Autonomous & Real-Time Mammalian Bioreporter System

Mammalian bioreporter systems, such as firefly luciferase (FLuc) and green fluorescent protein (GFP), require lysis and substrate addition or exogenous excitation, respectively. Thus, FLuc, GFP and similar reporters, are not able to function as stand-alone, real-time reporters in vivo. The bacterial luciferase gene (lux) is the only bioreporter system that can generate bioluminescent light signals exclusive of supplementary substrate additions or cell destruction. To produce a detectable light signal, this reporter system utilizes three endogenous substrates including molecular oxygen, reduced riboflavin phosphate (FMNH2), and a long chain aliphatic aldehyde. The lux operon consists of five genes (luxCDABE). The luciferase protein formed from the products of the luxA and luxB genes, first binds FMNH2, followed by oxygen, and then aldehyde re-synthesized by luxCDE gene products. The formed complex then slowly oxidizes to generate light at a wavelength of 490 nm which can be detected.

Although lux is a bacterial bioreporter system, it has been successfully expressed in mammalian cells, and has been developed for use as a biomedical detection tool for toxicity screening and visualization of tumors in small animal models. Compared with the traditional mammalian bioluminescent and fluorescent reporter system, the modified bacterial full-lux reporter system has its own advantages.
1) It does not need supplementary substrate additions or exogenous manipulations.
2) It autonomously and continuously produces bioluminescent light signal which enables to image directly within a living host in real-time.
3) It has negligible background.

While focusing its advantages, the disadvantages of this reporter system should also be considered.
1) It involves multiple genes which poses great difficulties to achieve co-expression.
2) It is demonstrated that the bioluminescent signal produced by the lux reporter system is several orders of magnitude lower than that of the more common firefly luciferase reporter. Thus, the lux reporter system requires more cells to produce a significantly detectable signal in both in vitro cell cultures and in vivo small animals.

To fully exploit the advantages of the lux reporter system, all five genes must be expressed simultaneously. However, expression of wild type lux genes in mammalian cells is quite low and thus need modification. RGBiotech offers plasmids containing the codon-optimized bacterial luciferase gene cassettes that allow the expression of the full luxCDABE. These plasmids can be used for generating mammalian stable cell lines that stably co-expressing all five genes of the bacterial luciferase gene (lux). Please contact us at admin@rgbiotech.com for more information.

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