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.

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...

<ol>
	<li>Chiba-Falek, O., Nussbaum, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+allelic+variation+at+the+NACP-Rep1+repeat+upstream+of+the+&#945;-synuclein+gene+(SNCA)+on+transcription+in+a+cell+culture+luciferase+reporter+system.">Effect of allelic variation at the NACP-Rep1 repeat upstream of the &#945;-synuclein gene (SNCA) on transcription in a cell culture luciferase reporter system.</a> Hum. Mol. Genet. 10 (26), 3101-3109 (2001).</li><li>Chiba-Falek, O., Touchman, J. W., Nussbaum, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+intra-allelic+variation+at+NACP-Rep1+in+the+alpha-synuclein+gene.">Functional analysis of intra-allelic variation at NACP-Rep1 in the alpha-synuclein gene.</a> 113 (5), 426-431 (2003).</li><li>Scherzer, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GATA+transcription+factors+directly+regulate+the+Parkinson's+disease-linked+gene+&#945;-synuclein.">GATA transcription factors directly regulate the Parkinson's disease-linked gene &#945;-synuclein.</a> Proc. Natl. Acad. Sci. USA. 105 (31), 10907-10912 (2008).</li><li>Spillantini, M. G., Schmidt, M. L., Lee, V. M. Y., Trojanowski, J. Q., Jakes, R., Goedert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#945;-Synuclein+in+Lewy+Bodies.">&#945;-Synuclein in Lewy Bodies.</a> Nature. 388 (6645), 839-840 (1997).</li><li>Pankratz, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meta-analysis+of+Parkinson's+disease:+identification+of+a+novel+locus,+RIT2.">Meta-analysis of Parkinson's disease: identification of a novel locus, RIT2.</a> Ann. Neurol. 71 (3), 370-384 (2012).</li><li>Satake, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+association+study+identifies+common+variants+at+four+loci+as+genetic+risk+factors+for+Parkinson's+disease.">Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease.</a> Nat. Genet. 41 (12), 1303-1307 (2009).</li><li>Sim&#243;n-S&#225;nchez, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+association+study+reveals+genetic+risk+underlying+Parkinson's+disease.">Genome-wide association study reveals genetic risk underlying Parkinson's disease.</a> Nat. Genet. 41 (12), 1308-1312 (2009).</li><li>Polymeropoulos, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+in+the+alpha-synuclein+gene+identified+in+families+with+Parkinson's+disease.">Mutation in the alpha-synuclein gene identified in families with Parkinson's disease.</a> Science. 276 (5321), 2045-2047 (1997).</li><li>Ib&#225;&#241;ez, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causal+relationship+between+a-synuclein+gene+duplication+and+familial+Parkinson's+disease.">Causal relationship between a-synuclein gene duplication and familial Parkinson's disease.</a> Lancet. 364 (9440), 1169-1171 (2004).</li><li>Chartier-Harlin, M. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a-Synuclein+locus+duplication+as+a+cause+of+familial+Parkinson's+disease.">a-Synuclein locus duplication as a cause of familial Parkinson's disease.</a> Lancet. 364 (9440), 1167-1169 (2004).</li><li>Singleton, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a-Synuclein+locus+triplication+causes+Parkinson's+disease.">a-Synuclein locus triplication causes Parkinson's disease.</a> Science. 302 (5646), 841 (2003).</li><li>Ahn, T. -. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a-Synuclein+gene+duplication+is+present+in+sporadic+Parkinson+disease.">a-Synuclein gene duplication is present in sporadic Parkinson disease.</a> Neurology. 70 (1), 43-49 (2008).</li><li>Boyce, F. M., Bucher, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus-mediated+gene+transfer+into+mammalian+cells.">Baculovirus-mediated gene transfer into mammalian cells.</a> Proc. Nat. Acad. USA. 93 (6), 2348-2352 (1996).</li><li>Montigny, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parameters+influencing+high-efficiency+transfection+of+bacterial+artificial+chromosomes+into+cultured+mammalian+cells.">Parameters influencing high-efficiency transfection of bacterial artificial chromosomes into cultured mammalian cells.</a> Biotechniques. 35, 796-807 (2003).</li><li>Gorman, C. M., Moffat, L. F., Howard, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+genomes+which+express+chloramphenicol+acetyltransferase+in+mammalian+cells.">Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells.</a> Mol. Cell. Biol. 2 (9), 1044-1051 (1982).</li><li>Li, J. J., Herskowitz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+ORC6,+a+Component+of+the+Yeast+Origin+Recognition+Complex+by+a+One-Hybrid+System.">Isolation of ORC6, a Component of the Yeast Origin Recognition Complex by a One-Hybrid System.</a> Science. 262, 1870-1874 (1993).</li><li>Dejardin, J., Kingston, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Proteins+Associated+with+Specific+Genomic+Loci.">Purification of Proteins Associated with Specific Genomic Loci.</a> Cell. 136, 175-186 (2009).</li><li>Shen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+map+of+the+cis-regulatory+sequences+in+the+mouseGenome.">A map of the cis-regulatory sequences in the mouseGenome.</a> Nature. 488, 116-120 (2012).</li><li>Fan, F., Wood, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+assays+for+high-throughput+screening.">Bioluminescent assays for high-throughput screening.</a> Assay Drug Dev. Technol. 5 (1), 127-136 (2007).</li><li>Hampf, M., Gossen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+combined+Photinus+and+Renilla+luciferase+quantification+compatible+with+protein+assays.">A protocol for combined Photinus and Renilla luciferase quantification compatible with protein assays.</a> Anal. Biochem. 356 (1), 94-99 (2006).</li><li>Auld, D. S., Thorne, N., Maguire, W. F., Inglese, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+PTC124+activity+in+cell-based+luciferase+assays+of+nonsense+codon+suppression.">Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression.</a> Proc. Natl. Acad. Sci. USA. 106 (9), 3585-3590 (2009).</li><li>Pearlberg, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screens+using+RNAi+and+cDNA+expression+as+surrogates+for+genetics+in+mammalian+tissue+culture+cells.">Screens using RNAi and cDNA expression as surrogates for genetics in mammalian tissue culture cells.</a> Cold Spring Harb.Symp. Quant. Biol. 70, 449-459 (2005).</li></ol>

