Advantages of firefly luciferase as a reporter gene: application to the interleukin-2 gene promoter.
The chloramphenicol acetyltransferase (CAT) gene is widely used in recombinant constructs employed to study promoter and enhancer control of gene expression. However, CAT-based assays require a laborious, multi-step procedure for quantitation of promoter activity. We have applied the recently described firefly luciferase (LUC) reporter gene to the study of the interleukin-2 (IL2) promoter and have further defined the properties of this reporter gene system. We find that IL2-LUC constructs have multiple advantages over IL2-CAT constructs. The LUC assay is highly sensitive and requires 1/10 the cells used in the CAT system. A final quantitative measure of promoter activity can be obtained within 25 h following transfection with IL2-LUC, compared to 108-160 h with IL2-CAT. Light emission significantly (fourfold) above background is detectable 3 h after induction in a direct assay of extracts from transfected cells. We have described the variability of the assay, the minimum number of transfected cells required to detect light, the stability of luciferase in cell extracts, the effect of Triton X-100 on the assay, and a rapid cell lysis procedure. The luciferase system is a simple, rapid, and sensitive method for the study of promoter activity in transfected cells, particularly for weakly expressed genes such as IL2 which give low activity in the CAT assay.
Williams
T M
TM
Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia 19104-6082.
Burlein
J E
JE
Ogden
S
S
Kricka
L J
LJ
Kant
J A
JA
D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular; D001517:Coleoptera / Q000201:enzymology; D005796:Genes; D007376:Interleukin-2 / Q000235:genetics*; D008156:Luciferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic; D014162:Transfection
D007376:Interleukin-2; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase