A new additional reporter enzyme, dinoflagellate luciferase, for monitoring of gene expression in mammalian cells.
Dinoflagellate luciferase (DL) catalyses the oxidation of dinoflagellate luciferin by molecular oxygen, resulting in an electronically excited species that emits blue light (lambda(max)=474 nm). Luciferase has three catalytic domains in its single polypeptide chain (M(r)=ca. 140 kDa), and each domain (about 40 kDa) is enzymatically active when expressed individually in recombinant fusion proteins in E. coli. Thus, DL should be useful as a reporter enzyme in studies of gene expression in mammalian cells. Expression plasmids consisting of one domain of luciferase (dDL) cDNA linked to different several promoters were introduced into a series of mammalian cell lines. Following transfection, dDL activities in cell extracts were determined by a rapid light emission assay of luciferase activity. For dual and multiple reporter assays, it is possible to exchange dDL for the firefly or renilla luciferases, and use the new luciferase for control or target reporter genes. Thus, the triple-reporter assay can identify three transcriptional activities of different genes at the same time. This work establishes the DL gene as a new efficient marker of gene expression in mammalian cells.
Suzuki
Chie
C
Toyo B-net Corporation, 3-5-1, Wadai, Tsukuba 432-8011, Japan.
Nakajima
Yoshihiro
Y
Akimoto
Hidetoshi
H
Wu
Chun
C
Ohmiya
Yoshihiro
Y
D000818:Animals; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D004141:Dinoflagellida / Q000201:enzymology*; D048888:Fireflies / Q000201:enzymology; D005786:Gene Expression Regulation / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements; D051379:Mice; D041681:NIH 3T3 Cells; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014162:Transfection
D011993:Recombinant Fusion Proteins; D008156:Luciferases