The ultimate goal for this research is to provide a new platform technology for a fully autonomous, reagentless bioluminescent (lux) reporter gene system for cancer diagnostics, in vivo imaging, tissue-based biosensing, and high throughput screening in mammalian cells. Reporter gene technology for in vitro and in vivo imaging employing fluorescent proteins (such as GFP), firefly luciferase (luc), or aqueorin has made tremendous advances in recent years. However, for even broader imaging and biosensing applications, each of these reporter systems has intrinsic limitations attributable to factors such as high background, cytotoxicity, or requirements for exogenous reagent additions. Our recent developments have demonstrated complete expression of the bacterial bioluminescence system (luxCDABE) in lower eukaryotes as well as expression of """"""""humanized"""""""" bacterial luciferase (luxAB) in human cell lines. These developments provide a clear path for overcoming limitations of other reporter systems and the creation of a new capability for imaging in vivo gene expression in early diagnosis, therapeutic efficacy and disease re-occurrence in mammalian systems. This capability builds upon the lux reporter gene system which provides endogenous synthesis and recycling of all biochemical substrates required for fully auto-catalytic light production as a gene expression response. In our efforts to date, a bioinformatics analysis and recursive PCR approach were used to re-engineer the bacterial luciferase, luxAB genes, of Photorhabdus luminescens for codon optimized expression in HEK293 cells. This work demonstrated in vivo production of transcript and mature protein with high levels of bioluminescence demonstrated in a cell-free assay with added n-decanal. The specific goal of this proposed research is to construct a stable mammalian cell line capable of autonomous bioluminescence from expression of the complete lux operon (luxCDABE) and to elucidate the value of this reporter by constitutive autonomous bioluminescence imaging in a colorectal cancer cell model. The working hypothesis of this research is that a human cancer cell line (HCT-116) can be engineered to efficiently express the complete bacterial bioluminescence reaction and be monitored in vivo. The ultimate goal for this research is to provide a new platform technology for a fully autonomous, reagentless bioluminescent (lux) reporter gene system for cancer diagnostics, in vivo imaging, tissue-based biosensing, and high throughput screening in mammalian cells. This technology will provide the capability for imaging in vivo gene expression in early diagnosis, therapeutic efficacy and disease re-occurrence in whole animal models. ? ? ?
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