title: High-throughput functional screening using a homemade dual-glow luciferase assay.
abstract: We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson's disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.
authors: Department of Neurology, Massachusetts General Hospital.
authors: Jessica M
authors: JM
authors: Baker
authors: Department of Neurology, Massachusetts General Hospital; fmboyce@rics.bwh.harvard.edu.
authors: Frederick M
authors: FM
authors: Boyce
mesh_terms: D017930:Genes, Reporter; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D049410:Luciferases, Renilla / Q000737:chemistry* / Q000235:genetics*; D008163:Luminescent Measurements / Q000379:methods*; D009474:Neurons / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation; D014162:Transfection / Q000379:methods*; D051844:alpha-Synuclein / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D051844:alpha-Synuclein; D049410:Luciferases, Renilla