Published: June 1st, 2014
We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.
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.
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. We accomplish this using a high-throughput, dual-luciferase, in vitro reporter screen to deconstruct alpha-synuclein ....
For an experimental overview, see Figure 1.
1. Prepare Reporter Plasmids Containing the Alpha-Synuclein Promoter
Regulatory elements of the alpha-synuclein gene (Figure 2) span approximately 10 kb, from the upstream NACP (non-A beta component of amyloid peptide) dinucleotide repeat sequence1,2 through intron 2 3. We include a portion of this region in our reporter construct. We found that plasmids containing intr.......
Typical luciferase values, F:R ratios and induction values for a single half-plate are shown in Figure 4. Note the hit in well H2, a 32-fold inducer. Genes causing excessive toxicity (for example, well E3), or wells that were poorly transfected, will produce low values for both firefly and Renilla luciferases, but an average induction value. Genes causing non-specific induction of luciferase activity, perhaps via interaction with the pGL4 backbone, will induce firefly and Renilla
Alpha-synuclein has been implicated in Parkinson's disease (PD) as a component of Lewy bodies4, intracellular inclusions considered pathognomonic for the disease. Numerous genome-wide association studies have linked single nucleotide polymorphisms in alpha-synuclein with increased risk for sporadic PD5,6,7. Although less common than sporadic PD, familial PD may be also caused by mutations in alpha-synuclein8, as well as duplication and triplication of the alpha-synuclein locus9,1.......
We thank John Darga of the MGH DNA Core for preparation of the DNA screening library. Christopher Chigas of Perkin Elmer provided invaluable support for our Wallac 1420 luminometer. Steven Ciacco and Martin Thomae of Agilent provided support for the Bravo robot. We thank Ron Johnson and Steve Titus at the NIH for generously providing their dual-glow luciferase assay protocol.....
|QIAQuick PCR Purification Kit
|PureLink Quick Gel Extraction Kit
|PureLink PCR Micro Kit
|PureLinkHiPure Plasmid Miniprep Kit
|PureLinkHiPure Plasmid Maxiprep Kit
|Robot Pipet Tips with Filter
|Robot Pipet Tips without Filter
|Clear-bottomed 96-well Plates
|Polystyrene V-bottomed 96-well Plate
|Polypropylene V-bottomed 96-well Plate
|Adhesive Plate Covers
|Phusion DNA Polymerase
|New England Biolabs
|Calf Intestine Alkaline Phosphatase
|T4 DNA Ligase
|New England Biolabs
|ElectroMAX Stbl4 Competent Cells
|Subcloning Efficiency DH5α Competent Cells
|DMEM without Phenol Red
|Triton X-100 Pure Liquid
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