Name: high-throughput functional screening using a homemade dual-glow luciferase assay
Uploaded: 2014-06-01T14:00:00
Description: Watch this Scientific Journal Video about High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay at JoVE.com

Vi takker John Darga af MGH DNA Core til forberedelse af DNA screening biblioteket. Christopher Chigas af Perkin Elmer uvurderlig støtte til vores Wallac 1420 luminometer. Steven Ciacco og Martin Thomae af Agilent ydet støtte til Bravo robot. Vi takker Ron Johnson og Steve Titus på NIH for generøst at give deres dual-glød luciferaseassayet protokol.

For en eksperimentel overblik, se figur 1. 1.. Forbered Reporter plasmider indeholdende Alpha-Synuclein Promoter Regulatoriske elementer af alfa-synuclein-genet (figur 2) strækker sig over omkring 10 kb, fra den opstrøms NACP (ikke-A-beta komponent amyloidpeptid) dinukleotid gentagelsessekvens 1,2 gennem intron 2 3. Vi inkluderer en del af denne region i vores reporter konstrukt. Vi fandt, at plasmider indeh...

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Alpha-synuclein har været impliceret i Parkinsons sygdom (PD) som en komponent af Lewy organer 4, intracellulære inklusioner anses pathognomonic for sygdommen. Talrige genom-dækkende forening undersøgelser har knyttet enkelt nukleotid polymorfier i alfa-synuclein med øget risiko for sporadisk PD 5,6,7. Selvom mindre udbredt end sporadiske PD kan familiær PD også blive forårsaget af mutationer i alfa-synuclein 8 samt overlapning og tre eksemplarer af alfa-synuclein locus 9,10...

Evnen til at identificere de vigtigste genetiske regulatoriske elementer og de faktorer, der virker på dem, er fundamental for udforskning af mange biologiske processer. Men at identificere faktorer, der regulerer ekspression af gener i sjældne celletyper, såsom specifikke neuronale populationer, kan være udfordrende. Her præsenterer vi en protokol til identifikation af nye transskriptionsregulatorer af alfa-synuclein (SNCA), et gen associeret med Parkinsons sygdom og udtrykkes i dopaminerge neuroner i substantianigra pars compacta region i midthjernen. Vi opnår dette ved hjælp af en high-throughput, dual-luciferase, in vitro-reporter skærmen for at dekonstruere alfa-synuclein udtryk i 293T-celler. Alfa-synuclein-promotoren først klonet ind i et reporterplasmid, der indeholder ildflue-luciferasegenet. Et kommercielt tilgængeligt plasmid indeholdende Renilla luciferase under kontrol af en konstitutivt aktiv promotor tjener som en intern kontrol. These reporterkonstruktioner er co-transficeret ind i 293T-celler i mikroplader med plasmider fra et DNA-ekspressionsbibliotek, således at hver brønd transficeret med et enkelt bibliotek plasmid. Efter 48 timer, luciferaseaktiviteten for hver reporter måles sekventielt ved hjælp af en dual-glød assay. Den relative ekspression af alfa-synuclein som reaktion på hvert bibliotek gen er udledt af forholdet ildflue: Renilla-luciferase-aktivitet i hver brønd (F: forholdet R) efter plade-baseret normalisering. Denne protokol giver mulighed for at screene et stort antal gener (~ 2.500 per uge) for deres evne til transaktivere en reporter gen ved hjælp af minimal arbejdskraft (1-2 personer) og minimale omkostninger (omkring $ 3 reagens pris pr mikroplade assay). Transkriptionelle regulatorer af neuronale gener (fx alfa-synuclein), som er vanskelige at undersøge i dyrkede neuroner refraktære over for genetisk manipulation, kan sammenlignes side-by-side i denne direkte funktionelt assay. