Three-dimensional distribution of enhanced yellow fluorescent protein (eYFP)-expressing neurons (yellow) and enhanced green fluorescent protein (eGFP)-positive microglia (green) in the neocortex of a Thy1-eYFP×Cx3cr1-eGFP mouse implanted with an open skull window: Movie 87_1.

In this study we sought to develop a method for the co-localization of proteins in living cells utilizing the enhanced green fluorescent protein (EGFP) and a redshifted EGFP variant, EYFP (enhanced yellow fluorescent protein). EYFP was expressed as an unsubstituted molecule while EGFP was fused to NF1 (EGFP-NF1), a transcription factor found exclusively in the nucleus. The Leica TCS SP laser scanning confocal microscope was used. This microscope allows the user to monitor the emitted light at defined wavelengths owing to the presence of a monochrometer in the emission light path. pEGFP-NF1 and pEYFP were co-expressed in the same cell and excited with the 476–nm and 488–nm argon laser lines. To separate the EYFP and EGFP fluorescence, EGFP-NF1 emission was recorded between 496 and 505 nm. These wavelengths are on the left shoulder of the EGFP emission peak and exclude most of the EYFP fluorescence. The EYFP emission was followed between 670 and 754 nm, utilizing the tail of EYFP emission that extends well beyond that for EGFP. Under these conditions we obtained excellent discrimination between EYFP fluorescence and EGFP-NF1 emission. These observations demonstrate that EYFP- and EGFP-substituted chimeras can be used for simultaneous detection in living cells.

Download Full-text

Use of Green Fluorescent Protein Color Variants Expressed on Stable Broad-Host-Range Vectors to Visualize Rhizobia Interacting with Plants

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2000.13.11.1163 ◽

2000 ◽

Vol 13 (11) ◽

pp. 1163-1169 ◽

Cited By ~ 89

Author(s):

Nico Stuurman ◽

Cristina Pacios Bras ◽

Helmi R. M. Schlaman ◽

André H. M. Wijfjes ◽

Guido Bloemberg ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Host Range ◽

Laser Scanning ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Confocal Laser ◽

Broad Host Range ◽

Enhanced Yellow Fluorescent Protein ◽

Green Fluorescent ◽

Color Variants

We developed two sets of broad-host-range vectors that drive expression of the green fluorescent protein (GFP) or color variants thereof (henceforth collectively called autofluorescent proteins [AFPs]) from the lac promoter. These two sets are based on different replicons that are maintained in a stable fashion in Escherichia coli and rhizobia. Using specific filter sets or a dedicated confocal laser scanning microscope setup in which emitted light is split into its color components through a prism, we were able to unambiguously identify bacteria expressing enhanced cyan fluorescent protein (ECFP) or enhanced yellow fluorescent protein (EYFP) in mixtures of the two. Clearly, these vectors will be valuable tools for competition, cohabitation, and rescue studies and will also allow the visualization of interactions between genetically marked bacteria in vivo. Here, we used these vectors to visualize the interaction between rhizobia and plants. Specifically, we found that progeny from different rhizobia can be found in the same nodule or even in the same infection thread. We also visualized movements of bacteroids within plant nodule cells.

Download Full-text

Enhanced Green Fluorescent Protein (GFP) fluorescence after polyelectrolyte caging

Optics Express ◽

10.1364/oe.14.009815 ◽

2006 ◽

Vol 14 (21) ◽

pp. 9815 ◽

Cited By ~ 5

Author(s):

Alberto Diaspro ◽

Silke Krol ◽

Barbara Campanini ◽

Fabio Cannone ◽

Giuseppe Chirico

Keyword(s):

Green Fluorescent Protein ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Gfp Fluorescence ◽

Green Fluorescent

Download Full-text

Development and Characterization of a cDNA-Launch Recombinant Simian Hemorrhagic Fever Virus Expressing Enhanced Green Fluorescent Protein: ORF 2b’ Is Not Required for In Vitro Virus Replication

Viruses ◽

10.3390/v13040632 ◽

2021 ◽

Vol 13 (4) ◽

pp. 632

Author(s):

Yingyun Cai ◽

Shuiqing Yu ◽

Ying Fang ◽

Laura Bollinger ◽

Yanhua Li ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Cell Lines ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Infectious Clone ◽

Hemorrhagic Fever ◽

Simian Hemorrhagic Fever Virus ◽

Hemorrhagic Fever Virus ◽

Green Fluorescent

Simian hemorrhagic fever virus (SHFV) causes acute, lethal disease in macaques. We developed a single-plasmid cDNA-launch infectious clone of SHFV (rSHFV) and modified the clone to rescue an enhanced green fluorescent protein-expressing rSHFV-eGFP that can be used for rapid and quantitative detection of infection. SHFV has a narrow cell tropism in vitro, with only the grivet MA-104 cell line and a few other grivet cell lines being susceptible to virion entry and permissive to infection. Using rSHFV-eGFP, we demonstrate that one cricetid rodent cell line and three ape cell lines also fully support SHFV replication, whereas 55 human cell lines, 11 bat cell lines, and three rodent cells do not. Interestingly, some human and other mammalian cell lines apparently resistant to SHFV infection are permissive after transfection with the rSHFV-eGFP cDNA-launch plasmid. To further demonstrate the investigative potential of the infectious clone system, we introduced stop codons into eight viral open reading frames (ORFs). This approach suggested that at least one ORF, ORF 2b’, is dispensable for SHFV in vitro replication. Our proof-of-principle experiments indicated that rSHFV-eGFP is a useful tool for illuminating the understudied molecular biology of SHFV.

Download Full-text