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This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.
Date Published: 6/01/2014, Issue 88; doi: 10.3791/50282 Keywords: Cellular Biology, Issue 88, Luciferases, Gene Transfer Techniques, Transfection, High-Throughput Screening Assays, Transfections, Roboticshigh-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 h CMV promoter directing 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 The ability to identify key genetic regulatory elements and the factors that act on them is fundamental for the exploration of numerous biological processes.
However, identifying factors that regulate expression of genes in rare cell types, such as specific neuronal populations, can be challenging.
Here, we present a protocol for identifying novel transcriptional regulators of alpha-synuclein (SNCA), a gene associated with Parkinson's disease and expressed in dopaminergic neurons in the substantianigra pars compacta region of the midbrain.