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Mammalian Luciferase Detection Vectors and Applications
Author(s) -
Naleway John Joseph,
McGibney Mischa Taylor,
Coleman Daniel John,
Cook Gabriele Maria
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1028.1
Subject(s) - luciferase , vibrio harveyi , microbiology and biotechnology , expression vector , decanal , biology , bioluminescence , plasmid , complementary dna , biochemistry , gene , chemistry , transfection , vibrio , bacteria , genetics , recombinant dna , food science
The light‐emitting reaction of the marine bioluminescent bacterium Vibrio harveyi is catalyzed by the bacterial luciferase enzyme which exists as an alpha‐beta heterodimer encoded by the luxA and luxB genes with subunit molecular weights of 42K and 37K respectively. The enzyme catalyzes a reaction with FMNH2, oxygen and a long‐chain aldehyde as substrates to yield visible light at 490 nm. A new luciferase marker gene detection system has been developed based upon this bacterial luciferase isolated from Vibrio harveyi . Sequences encoding the two luciferase subunits, luxA and luxB have been cloned into two separate vectors. These vectors also include a CMV promoter for expression in mammalian cells as well as an ampicillin resistance gene (amp) for selection and amplification, the SV40 polyadenylation sequence and the SD/SA‐RNA splice donor and acceptor sequence for maximum expression. The vectors were tested for light emission parameters in a live mammalian cell format and with cell lysate samples from NIH3T3 murine fibroblasts in conjunction with transfection using vector GC:11726 Mus musculus biliverdin reductase B (flavin reductase (NADPH)) sequence for production of the reduced flavin mononucleotide (FMNH2) cofactor intracellularly. These systems are being developed to monitor regulation of expression for two independent vector constructs, upon the dual expression.