Marcel

Reporter-Based Assays. Most of the transcription factors are modular, consisting of a DNA-binding domain and activation domain. These domains can be interchanged between different factors and still retain their functional properties. A reporter gene construct consists of an inducible transcriptional control element driving the expression of a reporter gene. Reporter genes code for proteins that possess unique enzyme activities, and the activity assays are adaptable to HTS. The test DNA is ligated upstream of the coding region of the reporter gene to generate a chimeric gene in which the regulatory element controls the expression of a reporter gene [65]. Functional enhancer elements that can augment transcription are placed upstream of the promoter. The reporter gene con struct with an inducible transcriptional element driving the expression of the reporter gene will regulate the reporter protein synthesis in response to receptor activation. Several reporter gene vectors are commercially available with a promoter or enhancer, proper cloning sites, reporter gene, intron, poly A site, antibiotic resistance, and prokaryotic origin of replication (Fig. 6). The fusion reporter gene constructs are expressed into mammalian cells by a variety of transfection methods, the most popular being calcium phosphate, DEAE-dextran, lipofect-amine, and electroporation methods [65].

Several reporter genes are available with a choice of many different vectors for appropriate cell line. The popular reporter systems are firefly luciferase, secreted alakaline phosphatase (SEAP), chloramiphenicol acetyltransferase (CAT), P-galactosidase, P-lactamase, and green fluorescent protein (GFP).

The firefly luciferase is a 62-kDa protein that is active as a monomer. The overall reaction catalyzed by luciferase is d-luciferin + ATP + O2 ^ oxyluciferin (4)

Luciferin is oxidized producing a flash of light (tm = 0.3 s) with highest bioluminescence quantum yield of 0.82. The signal is read in a luminometer wherein the reagent is injected into each well before reading the signal. Luminometer with single injector and single well reader takes a long time to read a 96-well

Figure 6 Map of a generic reporter vector. Ori represents the bacterial origin of replication. Promoter and enhancer attached to the reporter gene are cloned into the cloning site. A poly (A) sequence is positioned downstream of the reporter sequence to allow in vitro synthesis.

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