title: Coexpression of luxA and luxB genes of Vibrio fischeri in NIH3T3 mammalian cells and evaluation of its bioluminescence activities.
abstract: Expression of bacterial luciferase enzyme (lux) in eukaryotic cells would provide a new bioreporter system for in vivo imaging and diagnostics technology. In spite of this, until now only a few efforts have been made to express bacterial luciferase enzyme in eukaryotic cells. We attempted to synthesize an expression construct of luxA and luxB genes from Vibrio fischeri. The luxA and luxB genes were cloned into the MCS of pTZ57R via the 5' kpnI, BamHI and BamHI, EcoRI restriction sites to generate pTZ57R/luxA and pTZ57R/luxB respectively, then newly synthesized constructs were cleaved with the same enzymes and respectively cloned into the pcDNA3.1(+) (Hyg) and pcDNA3.1(+) (Neo) expression vectors to create pcDNA3.1(+) (Hyg)/luxA and pcDNA3.1(+) (neo)/luxB. Recombinant constructs were cotransfected to the NIH3T3 cell line. Gene expression was confirmed by reverse transcription-polymerase chain reaction, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting; in addition, bioluminescence characteristics of transfected NIH3T3 cell lines were evaluated by decanal supplement. In conclusion, in the current research, separate vector systems were constructed, which are composed of bacterial luciferase genes (luxA and luxB) that accordingly have not already been reported. These results hold promise toward the potential development of an autonomous light-generating lux reporter system in eukaryotic cells.
authors.affiliation: Department of Genetics, Islamic Azad University, Zanjan, Iran.
authors.forename: Golnaz Asaadi
authors.initials: GA
authors.lastname: Tehrani
authors.forename: Sina
authors.initials: S
authors.lastname: Mirzaahmadi
authors.forename: Mojgan
authors.initials: M
authors.lastname: Bandehpour
authors.forename: Bahram
authors.initials: B
authors.lastname: Kazemi
mesh_terms: D048908:Aliivibrio fischeri / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D015870:Gene Expression; D005798:Genes, Bacterial; D049449:Luminescence; D051379:Mice; D041681:NIH 3T3 Cells; D010957:Plasmids; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D017931:DNA Primers
keywords: Bioluminescence; Bioreporter; Vibrio fischeri; luciferase