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Autobioluminescent Human Cell Lines as Biomarkers for Localization and Toxicology Screening

490 BioTech


Both fluorescent and substrate-induced bioluminescent gene expression strategies have been employed extensively to visualize the location and health of human cell lines in cell culture and small animal host settings. While these methods have proven useful for a wide variety of experimental designs, they are similarly limited in their ability to generate continuous signal output due to their dependence on exogenous excitation in order to emit their respective fluorescent or bioluminescent signals. These requisite exogenous stimulations, whether they are luminescent or chemical in nature, can reduce the efficiency of data collection through the introduction of errors stemming from increased background fluorescence detection or inconsistent substrate administration and uptake. Similarly, this stimulating treatment may have unintentional effects on the cells under study, leading to perturbed or erroneous results. In an effort to alleviate the uncertainties associated with currently available fluorescent and bioluminescent reporter systems, we have re-engineered the bacterial luciferase gene cassette to function within a human cell line. Unlike alternative reporter systems, the bacterial luciferase gene cassette consists of six genes (luxCDABEfrp) that when expressed
simultaneously produce a bioluminescent signal in the visible range using only substrates endogenous to the host cell. A human kidney cell line stably expressing these genes was demonstrated to be detectable down to a population size of 15,000 cells and displayed quantitative correlation between bioluminescent output and population size (r-squared value of 0.95) when imaged in a 24 well culture plate. Unlike the same cell line expressing firefly
luciferase, bioluminescent production remained relatively constant over a 24 hr imaging period, displaying a range of 4,500 photons/sec/cm2/steradian under these imaging conditions. When injected subcutaneously into a nude mouse model, as few as 25,000 cells were visible through the host tissue, and bioluminescent output again correlated with cell population size (r-squared value of 0.99). By exposing these constitutively bioluminescent cells to a known toxic compound, it was possible to observe a decrease in luminescent
production at doses previously shown to be toxic in the literature and to monitor these changes in a near real-time fashion. Cells under toxic challenge could be visualized passing through three stages: a preliminary stage where the toxicant had not yet affected cellular health and bioluminescent production remained strong, an intermediate stage where the cells were attempting to counteract the effects of the toxicant and bioluminescence oscillated between normal and decreased levels, and a final stage where cells had succumbed to toxicant treatment and bioluminescence remained significantly (p < 0.05) below that of untreated cells. Because of its ability to autonomously and continuously produce a bioluminescent signal without exogenous excitation, this re-engineered bacterial luciferase
gene cassette presents an alternative method for localization and evaluation of human cell lines, both in cell culture and small animal models, that can be utilized with existing equipment and imaging protocols.

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Dan Close1,3, Steven Ripp2,3, Stacey Patterson3, and Gary Sayler1,2,3
1Joint Institute for Biological Sciences, The University of Tennessee, Knoxville, TN 37996
2Center for Environmental Biotechnology, The University of Tennessee, Knoxville, TN 37996
3490 Biotech, Inc., Knoxville, TN 37996

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