Gfp reporter virus

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J Biotechnol. Functional dissection of VP16, the trans -activator of herpes simplex virus immediate early gene expression. Support Center Support Center. It is likely that GFP will be useful as a reporter gene in transgenic plants as well as in virus-infected tissue. Abstract The gene encoding green fluorescent protein GFP of Aequorea victoria was introduced into the expression cassette of a virus vector based on potato virus X PVX.

Publication types Research Support, Non-U. Agrobacterium tumefaciens infiltration was performed as described previously LOV proteins were expressed as described previously 15 using E. Amino acid substitutions were introduced by using the QuikChange Site-directed Mutagenesis kit Stratagene. All amino acid changes were verified by DNA sequencing. Cells were attached to fibronectin-coated cover slips 24 h after transfection.

Proteins were extracted in lysis buffer 0. Fluorescence excitation and emission spectra were recorded by using a PerkinElmer LS luminescence spectrometer. Absorption spectra were measured by using a Shimadzu MultiSpec diode array spectrometer. For qualitative in vivo fluorescence measurements, equal densities of E.

For quantitative in vivo fluorescence measurements, E. For fluorescence quantum yield determination, FMN Sigma was used as a reference standard as reported previously 12 , LOV fluorescence was imaged by using a Leica SP2 confocal laser-scanning microscope with an excitation wavelength of nm. Fluorescence emission was detected between and nm.

GFP and YFP were excited at nm and their emission collected between — nm and — nm, respectively. For time series measurements, tissue was irradiated briefly to allow image focusing and minimize photobleaching. An initial image was recorded to select a region of interest before a series of images was collected automatically. Fluorescence was quantified by using Leica LCS software.

H2B-iLOV construct with the following settings: 1 pre-bleach scan, 40 bleaching scans every 6 s, 5 post-bleach scans every 60 s. We thank Brian Smith for his help in creating Fig. The authors declare no conflict of interest. This article contains supporting information online at www. National Center for Biotechnology Information , U. Published online Dec 5. Savenkov , d Alison G. Roberts , a Karl J.

Oparka , b and John M. Christie c, 2. Eugene I. Alison G. Karl J. John M. Author information Article notes Copyright and License information Disclaimer. E-mail: ku. Edited by Philip N. Received Aug 1. This article has been cited by other articles in PMC.

Abstract Fluorescent proteins FPs based on green fluorescent protein GFP are widely used throughout cell biology to study protein dynamics, and have extensive use as reporters of virus infection and spread. Keywords: fluorescence imaging, molecular evolution, photoreceptor, LOV domain.

Open in a separate window. Materials and Methods Plant Material. Virus Plasmid Constructs. Preparation of Infectious Transcripts and Plant Inoculation. Recombinant Protein Expression and Purification. Mammalian Cell Culture and Expression. Spectroscopic Analysis. Fluorescence Imaging. Supplementary Material Supporting Information: Click here to view.

Footnotes The authors declare no conflict of interest. References 1. Advances in fluorescent protein technology.

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Nat Rev Microbiol. Tsien RY. In addition, a significant proportion of NK and B cells were GFP positive, suggesting active infection of these cells. We next tested the effects of the influenza virus inhibitors oseltamivir and amantadine on the kinetics of in vivo infection progression. Treatment with oseltamivir dramatically reduced influenza infection in all cell types, whereas, surprisingly, amantadine treatment more efficiently blocked infection in B and NK cells.