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

Understanding gene regulation is of fundamental importance to virtually all biological systems. A gene is regulated through cis-acting regulatory DNA se-quences (elements) in conjunction with trans-acting regulatory proteins. The cis-acting regulatory elements involved in transcription include, for example, the promoter, enhancer, silencer, spe-cific activator binding sites, and spe-cific repressor binding sites, whereas the trans-acting 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 fluores-cent protein (GFP) (1–11). Luciferase reporter vectors have become the vec-tors 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 er-roneous results due to the variability in transfection efficiency (intracellular copy number) in different samples of transfected cells. Many individual factors can affect the transfection effi-ciency of a plasmid, including contami-nating 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