Improved eukaryotic promoter-detection vector carrying two luciferase reporter genes.

BENCHMARKS Understanding gene regulation is of fundamental importance to virtually all biological systems. A gene is regulated through cis-acting regulatory DNA sequences (elements) in conjunction with transacting regulatory proteins. The cis-acting regulatory elements involved in transcription include, for example, the promoter, enhancer, silencer, specific activator binding sites, and specific repressor binding sites, whereas the transacting regulatory proteins include basic transcription factors, acti-vators, repressors, coactivators, and co-repressors. For the quantitative analysis of transcriptional regulatory elements, a variety of reporter vectors have been developed based on firefly luciferase and Renilla luciferase, chlorampheni-col acetyltransferase (CAT), β-galac-tosidase, β-glucuronase, β-lactamase, alkaline phosphatase, human growth hormone (hGH), and green fluorescent protein (GFP) (1–11). Luciferase reporter vectors have become the vectors of choice for most investigations because of the availability of rapid and sensitive luciferase assay systems (1). Most of the currently available eu-karyotic promoter-detection vectors carry only a single reporter gene. The use of such a vector can generate erroneous results due to the variability in transfection efficiency (intracellular copy number) in different samples of transfected cells. Many individual factors can affect the transfection efficiency of a plasmid, including contaminating nucleases, endotoxins, and salts in DNA preparations. Dispensation of variable amounts of DNA during pipet-ting of small quantities of DNA can also influence transfection efficiency. The size of a plasmid also plays a role in transfection because some smaller plas-mids can enter cells more efficiently. To minimize the error due to variable trans-fection efficiency, current protocols generally use a second control plasmid to normalize the data for the test plas-mid. The normalized value of the test plasmid data is usually expressed as a percentage of the control plasmid data. However, the use of a control plasmid cannot completely eliminate variability in transcription efficiency because of contamination of individual samples and the variable quantity of individual plasmid from sample to sample. To solve this problem, which is associated with the use of two plasmids, Park (1) developed a promoter-detection vector, pJDL cmv , which carries both the firefly and Renilla luciferase genes. However, the two luciferase genes in this vector transcribe in the same clockwise direction, thus possibly expressing any fragment cloned upstream of the firefly luciferase gene due to " leaky " transcription from the cytomegalovirus (CMV) promoter driving the Renilla luciferase gene. For instance, Giannakis et al. (12) have described readthrough Figure 1. Structure of the eukaryotic promoter-detection vector pFRL2. pFRL1 (not shown) was constructed …