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Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 9/30/2001 Publication Date: N/A Citation: N/A Interpretive Summary: The regulation of human genes is essential to maintain physiological normality in human cells. Human genomic regulation is sophisticatedly orchestrated by numerous genetic factors. Gene promoter is one of the most important genetic factors. Genotype changes in gene promoters have been reported to be associated with abnormal cell growth such as cancer. The promoters of human genes have been intensively investigated for deciphering the mechanisms of their characteristic expression. Reporter vectors are essential for the quantitative analysis of gene promoters that potentially regulate gene expression. Several types of reporter vectors have been developed, and luciferase reporter vector is a most popular reporter vector, due to its rapid, sensitive, and reproducible assay system. However, the transfection of only luciferase vector cannot provide normalized values of the activities of gene promoters without simultaneous transfection of a second reporter vector to measure the efficiency. In this report, a novel report vector (pJDL) was constructed to contain two luciferase genes (Photinus pyralis and Renilla reniformis luciferases) regulated by two different promoters for the first time, to measure simultaneously promoter activity as well as transfection efficiency. The invention of this novel reporter vector is very useful for molecular and cellular biologists to save significant time and cost in both basic and applied transfection studies. Technical Abstract: Both promoter activity and transfection efficiency can be measured at the same time with a new reporter vector (pJDL) containing both Photinus pyralis and Renilla reniformis luciferase genes. Multiple cloning sites were generated immediately upstream of Photinus pyralis luciferase to clone various DNA fragments. Cytomegalovirus immediate early promoter was cloned upstream of Renilla reniformis luciferase gene to measure transfection efficiency. DNA fragments of two promoters from the human glutaredoxin and ribonucleotide reductase R2 subunit were cloned upstream of Photinus pyralis luciferase of pJDL. These chimeric pJDL reporter vectors were transfected to HeLa and NT cells to measure simultaneously the promoter activities as well as transfection efficiency. The promoter activities and transfection efficiency were determined by measuring the activities of Photinus pyralis and Renilla reniformis luciferases, respectively, using the dual luciferase reporter assay system. The promoter activities normalized with transfection efficiency were almost identical to the previously reported activities. These data suggest that a novel reporter vector can not only substitute for other luciferase vectors, but also determine both promoter activity and transfection efficiency simultaneously in an assay system, thereby saving significant time and cost in both basic and applied transfection studies. |
