Traditionally, human toxicant bioavailability screening has been forced to proceed in either a high throughput fashion
using prokaryotic or lower eukaryotic targets with minimal applicability to humans, or in a more expensive, lower
throughput manner that uses fluorescent or bioluminescent human cells to directly provide human bioavailability data.
While these efforts are often sufficient for basic scientific research, they prevent the rapid and remote identification of
potentially toxic chemicals required for modern biosecurity applications. To merge the advantages of high throughput,
low cost screening regimens with the direct bioavailability assessment of human cell line use, we re-engineered the
bioluminescent bacterial luciferase gene cassette to function autonomously (without exogenous stimulation) within
human cells. Optimized cassette expression provides for fully endogenous bioluminescent production, allowing
continuous, real time monitoring of the bioavailability and toxicology of various compounds in an automated fashion.
To access the functionality of this system, two sets of bioluminescent human cells were developed. The first was
programed to suspend bioluminescent production upon toxicological challenge to mimic the non-specific detection of a
toxicant. The second induced bioluminescence upon detection of a specific compound to demonstrate autonomous
remote target identification. These cells were capable of responding to μM concentrations of the toxicant n-decanal, and
allowed for continuous monitoring of cellular health throughout the treatment process. Induced bioluminescence was
generated through treatment with doxycycline and was detectable upon dosage at a 100 ng/ml concentration. These
results demonstrate that leveraging autonomous bioluminescence allows for low-cost, high throughput direct assessment
of toxicant bioavailability.
KEYWORDS: Imaging systems, Green fluorescent protein, Signal detection, Tissues, In vivo imaging, In vitro testing, Preclinical imaging, Animal model studies, Bioluminescence, Magnesium
Bioluminescent and fluorescent reporter systems have enabled the rapid and continued growth of the optical imaging field over the last two decades. Of particular interest has been noninvasive signal detection from mammalian tissues under both cell culture and whole animal settings. Here we report on the advantages and limitations of imaging using a recently introduced bacterial luciferase (lux) reporter system engineered for increased bioluminescent expression in the mammalian cellular environment. Comparison with the bioluminescent firefly luciferase (Luc) system and green fluorescent protein system under cell culture conditions demonstrated a reduced average radiance, but maintained a more constant level of bioluminescent output without the need for substrate addition or exogenous excitation to elicit the production of signal. Comparison with the Luc system following subcutaneous and intraperitoneal injection into nude mice hosts demonstrated the ability to obtain similar detection patterns with in vitro experiments at cell population sizes above 2.5 × 104 cells but at the cost of increasing overall image integration time.
KEYWORDS: Bioluminescence, Proteins, In vivo imaging, Kidney, Imaging systems, Green fluorescent protein, Sensors, Biotechnology, Signal generators, Manufacturing
Current mammalian bioreporters using either firefly luciferase (luc) or GFP constructs require lysis and/or exogenous excitation to evoke a measurable response. Consequently, these cells cannot serve as continuous, on-line monitoring devices for in vivo imaging. Bacterial luciferase, lux, produces a photonic reaction that is cyclic, resulting in autonomous signal generation without the requirement for exogenous substrates or external activation. Therefore, lux-based bioluminescent bioreporters are the only truly autonomous light-generating sensors in existence. Unfortunately, the bacterial lux system has not yet been efficiently expressed in mammalian cells. In this research, three approaches for optimal expression of the a and b subunits of the bacterial luciferase protein were compared and reporter signal stability was evaluated from stably transfected human embryonic kidney cells. Maximum light levels were obtained from cells expressing the luciferase subunits linked with an internal ribosomal entry site (IRES). Cells harboring this construct produced bioluminescence equaling 2.6 X 106 photons/sec compared to 7.2 X 104 photons/sec obtained from cells expressing the luciferase from a dual promoter vector and 3.5 X 104 photons/sec from a Lux fusion protein. Furthermore, the bioluminescence levels remained stable for more than forty cell passages (5 months) in the absence of antibiotic selection. After this time, bioluminescence signals dropped at a rate of approximately 5% per cell passage. These data indicate that mammalian cell lines can be engineered to efficiently express the bacterial lux system, thus lending themselves to possible long-term continuous monitoring or imaging applications in vivo.
Microorganisms pose numerous problems when present in human occupied enclosed environments. Primary among these are health related hazards, manifested as infectious diseases related to contaminated drinking water, food, or air circulation systems or non-infectious allergy related complications associated with microbial metabolites (sick building syndrome). As a means towards rapid detection of microbial pathogens, we are attempting to harness the specificity of bacterial phage for their host with a modified quorum sensing amplification signal to produce quantifiable bioluminescent (lux) detection on a silicon microluminometer. The bacteriophage itself is metabolically inactive, only achieving replicative capabilities upon infection of its specific host bacterium. Bacteriophage bioluminescent bioreporters contain a genomically inserted luxI component. During an infection event, the phage genes and accompanying luxI construct are taken up by the host bacterium and transcribed, resulting in luxI expression and subsequent activation of a homoserine lactone inducible bioluminescent bioreporter. We constructed a vector carrying the luxI gene under the control of a strong E. coli promoter and cloned it into E. coli. We have shown that it can induce luminescence up to 14,000 counts per second when combined with the bioreporter strain. In their final embodiment, these sensors will be fully independent microelectronic monitors for microbial contamination, requiring only exposure of the biochip to the sample, with on-chip signal processing downloaded directly to the local area network of the environmental control system.
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