Expression of a Humanized Viral 2A-Mediated lux Operon Efficiently Generates Autonomous Bioluminescence in Human Cells

Xu, Tingting; Ripp, Steven; Sayler, Gary S.; Close, Dan M.

doi:10.1371/journal.pone.0096347

Expression of a Humanized Viral 2A-Mediated lux Operon Efficiently Generates Autonomous Bioluminescence in Human Cells

Journal Article · Fri May 02 00:00:00 EDT 2014 · PLoS ONE

DOI:https://doi.org/10.1371/journal.pone.0096347· OSTI ID:1627696

Xu, Tingting ^[1]; Ripp, Steven ^[2]; Sayler, Gary S. ^[3]; Close, Dan M. ^[2]

Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). The Joint Institute for Biological Sciences; DOE/OSTI
490 BioTech, Inc., Knoxville, TN (United States)
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). The Joint Institute for Biological Sciences

Expression of autonomous bioluminescence from human cells was previously reported to be impossible, suggesting that all bioluminescent-based mammalian reporter systems must therefore require application of a potentially influential chemical substrate. While this was disproven when the bacterial luciferase (lux) cassette was demonstrated to function in a human cell, its expression required multiple genetic constructs, was functional in only a single cell type, and generated a significantly reduced signal compared to substrate-requiring systems. Here we investigate the use of a humanized, viral 2A-linked lux genetic architecture for the efficient introduction of an autobioluminescent phenotype across a variety of human cell lines. The lux cassette was codon optimized and assembled into a synthetic human expression operon using viral 2A elements as linker regions. Human kidney, breast cancer, and colorectal cancer cell lines were both transiently and stably transfected with the humanized operon and the resulting autobioluminescent phenotype was evaluated using common imaging instrumentation. Autobioluminescent cells were screened for cytotoxic effects resulting from lux expression and their utility as bioreporters was evaluated through the demonstration of repeated monitoring of single populations over a prolonged period using both a modified E-SCREEN assay for estrogen detection and a classical cytotoxic compound detection assay for the antibiotic Zeocin. Furthermore, the use of self-directed bioluminescent initiation in response to target detection was assessed to determine its amenability towards deployment as fully autonomous sensors. In all cases, bioluminescent measurements were supported with traditional genetic and transcriptomic evaluations. Our results demonstrate that the viral 2A-linked, humanized lux genetic architecture successfully produced autobioluminescent phenotypes in all cell lines tested without the induction of cytotoxicity. This autobioluminescent phenotype allowed for repeated interrogation of populations and self-directed control of bioluminescent activation with detection limits and EC₅₀ values similar to traditional reporter systems, making the autobioluminescent cells amenable to automated monitoring and significantly reducing the time and cost required to perform bioluminescent workflows.

View Accepted Manuscript (DOE)

Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: Alternative Research & Development Foundation; Army Defense University Research Instrumentation Program; National Institutes of Health (NIH); National Science Foundation (NSF); USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 1627696

Journal Information:: PLoS ONE, Journal Name: PLoS ONE Journal Issue: 5 Vol. 9; ISSN 1932-6203

Publisher:: Public Library of ScienceCopyright Statement

Country of Publication:: United States

Language:: English

References (31)

Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein. Ryan, M. D.; Drew, J. The EMBO Journal, Vol. 13, Issue 4 https://doi.org/10.1002/j.1460-2075.1994.tb06337.x	journal	February 1994
Strengths and weaknesses of in vitro assays for estrogenic and androgenic activity Soto, Ana M.; Maffini, Maricel V.; Schaeberle, Cheryl M. Best Practice & Research Clinical Endocrinology & Metabolism, Vol. 20, Issue 1 https://doi.org/10.1016/j.beem.2005.09.001	journal	March 2006
Biosynthesis of aliphatic aldehydes for the bacterial bioluminescent reaction stimulation by ATP and NADPH Meighen, E. A. Biochemical and Biophysical Research Communications, Vol. 87, Issue 4 https://doi.org/10.1016/s0006-291x(79)80018-2	journal	April 1979
Toxicity of the bacterial luciferase substrate, n -decyl aldehyde, to Saccharomyces cerevisiae and Caenorhabditis elegans Hollis, R. P.; Lagido, C.; Pettitt, J. FEBS Letters, Vol. 506, Issue 2 https://doi.org/10.1016/s0014-5793(01)02905-2	journal	September 2001
Recombinant Gaussia Luciferase. Overexpression, Purification, and Analytical Application of a Bioluminescent Reporter for DNA Hybridization Verhaegen, Monique; Christopoulos, Theodore K. Analytical Chemistry, Vol. 74, Issue 17 https://doi.org/10.1021/ac025742k	journal	September 2002
piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells Woltjen, Knut; Michael, Iacovos P.; Mohseni, Paria Nature, Vol. 458, Issue 7239 https://doi.org/10.1038/nature07863	journal	March 2009
Mathematical model of the Lux luminescence system in the terrestrial bacterium Photorhabdus luminescens Welham, Patricia A.; Stekel, Dov J. Mol. BioSyst., Vol. 5, Issue 1 https://doi.org/10.1039/b812094c	journal	January 2009
Molecular mechanisms of translation initiation in eukaryotes Pestova, T. V.; Kolupaeva, V. G.; Lomakin, I. B. Proceedings of the National Academy of Sciences, Vol. 98, Issue 13 https://doi.org/10.1073/pnas.111145798	journal	June 2001
Isolation and expression of a cDNA encoding Renilla reniformis luciferase. Lorenz, W. W.; McCann, R. O.; Longiaru, M. Proceedings of the National Academy of Sciences, Vol. 88, Issue 10 https://doi.org/10.1073/pnas.88.10.4438	journal	May 1991
Highly Efficient Multicistronic Lentiviral Vectors with Peptide 2A Sequences Ibrahimi, Abdelilah; Velde, Greetje Vande; Reumers, Veerle Human Gene Therapy, Vol. 20, Issue 8 https://doi.org/10.1089/hum.2008.188	journal	August 2009
Anhydrotetracycline, a novel effector for tetracycline controlled gene expression systems in eukaryotic cells Gossen, Manfred; Bujard, Hermann Nucleic Acids Research, Vol. 21, Issue 18 https://doi.org/10.1093/nar/21.18.4411	journal	January 1993
Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line Legler, J.; van den Brink, C. E.; Brouwer, A. Toxicological Sciences, Vol. 48, Issue 1 https://doi.org/10.1093/toxsci/48.1.55	journal	March 1999
Molecular biology of bacterial bioluminescence Meighen, E. A. Microbiological Reviews, Vol. 55, Issue 1 https://doi.org/10.1128/mr.55.1.123-142.1991	journal	March 1991
Enzymes and Genes from the lux Operons of Bioluminescent Bacteria Meighen, E. A. Annual Review of Microbiology, Vol. 42, Issue 1 https://doi.org/10.1146/annurev.mi.42.100188.001055	journal	October 1988
Development of 2A peptide-based strategies in the design of multicistronic vectors Szymczak, Andrea L.; Vignali, Dario AA Expert Opinion on Biological Therapy, Vol. 5, Issue 5 https://doi.org/10.1517/14712598.5.5.627	journal	May 2005
Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity Xu, Tingting; Close, Dan M.; Webb, James D. Journal of Visualized Experiments, Issue 80 https://doi.org/10.3791/50972	journal	October 2013
Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method Livak, Kenneth J.; Schmittgen, Thomas D. Methods, Vol. 25, Issue 4 https://doi.org/10.1006/meth.2001.1262	journal	December 2001
[1] Cloning firefly luciferase de Wet, Jeffrey R.; Wood, Keith V.; Helinski, Donald R. Methods in Enzymology https://doi.org/10.1016/0076-6879(86)33050-7	book	January 1986
Inducible vectors for expression in mammalian cells Yarranton, Geoffrey T. Current Opinion in Biotechnology, Vol. 3, Issue 5 https://doi.org/10.1016/0958-1669(92)90078-W	journal	October 1992
Biosynthesis of aliphatic aldehydes for the bacterial bioluminescent reaction stimulation by ATP and NADPH Meighen, E. A. Biochemical and Biophysical Research Communications, Vol. 87, Issue 4 https://doi.org/10.1016/S0006-291X(79)80018-2	journal	April 1979
Toxicity of the bacterial luciferase substrate, n -decyl aldehyde, to Saccharomyces cerevisiae and Caenorhabditis elegans Hollis, R. P.; Lagido, C.; Pettitt, J. FEBS Letters, Vol. 506, Issue 2 https://doi.org/10.1016/S0014-5793(01)02905-2	journal	September 2001
The fusion Vibrio campbellii luciferase as a eukaryotic gene reporter Tinikul, Ruchanok; Thotsaporn, Kittisak; Thaveekarn, Wichit Journal of Biotechnology, Vol. 162, Issue 2-3 https://doi.org/10.1016/j.jbiotec.2012.08.018	journal	December 2012
Bacterial luciferase: a carbon-13, nitrogen-15, and phosphorus-31 nuclear magnetic resonance investigation Vervoort, Jacques; Muller, Franz; O'Kane, Dennis J. Biochemistry, Vol. 25, Issue 24 https://doi.org/10.1021/bi00372a041	journal	December 1986
Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide–based retroviral vector Szymczak, Andrea L.; Workman, Creg J.; Wang, Yao Nature Biotechnology, Vol. 22, Issue 5 https://doi.org/10.1038/nbt957	journal	April 2004
Analyzing real-time PCR data by the comparative CT method Schmittgen, Thomas D.; Livak, Kenneth J. Nature Protocols, Vol. 3, Issue 6 https://doi.org/10.1038/nprot.2008.73	journal	June 2008
Mathematical model of the Lux luminescence system in the terrestrial bacterium Photorhabdus luminescens Welham, Patricia A.; Stekel, Dov J. Mol. BioSyst., Vol. 5, Issue 1 https://doi.org/10.1039/B812094C	journal	January 2009
Development and Characterization of a Cell Line That Stably Expresses an Estrogen-Responsive Luciferase Reporter for the Detection of Estrogen Receptor Agonist and Antagonists Wilson, V. S. Toxicological Sciences, Vol. 81, Issue 1 https://doi.org/10.1093/toxsci/kfh180	journal	May 2004
Comparison of human optimized bacterial luciferase, firefly luciferase, and green fluorescent protein for continuous imaging of cell culture and animal models Close, Dan M.; Hahn, Ruth E.; Patterson, Stacey S. Journal of Biomedical Optics, Vol. 16, Issue 4 https://doi.org/10.1117/1.3564910	journal	January 2011
Molecular biology of bacterial bioluminescence. Meighen, E. A. Microbiological Reviews, Vol. 55, Issue 1 https://doi.org/10.1128/MMBR.55.1.123-142.1991	journal	January 1991
The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic environmental pollutants. Soto, A. M.; Sonnenschein, C.; Chung, K. L. Environmental Health Perspectives, Vol. 103, Issue suppl 7 https://doi.org/10.1289/ehp.95103s7113	journal	October 1995
Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line Close, Dan M.; Patterson, Stacey S.; Ripp, Steven PLoS ONE, Vol. 5, Issue 8 https://doi.org/10.1371/journal.pone.0012441	journal	August 2010

