Spectral-resolved gene technology for multiplexed bioluminescence and high-content screening.
The availability of new bioluminescent proteins, obtained by cDNA cloning and mutagenesis of wild-type genes, expanded the applicability of these reporters from the perspective of using more proteins emitting at different wavelengths in the same cell-based assay. By spectrally resolving the light emitted by different reporter proteins, it is in fact possible to simultaneously monitor multiple targets. A new luciferase isolated from Luciola italica has been recently cloned, and thermostable red- and green-emitting mutants were obtained by random and site-directed mutagenesis. Different combinations of luciferases were used in vitro as purified proteins and expressed in bacterial and mammalian cells to test their suitability for multicolor assays. A mammalian triple-color reporter model system was then developed using a green-emitting wild-type Photinus pyralis luciferase, a red thermostable mutant of L. italica luciferase, and a secreted Gaussia princeps luciferase (GLuc) to monitor the two main pathways of bile acid biosynthesis. The two firefly luciferases were used to monitor cholesterol 7-alpha hydroxylase and sterol 27-hydroxylase, while secreted constitutively expressed GLuc was used as an internal vitality control. By treating the cells with chenodeoxycholic acid, it was possible to obtain dose-dependent inhibitions of the two specific signals together with a constant production of GLuc, which allowed for a dynamic evaluation of the metabolic activity of the cells. This is the first triple-color mammalian reporter assay that combines secreted and nonsecreted luciferases requiring different substrates, thus avoiding reciprocal interference between different BL signals. This approach is suitable for high content analysis of gene transcription in living cells to shorten the time for screening assays, increasing throughput and cost-effectiveness.
Michelini
Elisa
E
Department of Pharmaceutical Sciences, University of Bologna, and Center for Applied Biomedical Research (CRBA), S. Orsola-Malpighi Hospital, Via Massarenti 9, 40138 Bologna, Italy.
Cevenini
Luca
L
Mezzanotte
Laura
L
Ablamsky
Danielle
D
Southworth
Tara
T
Branchini
Bruce
B
Roda
Aldo
A
D002452:Cell Count; D002460:Cell Line; D002470:Cell Survival; D002635:Chenodeoxycholic Acid / Q000494:pharmacology; D053493:Cholestanetriol 26-Monooxygenase / Q000235:genetics; D002790:Cholesterol 7-alpha-Hydroxylase / Q000235:genetics; D003116:Color; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000032:analysis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
D008164:Luminescent Proteins; D002635:Chenodeoxycholic Acid; D008156:Luciferases; D002790:Cholesterol 7-alpha-Hydroxylase; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase