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

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.

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Green Fluorescent Protein As a Cell-Labeling Tool and a Reporter of Gene Expression in Transgenic Rainbow Trout

Marine Biotechnology ◽

10.1007/pl00011801 ◽

1999 ◽

Vol 1 (5) ◽

pp. 448-457 ◽

Cited By ~ 20

Author(s):

Yutaka Takeuchi ◽

Goro Yoshizaki ◽

Toshio Takeuchi

Keyword(s):

Gene Expression ◽

Rainbow Trout ◽

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Cell Labeling ◽

A Cell ◽

Green Fluorescent

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Out with the Old, In with the New

Microscopy Today ◽

10.1017/s1551929500052251 ◽

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.

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

Phytopathology ◽

10.1094/phyto-97-5-0544 ◽

2007 ◽

Vol 97 (5) ◽

pp. 544-550 ◽

Cited By ~ 17

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.

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A Cell-Based Functional Assay Using a Green Fluorescent Protein-Based Calcium Indicator dCys-GCaMP

Assay and Drug Development Technologies ◽

10.1089/adt.2014.584 ◽

2014 ◽

Vol 12 (6) ◽

pp. 342-351 ◽

Cited By ~ 6

Author(s):

Bin Cai ◽

Xia Chen ◽

Fang Liu ◽

Jun Li ◽

Lijuan Gu ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Functional Assay ◽

Calcium Indicator ◽

A Cell ◽

Green Fluorescent

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Cell Cycle-Dependent Changes in Microtubule Dynamics in Living Cells Expressing Green Fluorescent Protein-α Tubulin

Molecular Biology of the Cell ◽

10.1091/mbc.12.4.971 ◽

2001 ◽

Vol 12 (4) ◽

pp. 971-980 ◽

Cited By ~ 228

Author(s):

Nasser M. Rusan ◽

Carey J. Fagerstrom ◽

Anne-Marie C. Yvon ◽

Patricia Wadsworth

Keyword(s):

Cell Cycle ◽

Green Fluorescent Protein ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Dynamic Instability ◽

Microtubule Dynamics ◽

A Cell ◽

Cycle Dependent ◽

Green Fluorescent ◽

Quantitative Immunoblotting

LLCPK-1 cells were transfected with a green fluorescent protein (GFP)-α tubulin construct and a cell line permanently expressing GFP-α tubulin was established (LLCPK-1α). The mitotic index and doubling time for LLCPK-1α were not significantly different from parental cells. Quantitative immunoblotting showed that 17% of the tubulin in LLCPK-1α cells was GFP-tubulin; the level of unlabeled tubulin was reduced to 82% of that in parental cells. The parameters of microtubule dynamic instability were compared for interphase LLCPK-1α and parental cells injected with rhodamine-labeled tubulin. Dynamic instability was very similar in the two cases, demonstrating that LLCPK-1α cells are a useful tool for analysis of microtubule dynamics throughout the cell cycle. Comparison of astral microtubule behavior in mitosis with microtubule behavior in interphase demonstrated that the frequency of catastrophe increased twofold and that the frequency of rescue decreased nearly fourfold in mitotic compared with interphase cells. The percentage of time that microtubules spent in an attenuated state, or pause, was also dramatically reduced, from 73.5% in interphase to 11.4% in mitosis. The rates of microtubule elongation and rapid shortening were not changed; overall dynamicity increased 3.6-fold in mitosis. Microtubule release from the centrosome and a subset of differentially stable astral microtubules were also observed. The results provide the first quantitative measurements of mitotic microtubule dynamics in mammalian cells.

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