Symposium 1997: Abstract for HG Wada et al

Symposium 1997: Abstracts

Measurement of Firefly and Renilla Luciferase in the FLIPR

HG Wada¹, IL Dale², EM Tucker², CJ Oakley², BD Neagle², KS Schroeder¹, M Sullivan² and E Sullivan²

¹Molecular Device Corporation, 1311 Orleans Drive, Sunnyvale, CA, USA,
²Astra Charnwood, Bakewell Road, Loughborough,UK

Reporter gene assays have proven to be an important tool in the analysis of gene transcription and have been used to identify regulatory sequences involved in specific gene regulation and in high throughput screening to identify molecules that modulate gene specific transcription. The luciferase enzymes whose genes have been cloned from firefly Photinus pyralis and the sea pansy Renilla reniformis are commercially available and are used widely as reporter genes. We have measured the activities of these enzymes using the Dual Luciferase^TM Reporter (DLR) assay system (Promega) in a 96 well micotiter plate format with a luminometer (Dynatech) which will dispense substrates and measure one well at a time. This is very time consuming and is not suitable for screening large numbers of compounds.

We have investigated the use of the Fluorescent Imaging Plate Reader (FLIPR) from Molecular Devices Corporation to measure levels of both firefly and Renilla luciferases. This instrument has an automatic 96 well dispenser and an integrated CCD camera. Initially to determine whether it was possible to measure luminescence in the FLIPR we used firefly luciferase (Promega) and recombinant Renilla luciferase that we expressed in E. coli. A large luminescence signal was detected for both enzymes with a lower limit of detection for the firefly luciferase calculated at less than 2 pg.

Having shown that the FLIPR detected isolated preparations of both luciferase enzymes we then looked at its ability to detect the activities of the enzymes in transiently transfected CHO cells. Cells were transfected with individual mammalian expression plasmids containing either the firefly luciferase gene or the Renilla luciferase, dual tranfections were also carried out.

Transfected cells were lysed and assayed for firefly and Renilla luciferases using the DLR system reagents. The signal obtained from the firefly and Renilla luciferases showed a dose dependency on the amount of DNA transfected and the number of the cells added per well. The maximum signal obtained with firefly luciferase expressed in CHO cells was 55,000 counts compared with cells carrying no plasmid that produced background counts of 200-300 counts. The firefly luciferase quenching reagent in the DLR system completely inhibited the firefly luciferase activity. The maximum signal for the Renilla luciferase was approximately 18,000 counts in the presence of the firefly luciferase quencher.

In conclusion we have shown that the FLIPR can measure both luciferases in the presence of one another in transfected cell lysates. It is therefore feasible to quantify firefly luciferase expression from a target promoter and Renilla luciferase expression from a control promoter in the same cell extract. The ability to dispense and measure luminescence in 96 wells simultaneously is of great benefit in high throughput screening.