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Comparison Of The Human Optimized Bacterial Luciferase Gene Cassette With Firefly Luciferase And Green Fluorescent Protein And Development Into A Real-Time Biosentinel For Toxic Chemicals

490 BioTech

Abstract:

Our group has previously demonstrated the ability to elicit autonomous bioluminescent production from the bacterial luciferase gene cassette (lux) from within the mammalian cellular environment. With an emission spectra that is virtually background-free and the ability to generate light autonomously, the sensitivity and real-time imaging capacity of bacterial luciferase far surpasses that of green fluorescent protein (GFP) and firefly luciferase (Luc).

To provide a frame of reference for use of this new technology, we have performed side-byside comparisons of the lux, Luc, and GFP reporter systems under both culture and small animal imaging conditions. A mammalian cell line was transfected with either the human optimized (ho) lux genes, holuc gene, or hogfp gene. These cell lines were then interrogated either in culture or following injection into a nude mouse model. Comparisons of the resulting signal strengths and dynamics were compared to evaluate their ability to function as reporters under these conditions. To validate the utility of the lux system as an autonomous reporter, it was challenged with the
known toxicant n-decanal and evaluated for its ability to report on bioavailability of the compound in real time.

At equivalent cellular population sizes the lux system was shown to be more sensitive than GFP (a minimum of 1.5 × 104 lux transfected cells visible compared to 5.0 × 105 GFP transfected cells visible) due to the reduction of detectable background signal during bioluminescent imaging. Larger cell populations are required to detect lux transfected cells than are required for the detection of Luc in mouse models, however, this can be overcome at population sizes above 2.5 × 104 by increasing integration time to 1 min. Additionally, the lux system retains the benefit of not requiring exogenous substrate input, allowing for repeated imaging and consistent correlation of the luminescent signal with cell population size. It is further demonstrated that the lux system is capable of detecting cytotoxicity quickly and in the μM concentration range for the known toxicant n-decanal.

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D.M. Close1,2, R.E Hahn1, S.A. Ripp1, S.J. Baek3, J. Sanseverino2 and G.S. Sayler1,2
1Center for Environmental Biotechnology, University of Tennessee, Knoxville
2Joint Institute for Biological Sciences, Oak Ridge National Lab Abstract
3College of Veterinary Medicine, University of Tennessee, Knoxville

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