Using a promoter that can drive expression at an appropriate level is crucial in designing constructs for gene expression. Promoters can be tested via transient or stable transfection. But transfection efficiency in such assays can be low, so promoters are commonly fused to heterologous reporter genes that encode enzymes that can be quantified using highly sensitive assays. The reporter protein’s activity or fluorescence within a transfected cell population is approximately proportional to the steady-state mRNA level. The May issue of Cold Spring Harbor Protocols includes updated versions of three commonly used assays for promoter strength.

The Luciferase Assay uses a gene from the firefly Photinus pyralis. This gene encodes a 61-kDa enzyme that oxidizes D-luciferin in the presence of ATP, oxygen, and Mg++, yielding a fluorescent product that can be quantified by measuring the released light with a luminometer. The luciferase assay is extremely rapid, simple, relatively inexpensive, sensitive, and possesses a broad linear range.

The Chloramphenicol Acetyltransferase Assay utilizes an Escherichia coli chloramphenicol acetyltransferase (CAT) reporter gene. CAT catalyzes the acetylation of [14C]chloramphenicol which is monitored by autoradiography following thin-layer chromatography (TLC). The percent conversion of [14C]chloramphenicol to acetyl-[14C]chloramphenicol can be measured by PhosphorImager analysis of the TLC plate, counting in a scintillation counter, or by densitometry analysis of an autoradiograph.

The Beta-Galactosidase Assay uses the E. coli lacZ gene which encodes a beta-galactosidase. Beta-gal activity is measured through a simple and inexpensive colorimetric assay. Cells are lysed and extracts are mixed with O-nitrophenyl-beta-D-galactopyranoside (ONPG), which results in a yellow product. The optical densities of the samples are then determined spectrophotometrically.