Cargando…
Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line
BACKGROUND: The bacterial luciferase (lux) gene cassette consists of five genes (luxCDABE) whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux ope...
Autores principales: | , , , , , |
---|---|
Formato: | Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2010
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2929204/ https://www.ncbi.nlm.nih.gov/pubmed/20805991 http://dx.doi.org/10.1371/journal.pone.0012441 |
_version_ | 1782185908919861248 |
---|---|
author | Close, Dan M. Patterson, Stacey S. Ripp, Steven Baek, Seung J. Sanseverino, John Sayler, Gary S. |
author_facet | Close, Dan M. Patterson, Stacey S. Ripp, Steven Baek, Seung J. Sanseverino, John Sayler, Gary S. |
author_sort | Close, Dan M. |
collection | PubMed |
description | BACKGROUND: The bacterial luciferase (lux) gene cassette consists of five genes (luxCDABE) whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH(2)) was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp) from Vibrio harveyi. FMNH(2) supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background. CONCLUSIONS/SIGNIFICANCE: The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies. |
format | Text |
id | pubmed-2929204 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2010 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-29292042010-08-30 Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line Close, Dan M. Patterson, Stacey S. Ripp, Steven Baek, Seung J. Sanseverino, John Sayler, Gary S. PLoS One Research Article BACKGROUND: The bacterial luciferase (lux) gene cassette consists of five genes (luxCDABE) whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH(2)) was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp) from Vibrio harveyi. FMNH(2) supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background. CONCLUSIONS/SIGNIFICANCE: The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies. Public Library of Science 2010-08-27 /pmc/articles/PMC2929204/ /pubmed/20805991 http://dx.doi.org/10.1371/journal.pone.0012441 Text en This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. https://creativecommons.org/publicdomain/zero/1.0/ This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. |
spellingShingle | Research Article Close, Dan M. Patterson, Stacey S. Ripp, Steven Baek, Seung J. Sanseverino, John Sayler, Gary S. Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line |
title | Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line |
title_full | Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line |
title_fullStr | Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line |
title_full_unstemmed | Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line |
title_short | Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line |
title_sort | autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux) in a mammalian cell line |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2929204/ https://www.ncbi.nlm.nih.gov/pubmed/20805991 http://dx.doi.org/10.1371/journal.pone.0012441 |
work_keys_str_mv | AT closedanm autonomousbioluminescentexpressionofthebacterialluciferasegenecassetteluxinamammaliancellline AT pattersonstaceys autonomousbioluminescentexpressionofthebacterialluciferasegenecassetteluxinamammaliancellline AT rippsteven autonomousbioluminescentexpressionofthebacterialluciferasegenecassetteluxinamammaliancellline AT baekseungj autonomousbioluminescentexpressionofthebacterialluciferasegenecassetteluxinamammaliancellline AT sanseverinojohn autonomousbioluminescentexpressionofthebacterialluciferasegenecassetteluxinamammaliancellline AT saylergarys autonomousbioluminescentexpressionofthebacterialluciferasegenecassetteluxinamammaliancellline |