Reporter genes firefly luciferase

Table 3.4.5 summarizes the advantages and drawbacks of the main reporter systems. Firefly luciferase (from Photinus pyralis lucFF gene)... 

Figure 1 The principles and variant parameters of lipofection. (i) Preparation of a lipofection reagent cationic liposomes were prepared from cationic lipids and helper (if required), (ii) Formation of positively charged lipoplexes by addition of DNA (e.g., reporter plasmid carrying the firefly luciferase gene) to the cationic liposomes, (iii) Transfection (lipofection) by incubation cells with the preformed lipoplexes. The efficiency of gene transfer (lipofection efficiency) can be determined from reporter gene amount or activity (e.g., luciferase activity). Most of the steps of a lipofection experiment can be varied and optimized (grey spots).

The most common reporter proteins (coded by reporter genes) employed in biosensor systems include firefly luciferase (lucFF gene) (Justus and Thomas, 1999), bacterial luciferase (luxCDABE gene) (Belkin et al., 1996 Vollmer et al., 1997 Min et al.,... 

The reporter gene is the luc operon from the firefly Photinus pyralis. This operon codes for an enzyme, luciferase, that catalyses the reaction between D-luciferin (the substrate), and oxygen, and requires ATP as a cofactor. This reaction produces the emission light that provides the measure of enzyme activity. The oxidation of one molecule of luciferin involves one ATP molecule and releases one photon (a stoichiometric reaction) (DeLuca and McElroy, 1974 Wood el al. 1989). The reaction can be summarized as follows ... 

Glow luminescence techniques have been used extensively with luciferases as reporter genes in cell-based assays. An overview of such assays is given in Section 10.3.2 Reporter Assays below. Luciferases are enzymes which catalyze bio-luminescent reactions. Two forms are used as reporters, one originating from the firefly (firefly luciferase) and the other from Rmilla (Renilla luciferase). Due to their different origins, the enzyme structures and their respective substrates are quite different While Rmilla luciferase catalyzes the oxidation of coelenterazine, the substrate of firefly luciferase is the beetle luciferin, which is oxidized in the presence of ATP and Mg as depicted in Fig. 17. 

Brasier, A. R., Tate, J. E., and Habener, J. F. (1989) Optimized use of firefly luciferase assay as a reporter gene in mammalian tell lines. Biotechnology 7(10), 1116-1122. 

Assays can be prepared with a reporter system containing, for example, the firefly luciferase gene. The reporter cells are coupled to receptor genes. When a ligand binds to the receptor, luminescence glow can be observed. In this way, the effects of the signaling events are evaluated. 

Plasmid pVTL2, which is another example of an extrachromosomal plasmid [83], was used to express the firefly luciferase gene under the control of various promoters. The luciferase gene can be replaced for other heterologous transgenes, which can be expressed from any D. discoideum promoter introduced into the multiple cloning site. The pVTL2 plasmid was reported to produce 10-50 copies per cell [83]. 

In a reporter gene assay, CKR-mediated activation of transcription factors can be quantified. A plasmid encoding the CKR of interest is cotransfected with a plasmid encoding a reporter protein of which expression can easily be detected, such as firefly luciferase (Flue) (or another bioluminescent protein or enzyme such as Gaussia luciferase (Glue) or P-galactosidase). Luciferase transcription is controUed by an inducible promoter containing multiple response elements for a specific transcription factor. Hence, activation (or inhibition) of this transcription factor in response to CKR activation leads to the expression of luciferase (Fig. 15A). 

---