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 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.


RESULTS
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
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.
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Dan M
DM
The Center for Environmental Biotechnology, The University of Tennessee, Knoxville, Tennessee, United States of America.
Patterson
Stacey S
SS
Ripp
Steven
S
Baek
Seung J
SJ
Sanseverino
John
J
Sayler
Gary S
GS
D000447:Aldehydes / Q000494:pharmacology; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D002452:Cell Count; D002460:Cell Line; D003062:Codon / Q000235:genetics; D038181:FMN Reductase / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008027:Light; D008156:Luciferases / Q000235:genetics*; D008163:Luminescent Measurements; D051379:Mice; D057054:Molecular Imaging; D055620:Optical Phenomena; D010088:Oxidoreductases / Q000235:genetics; D015203:Reproducibility of Results; D014733:Vibrio / Q000201:enzymology / Q000235:genetics*
D000447:Aldehydes; D001426:Bacterial Proteins; D003062:Codon; D010088:Oxidoreductases; D008156:Luciferases; D038181:FMN Reductase
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