Luciferases and in vivo Molecular Imaging.
Introduction
Multiple luciferases have been cloned and applications in molecular biology and in vivo optical imaging have been identified. This review focuses on features of luciferases and their correspondent substrates called luciferins, commonly used in in vivo molecular imaging. Luciferases and bioluminescence imaging offer a significant advantage, since as opposed to fluorescence imaging: 1) they do not require an external excitation source in order to produce light, and 2) animal tissues do not emit significant amounts of light; low autoluminescence background signals favors high signal to noise detection sensitivity.
Firefly luciferase is the most popular enzyme, followed by Renilla luciferase, Gaussia luciferase, Metridia Luciferase, Clickbeetle luciferase and Bacterial luciferase. Luciferases are oxidative enzymes that generate light in the presence of oxygen and usually ATP. The phenomenon of light emission by a living organism such as marine vertebrates and invertebrates, as well as microorganisms and terrestrial animals is called bioluminescence.
Bioluminescence is a naturally occurring form of chemiluminescence where energy is released by a chemical reaction in the form of light emission. In luminescent reactions, light is produced by the oxidation of a luciferin (an energy rich chemical pigment). This reaction is catalyzed when the luciferase enzyme acts upon the appropriate luciferin substrate. This reaction is highly energetically efficient: nearly all of the energy input into the reaction is transformed into light (quantum yield of 88% for firefly luciferase).
The commercially available mammalian-optimized luciferases fall into two classes: the benzothiazole luciferin-utilizing coleopteran beetle luciferases from terrestrial organisms, such as FLuc and CBLuc; and the coelenterazine-utilizing enzymes from marine organisms, such as hRluc, hGluc and Metluc. Prokaryotes can be labeled with Bacterial luciferase (Lux) from e.g. the soil bacterium Photorhabdus luminescens.
Each of these luciferase genes can be synthesized and inserted into organisms or transfected into cells. Light emission can be detected by means of a luminometer or a sensitive CCD-camera. With these CCD camera systems, bioluminescence whole animal imaging can be accomplished to allow for non-invasive visualization of disease or molecular events. Cells (tumor cells, WBC, stem cells) or disease organisms (bacterial, viral, parasites) are engineered to express luciferase and subsequent light emission from within the animal allows monitoring disease course, biological activity, biodistribution or therapeutic effects.
Luciferase reporter gene expression in eukaryotes can be used in an always on, constitutive fashion under viral promoters such as CMV, SV40 or housekeeping gene promoters such as Beta-actin, Ubiquitin C, GAPDH. Alternatively, luciferases can be utilized as an inducible reporter gene to monitor promoter transcriptional activity in cells that are transfected with a genetic construct containing the luciferase gene under the control of a promoter of interest. More sophisticated models such as functional reporter fusion proteins, and split luciferases for protein-protein interaction studies are also published. (Lyons et al., 2005, Luker et al., 2009).
to be continued in future newsletters...
1) Terrestrial luciferases
2) Marine Luciferases
3) Bacterial Luciferases
Recommended Product
Lenti Fire™ – Luc2 lentiviral vector - In Vivo Imaging Solutions.
Lenti-Fire™ combines the best in vivo optical imaging reporter, namely the enhanced firefly luciferase Luc2 construct from the pGL4 plasmid with the best gene delivery system, a third generation lentiviral vector. An Ubiquitin C promoter drives high constitutive expression of the transgene (Schorpp et al., 1996) since CMV promoter silencing can occur in certain cell lines. Luc2 is an enhanced firefly luciferase construct, codon optimized for mammalian cell cytoplasmic expression. Emission peaks at 610nm in mammalian cells. Regular D-luciferin is the substrate. Luc2 is the most sensitive reporter gene for deep tissue in vivo bioluminescence imaging. Because of the high transfection efficiency of a lenti, this product makes the creation of luciferase expressing cell lines easy.
Lenti-Fire™ product manual for additional info.
