target (lysozyme) at higher frequency than a library of monovalent mutants generated by random mutagenesis alone. Increased binding affinity was due to intramolecular avidity generated by linking binders targeting non-overlapping epitopes; individual binders of KD ~ 1...
June 6, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27184291/spatial-organization-of-heterologous-metabolic-system-in-vivo-based-on-tale
#16
Ling-Yun Zhu, Xin-Yuan Qiu, Ling-Yun Zhu, Xiao-Min Wu, Yuan Zhang, Qian-Hui Zhu, Dong-Yu Fan, Chu-Shu Zhu, Dong-Yi Zhang
For years, prokaryotic hosts have been widely applied in bio-engineering. However, the confined in vivo enzyme clustering of heterologous metabolic pathways in these organisms often results in low local concentrations of enzymes and substrates, leading to a low productive efficacy. We developed a new method to accelerate a heterologous metabolic system by integrating a transcription activator-like effector (TALE)-based scaffold system into an Escherichia coli chassis. The binding abilities of the TALEs to the artificial DNA scaffold were measured through ChIP-PCR...
2016: Scientific Reports
https://www.readbyqxmd.com/read/27140913/data-analysis-for-total-internal-reflection-fluorescence-microscopy
#17
Charles L Asbury
In the microscopes we use to analyze total internal reflection fluorescence (TIRF), the emitted fluorescence is split chromatically, using dichroic filters, into either two or three different colors ("channels"). In our two-color instrument, the green emission wavelengths (405-488 nm; for imaging green fluorescent protein [GFP]-tagged proteins) and far-red emission wavelengths (650-800 nm; for imaging Alexa-647-labeled microtubules) are projected onto the upper and lower halves, respectively, of a single camera...
2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/26988139/versatile-protein-tagging-in-cells-with-split-fluorescent-protein
#18
Daichi Kamiyama, Sayaka Sekine, Benjamin Barsi-Rhyne, Jeffrey Hu, Baohui Chen, Luke A Gilbert, Hiroaki Ishikawa, Manuel D Leonetti, Wallace F Marshall, Jonathan S Weissman, Bo Huang
In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair...
2016: Nature Communications
https://www.readbyqxmd.com/read/26976827/stabilization-of-a-prokaryotic-lat-transporter-by-random-mutagenesis
#19
Arturo Rodríguez-Banqueri, Ekaitz Errasti-Murugarren, Paola Bartoccioni, Lukasz Kowalczyk, Alex Perálvarez-Marín, Manuel Palacín, José Luis Vázquez-Ibar
The knowledge of three-dimensional structures at atomic resolution of membrane transport proteins has improved considerably our understanding of their physiological roles and pathological implications. However, most structural biology techniques require an optimal candidate within a protein family for structural determination with (a) reasonable production in heterologous hosts and (b) good stability in detergent micelles. SteT, the Bacillus subtilis L-serine/L-threonine exchanger is the best-known prokaryotic paradigm of the mammalian L-amino acid transporter (LAT) family...
April 2016: Journal of General Physiology
https://www.readbyqxmd.com/read/26857153/specific-cell-surface-labeling-of-gpcrs-using-split-gfp
#20
Wen-Xue Jiang, Xu Dong, Jing Jiang, Yu-Hong Yang, Ju Yang, Yun-Bi Lu, San-Hua Fang, Er-Qing Wei, Chun Tang, Wei-Ping Zhang
Specific cell surface labeling is essential for visualizing the internalization processes of G-protein coupled receptors (GPCRs) and for gaining mechanistic insight of GPCR functions. Here we present a rapid, specific, and versatile labeling scheme for GPCRs at living-cell membrane with the use of a split green fluorescent protein (GFP). Demonstrated with two GPCRs, GPR17 and CysLT2R, we show that two β-stands (β-stands 10 and 11) derived from a superfolder GFP (sfGFP) can be engineered to one of the three extracellular loop of a GPCR...
2016: Scientific Reports
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