Cited By (10)

Autonomous bioluminescence imaging of single mammalian cells with the bacterial bioluminescence system Gregor, Carola; Pape, Jasmin K.; Gwosch, Klaus C. Proceedings of the National Academy of Sciences, Vol. 116, Issue 52 https://doi.org/10.1073/pnas.1913616116	journal	December 2019
Imaging of Red-Shifted Light From Bioluminescent Tumors Using Fluorescence by Unbound Excitation From Luminescence Sônego, Fabiane; Bouccara, Sophie; Pons, Thomas Frontiers in Bioengineering and Biotechnology, Vol. 7 https://doi.org/10.3389/fbioe.2019.00073	journal	April 2019
The Expanding Toolbox of In Vivo Bioluminescent Imaging Xu, Tingting; Close, Dan; Handagama, Winode Frontiers in Oncology, Vol. 6 https://doi.org/10.3389/fonc.2016.00150	journal	June 2016
In Vivo Analysis of Protein–Protein Interactions with Bioluminescence Resonance Energy Transfer (BRET): Progress and Prospects Sun, Sihuai; Yang, Xiaobing; Wang, Yao International Journal of Molecular Sciences, Vol. 17, Issue 10 https://doi.org/10.3390/ijms17101704	journal	October 2016
Validation of a New Multicistronic Plasmid for the Efficient and Stable Expression of Transgenes in Microalgae Molina-Márquez, Ana; Vila, Marta; Rengel, Rocío International Journal of Molecular Sciences, Vol. 21, Issue 3 https://doi.org/10.3390/ijms21030718	journal	January 2020
Strongly enhanced bacterial bioluminescence with the ilux operon for single-cell imaging Gregor, Carola; Gwosch, Klaus C.; Sahl, Steffen J. Proceedings of the National Academy of Sciences, Vol. 115, Issue 5 https://doi.org/10.1073/pnas.1715946115	journal	January 2018
Improving Estrogenic Compound Screening Efficiency by Using Self-Modulating, Continuously Bioluminescent Human Cell Bioreporters Expressing a Synthetic Luciferase Xu, Tingting; Kirkpatrick, Andrew; Toperzer, Jody Toxicological Sciences, Vol. 168, Issue 2 https://doi.org/10.1093/toxsci/kfz004	journal	January 2019
Imaging of red-shifted photons from bioluminescent tumours using fluorescence by unbound excitation from luminescence Sônego, Fabiane; Bouccara, Sophie; Pons, Thomas Bioengineering and Biotechnology https://doi.org/10.1101/428771	journal	September 2018
Autonomous bioluminescence imaging of single mammalian cells with the bacterial bioluminescence system Gregor, Carola; Pape, Jasmin K.; Gwosch, Klaus C. Proceedings of the National Academy of Sciences https://doi.org/10.1101/798108	journal	October 2019
Real-time tracking of stem cell viability, proliferation, and differentiation with autonomous bioluminescence imaging Conway, Michael; Xu, Tingting; Kirkpatrick, Andrew BMC Biology, Vol. 18, Issue 1 https://doi.org/10.1186/s12915-020-00815-2	journal	July 2020

Similar Records

The Evolution of the Bacterial Luciferase Gene Cassette (lux) as a Real-Time Bioreporter

Journal Article · Tue Jan 10 19:00:00 EST 2012 · Sensors · OSTI ID:1628534

Catabolic gene expression is monitored by bioluminescence in bioreactor studies

Conference · Thu Dec 31 23:00:00 EST 1992 · OSTI ID:7096874

Catabolic gene expression is monitored by bioluminescence in bioreactor studies

Conference · Sun Feb 28 23:00:00 EST 1993 · OSTI ID:10124602

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
MTT assay
cell metabolism
gene expression
luciferase
operons
phenotypes
reactive oxygen species
transfection

Expression of a Humanized Viral 2A-Mediated lux Operon Efficiently Generates Autonomous Bioluminescence in Human Cells

Citation Formats

References (31)

Cited By (10)

Similar Records

Related Subjects