scholarly journals Out with the Old, In with the New

2003 ◽  
Vol 11 (1) ◽  
pp. 3-4
Author(s):  
Stephen W. Carmichael
Keyword(s):  
Electron Microscopy ◽  
Green Fluorescent Protein ◽  
Temporal Resolution ◽  
Fluorescent Protein ◽  
Fluorescence And Electron Microscopy ◽  
A Cell ◽  
Green Fluorescent ◽  
Microscopic Techniques ◽  
Biologic System ◽  
Over Time

Temporal resolution has long been a challenge to microscopists. Certainly, spatial resolution has occupied center stage, but we're all concerned about what happens over time in a biologic system, for example, a cell. Tags such as green fluorescent protein (GFP) have been used with confocal microscopy and other light microscopic techniques to achieve outstanding temporal resolution, but good spatial and temporal resolution have proven to be difficult to achieve simultaneously. This has been accomplished in a remarkable study by Guido Gaietta, Thomas Deerinck, Stephen Adams, James Bouwer, Oded Tour, Dale Laird, Gina Sosinsky, Roger Tsien, and Mark Ellisman, who demonstrated a pulse-chase technique that correlates with both fluorescence and electron microscopy.

Correlating cell cycle with apoptosis in a cell line expressing a tandem green fluorescent protein substrate specific for group II caspases

Cytometry ◽  
2001 ◽  
Vol 45 (3) ◽  
pp. 225-234 ◽  
Author(s):  
Christopher J. Donahue ◽  
Maxine Santoro ◽  
Donald Hupe ◽  
Jay M. Jones ◽  
Brian Pollok ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Green Fluorescent Protein ◽  
Cell Line ◽  
Fluorescent Protein ◽  
Protein Substrate ◽  
A Cell ◽  
Group Ii ◽  
Green Fluorescent

scholarly journals Establishment of a Stable Cell Line Coexpressing Dengue Virus-2 and Green Fluorescent Protein for Screening of Antiviral Compounds

2011 ◽  
Vol 17 (3) ◽  
pp. 283-292 ◽  
Author(s):  
V. Leardkamolkarn ◽  
W. Sirigulpanit
Keyword(s):  
Green Fluorescent Protein ◽  
Dengue Virus ◽  
Cell Line ◽  
Fluorescent Protein ◽  
Antiviral Drug ◽  
Stable Cell Line ◽  
Entry Site ◽  
Mouth Disease Virus ◽  
A Cell ◽  
Green Fluorescent

This study aimed to generate a stable cell line harboring subgenomic dengue virus replicon and a green fluorescent gene (DENV/GFP) for a cell-based model to screen anti-DENV compounds. The gene-encoding envelope protein of DENV-2 was deleted and then replaced with fragments of the GFP gene and a foot-and-mouth-disease virus 2A–derived cleavage site. The human cytomegalovirus immediate early and antisense hepatitis delta virus ribozyme sequences were added at the 5′- and 3′-ends. An internal ribosome entry site and neomycin resistance genes were placed upstream and next to the NS1 gene. The recombinant plasmids were propagated in a mammalian cell line. A stable cell line with the brightest green fluorescent protein and the highest viral protein and RNA expression was selected from six clones. The clone was then examined for effectiveness in an antiviral drug screening assay with compounds isolated from the local plants using two known antiviral agents as controls. Two novel flavones, PMF and TMF, were discovered having DENV-inhibitory properties. The data were validated by a conventional plaque titration assay. The results indicate that this newly developed cell line is efficient for use as a cell-based model for primary screening of anti-DENV compounds.

Synthesis and maturation of green fluorescent protein in a cell-free translation system

1996 ◽  
Vol 18 (12) ◽  
pp. 1447-1452 ◽  
Author(s):  
Vyacheslav A. Kolb ◽  
Eugeny V. Makeyev ◽  
William W. Ward ◽  
Alexander S. Spirin
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Translation System ◽  
Free Translation ◽  
A Cell ◽  
Green Fluorescent

scholarly journals Two Resistance Modes to Clover yellow vein virus in Pea Characterized by a Green Fluorescent Protein-Tagged Virus

2007 ◽  
Vol 97 (5) ◽  
pp. 544-550 ◽  
Author(s):  
Marcelo Andrade ◽  
Masanao Sato ◽  
Ichiro Uyeda
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Cell Movement ◽  
Recessive Gene ◽  
Yellow Vein ◽  
Mechanical Inoculation ◽  
Viral Movement ◽  
Clover Yellow Vein Virus ◽  
A Cell ◽  
Green Fluorescent