Alpha-synuclein has been implicated in Parkinson&#39;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...

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&#39;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 expression in 293T cells. The alpha-synuclein promoter is first cloned into a reporter plasmid containing the firefly luciferase gene. A commercially available plasmid containing the Renilla luciferase under the control of a constitutively active promoter serves as an internal control. These reporter constructs are co-transfected into 293T cells in microplates with plasmids from a DNA expression library, such that each well is transfected with a single library plasmid. After 48 hr, luciferase activity for each reporter is measured sequentially using a dual-glow assay. The relative expression of alpha-synuclein in response to each library gene is inferred by the ratio of firefly:Renilla luciferase activity in each well (F:R ratio) after plate-based normalization.
This protocol provides the ability to screen a large number of genes (~2,500 per week) for their ability to transactivate a reporter gene using minimal manpower (1-2 people) and minimal cost (about $3 reagent cost per microplate assay). Transcriptional regulators of neuronal genes (e.g., alpha-synuclein), which are difficult to study in cultured neurons refractory to genetic manipulation, can be compared side-by-side in this direct functional assay. We include in our protocol a detailed method for cloning and growth of plasmids containing the alpha-synuclein promoter, since we found that plasmids containing this region are unstable when grown with conventional procedures (see Discussion). We also include an inexpensive alternative to commercially available dual-luciferase reagents for high-throughput assays. This protocol can be easily adapted to target other regulatory elements of interest, or to any process requiring high-throughput transient transfections.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Material</td>
 </tr>
 <tr>
 <td>QIAQuick PCR Purification Kit</td>
 <td>Qiagen</td>
 <td>28106</td>
 <td></td>
 </tr>
 <tr>
 <td>PureLink Quick Gel Extraction Kit</td>
 <td>Invitrogen</td>
 <td>K2100-12</td>
 <td></td>
 </tr>
 <tr>
 <td>PureLink PCR Micro Kit</td>
 <td>Invitrogen</td>
 <td>K310250</td>
 <td></td>
 </tr>
 <tr>
 <td>PureLinkHiPure Plasmid Miniprep Kit</td>
 <td>Invitrogen</td>
 <td>K2100-03</td>
 <td></td>
 </tr>
 <tr>
 <td>PureLinkHiPure Plasmid Maxiprep Kit</td>
 <td>Invitrogen</td>
 <td>K2100-07</td>
 <td></td>
 </tr>
 <tr>
 <td>Bravo Robot</td>
 <td>Agilent</td>
 <td>-</td>
 <td></td>
 </tr>
 <tr>
 <td>Robot Pipet Tips with Filter</td>
 <td>Agilent</td>
 <td>19477-022</td>
 <td></td>
 </tr>
 <tr>
 <td>Robot Pipet Tips without Filter</td>
 <td>Agilent</td>
 <td>19477-002</td>
 <td></td>
 </tr>
 <tr>
 <td>Clear-bottomed 96-well Plates</td>
 <td>Corning</td>
 <td>3610</td>
 <td></td>
 </tr>
 <tr>
 <td>Reservoirs</td>
 <td>Axygen</td>
 <td>RES-SW96-HP-SI</td>
 <td></td>
 </tr>
 <tr>
 <td>Polystyrene V-bottomed 96-well Plate</td>
 <td>Greiner</td>
 <td>651101</td>
