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Title: Evaluation of Gaussia luciferase and foot-and-mouth disease virus 2A translational interrupter chimeras as polycistronic reporters for transgene expression

Abstract

Background: The Gaussia princeps luciferase is used as a stand-alone reporter of transgene expression for in vitro and in vivo expression systems due to the rapid and easy monitoring of luciferase activity. We sought to simultaneously quantitate production of other recombinant proteins by transcriptionally linking the Gaussia princeps luciferase gene to other genes of interest through the foot-and-mouth disease virus 2A translational interrupter sequence. Results: We produced six plasmids, each encoding a single open reading frame, with the foot-and-mouth disease virus 2A sequence placed either N-terminal or C-terminal to the Gaussia princeps luciferase gene. Two plasmids included novel Gaussia princeps luciferase variants with the position 1 methionine deleted. Placing a foot-and-mouth disease virus 2A translational interrupter sequence on either the N- or C-terminus of the Gaussia princeps luciferase gene did not prevent the secretion or luminescence of resulting chimeric luciferase proteins. We also measured the ability of another polycistronic plasmid vector with a 2A-luciferase sequence placed downstream of the foot-and-mouth disease virus P1 and 3C protease genes to produce of foot-and-mouth disease virus-like particles and luciferase activity from transfected cells. Incorporation of the 2A-luciferase sequence into a transgene encoding foot-and-mouth disease virus structural proteins retained luciferase activity and the abilitymore » to form virus-like particles. Conclusions: We demonstrated a mechanism for the near real-time, sequential, non-destructive quantitative monitoring of transcriptionally-linked recombinant proteins and a valuable method for monitoring transgene expression in recombinant vaccine constructs.« less

Authors:
ORCiD logo [1];  [2];  [2];  [2]
  1. Plum Island Animal Disease Center, Greenport, NY (United States). U.S. Department of Homeland Security Science and Technology Directorate; Plum Island Animal Disease Center Research Participation Program (PIADC), Oak Ridge, TN (United States). Oak Ridge Institute for Science and Education
  2. Plum Island Animal Disease Center, Greenport, NY (United States). U.S. Department of Homeland Security Science and Technology Directorate
Publication Date:
Research Org.:
Oak Ridge Inst. for Science and Education (ORISE), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1494693
Grant/Contract Number:  
AC05-06OR23100
Resource Type:
Accepted Manuscript
Journal Name:
BMC Biotechnology (Online)
Additional Journal Information:
Journal Name: BMC Biotechnology (Online); Journal Volume: 17; Journal Issue: 1; Journal ID: ISSN 1472-6750
Publisher:
BioMed Central
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Gaussia luciferase; Foot-and-mouth disease virus; 2A; Bicistronic; Polycistronic; Biomarker; Virus-like particles

Citation Formats

Puckette, Michael, Burrage, Thomas, Neilan, John G., and Rasmussen, Max. Evaluation of Gaussia luciferase and foot-and-mouth disease virus 2A translational interrupter chimeras as polycistronic reporters for transgene expression. United States: N. p., 2017. Web. doi:10.1186/s12896-017-0367-0.
Puckette, Michael, Burrage, Thomas, Neilan, John G., & Rasmussen, Max. Evaluation of Gaussia luciferase and foot-and-mouth disease virus 2A translational interrupter chimeras as polycistronic reporters for transgene expression. United States. doi:10.1186/s12896-017-0367-0.
Puckette, Michael, Burrage, Thomas, Neilan, John G., and Rasmussen, Max. Mon . "Evaluation of Gaussia luciferase and foot-and-mouth disease virus 2A translational interrupter chimeras as polycistronic reporters for transgene expression". United States. doi:10.1186/s12896-017-0367-0. https://www.osti.gov/servlets/purl/1494693.
@article{osti_1494693,
title = {Evaluation of Gaussia luciferase and foot-and-mouth disease virus 2A translational interrupter chimeras as polycistronic reporters for transgene expression},
author = {Puckette, Michael and Burrage, Thomas and Neilan, John G. and Rasmussen, Max},
abstractNote = {Background: The Gaussia princeps luciferase is used as a stand-alone reporter of transgene expression for in vitro and in vivo expression systems due to the rapid and easy monitoring of luciferase activity. We sought to simultaneously quantitate production of other recombinant proteins by transcriptionally linking the Gaussia princeps luciferase gene to other genes of interest through the foot-and-mouth disease virus 2A translational interrupter sequence. Results: We produced six plasmids, each encoding a single open reading frame, with the foot-and-mouth disease virus 2A sequence placed either N-terminal or C-terminal to the Gaussia princeps luciferase gene. Two plasmids included novel Gaussia princeps luciferase variants with the position 1 methionine deleted. Placing a foot-and-mouth disease virus 2A translational interrupter sequence on either the N- or C-terminus of the Gaussia princeps luciferase gene did not prevent the secretion or luminescence of resulting chimeric luciferase proteins. We also measured the ability of another polycistronic plasmid vector with a 2A-luciferase sequence placed downstream of the foot-and-mouth disease virus P1 and 3C protease genes to produce of foot-and-mouth disease virus-like particles and luciferase activity from transfected cells. Incorporation of the 2A-luciferase sequence into a transgene encoding foot-and-mouth disease virus structural proteins retained luciferase activity and the ability to form virus-like particles. Conclusions: We demonstrated a mechanism for the near real-time, sequential, non-destructive quantitative monitoring of transcriptionally-linked recombinant proteins and a valuable method for monitoring transgene expression in recombinant vaccine constructs.},
doi = {10.1186/s12896-017-0367-0},
journal = {BMC Biotechnology (Online)},
number = 1,
volume = 17,
place = {United States},
year = {2017},
month = {6}
}

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Figures / Tables:

Figure 1 Figure 1: a Construct layouts of bicistronic templates evaluated. b Nucleotide and amino acid sequences of Δ1D2A translational interrupter

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    Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.