This study characterized resistance in pea lines PI 347295 and PI 378159 to Clover yellow vein virus (ClYVV). Genetic cross experiments showed that a single recessive gene controls resistance in both lines. Conventional mechanical inoculation did not result in infection; however, particle bombardment with infectious plasmid or mechanical inoculation with concentrated viral inocula did cause infection. When ClYVV No. 30 isolate was tagged with a green fluorescent protein (GFP) and used to monitor infection, viral cell-to-cell movement differed in the two pea lines. In PI 347595, ClYVV replicated at a single-cell level, but did not move to neighboring cells, indicating that resistance operated at a cell-to-cell step. In PI 378159, the virus moved to cells around the infection site and reached the leaf veins, but viral movement was slower than that in the susceptible line. The viruses observed around the infection sites and in the veins were then recovered and inoculated again by a conventional mechanical inoculation method onto PI 378159 demonstrating that ClYVV probably had mutated and newly emerged mutant viruses can move to neighboring cells and systemically infect the plants. Tagging the virus with GFP was an efficient tool for characterizing resistance modes. Implications of the two resistance modes are discussed.

Localization of Green Fluorescent Protein Absorbance by Energy Filtered Transmission Electron Microscopy

2000 ◽  
Vol 6 (S2) ◽  
pp. 324-325
Author(s):  
J. A. Davis ◽  
R. G. Garces ◽  
J.-Y. Diao ◽  
F. P. Ottensmeyer
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Green Fluorescent Protein ◽  
Energy Loss ◽  
Energy Resolution ◽  
Fluorescent Protein ◽  
High Energy ◽  
High Energy Resolution ◽  
Transmission Electron ◽  
Green Fluorescent

Energy filtered transmission electron microscopy has the potential to provide high resolution, spatially resolved, atomic and chemical information. However, aberrations generated by the electron spectrometer blur the energy resolution and limit the atomic or molecular distributions that can be studied. Energy absorptions corresponding to the visible light range fall below an energy loss of 5 eV. The selection of electrons that have lost an amount of energy corresponding to chromophore absorption by the sample thus requires a spectrometer with a high energy resolution over the full image plane. A corrected prism-mirror-prism filter that has a resolution of 1.1 eV, sufficient to select these low energy loss electrons, was developed and installed by us in a Zeiss EM902. Its imaging capability was verified for a number of different chromophores. The chromophore currently under study is that of the green fluorescent protein (GFP).

Visualizing Green Fluorescent Protein and Fluorescence Associated with a Gold Conjugate in Thin Sections with Correlative Confocal and Electron Microscopy

2004 ◽  
Vol 10 (S02) ◽  
pp. 156-157 ◽  
Author(s):  
Paul. A Sims ◽  
Jeff Hardin
Keyword(s):  
Electron Microscopy ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Thin Sections ◽  
Gold Conjugate ◽  
Green Fluorescent ◽  
Confocal And Electron Microscopy

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

scholarly journals Better Protein Localization

2011 ◽  
Vol 19 (5) ◽  
pp. 8-10
Author(s):  
Stephen W. Carmichael ◽  
Philip Oshel
Keyword(s):  
Electron Microscopy ◽  
Green Fluorescent Protein ◽  
Cell Membrane ◽  
Fluorescence Microscopy ◽  
Light Microscopy ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
On Line ◽  
Specific Proteins ◽  
Green Fluorescent

Localizing specific proteins within cells, tissues, and organisms has been a goal of microscopists for generations. In the early 1990s, a breakthrough was made when a molecule originally derived from a jellyfish was introduced as a probe for fluorescence microscopy. This molecule is green fluorescent protein (GFP), and it has become well known for its usefulness in localizing proteins at the level of the light microscope. It is also well known that electron microscopy (EM) offers far superior spatial resolution over light microscopy, but the application of probes to localize specific proteins has required antibodies conjugated with colloidal metals (such as gold). Delivery of antibodies into the cell commonly requires detergents to permeabilize the cell membrane, which compromises the ultrastructural detail. Another breakthrough was recently published on-line by Xiaokun Shu, Varda Lev-Ram, Thomas Deerinck, Yingchuan Qi, Ericka Ramko, Michael Davidson, Yishi Jin, Mark Ellisman, and Roger Tsien: they have developed a method similar to using GFP for light microscopy, but for specifically tagging proteins at the EM level.

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