 <td></td>
 </tr>
 <tr>
 <td>Polypropylene V-bottomed 96-well Plate</td>
 <td>Greiner</td>
 <td>651201</td>
 <td></td>
 </tr>
 <tr>
 <td>Adhesive Plate Covers</td>
 <td>CryoStuff</td>
 <td>#FS100</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>Phusion DNA Polymerase</td>
 <td>New England Biolabs</td>
 <td>M0530L</td>
 <td></td>
 </tr>
 <tr>
 <td>BAC 2002-D6</td>
 <td>Invitrogen</td>
 <td>2002-D6</td>
 <td></td>
 </tr>
 <tr>
 <td>Calf Intestine Alkaline Phosphatase</td>
 <td>Roche</td>
 <td>10713023001</td>
 <td></td>
 </tr>
 <tr>
 <td>T4 DNA Ligase</td>
 <td>New England Biolabs</td>
 <td>M0202L</td>
 <td></td>
 </tr>
 <tr>
 <td>pGL4.10</td>
 <td>Promega</td>
 <td>E6651</td>
 <td></td>
 </tr>
 <tr>
 <td>pGL4.74</td>
 <td>Promega</td>
 <td>E6931</td>
 <td></td>
 </tr>
 <tr>
 <td>ElectroMAX Stbl4 Competent Cells</td>
 <td>Invitrogen</td>
 <td>11635-018</td>
 <td></td>
 </tr>
 <tr>
 <td>Subcloning Efficiency DH5&alpha; Competent Cells</td>
 <td>Invitrogen</td>
 <td>18265-017</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM without Phenol Red</td>
 <td>Invitrogen</td>
 <td>31053-028</td>
 <td></td>
 </tr>
 <tr>
 <td>Optifect</td>
 <td>Invitrogen</td>
 <td>12579017</td>
 <td></td>
 </tr>
 <tr>
 <td>Ciprofloxacin</td>
 <td>CellGro</td>
 <td>61-277-RF</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris-Hydrochloride Powder</td>
 <td>Sigma</td>
 <td>93287</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris-Base Powder </td>
 <td>Sigma</td>
 <td>93286</td>
 <td></td>
 </tr>
 <tr>
 <td>Triton X-100 Pure Liquid</td>
 <td>Fisher</td>
 <td>BP151-100</td>
 <td></td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>Invitrogen</td>
 <td>15508-013</td>
 <td></td>
 </tr>
 <tr>
 <td>Coenzyme A</td>
 <td>Nanolight</td>
 <td>309</td>
 <td></td>
 </tr>
 <tr>
 <td>ATP</td>
 <td>Sigma</td>
 <td>A6419</td>
 <td></td>
 </tr>
 <tr>
 <td>Luciferin</td>
 <td>Invitrogen</td>
 <td>L2912</td>
 <td></td>
 </tr>
 <tr>
 <td>h-CTZ</td>
 <td>Nanolight</td>
 <td>301</td>
 <td></td>
 </tr>
 <tr>
 <td>PTC124</td>
 <td>SelleckBiochemicals</td>
 <td>S6003</td>
 <td></td>
 </tr>
 </tbody>
</table>

high-throughput functional screening using a homemade dual-glow luciferase assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;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.

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</e...

Watch this Scientific Journal Video about High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay at JoVE.com

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

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 ...

high-throughput-functional-screening-using-homemade-dual-glow

Research

JoVE Journal

Biology

23.2K visualizações.  Massachusetts General Hospital.  Apresentamos um método de triagem rápida e de baixo custo para a identificação de reguladores de transcrição usando de alto rendimento transfections robóticos e um ensaio de luciferase dual-brilho caseiro. Este protocolo gera rapidamente dados funcionais diretos side-by-side para milhares de genes e é facilmente modificável para atingir qualquer gene de interesse. 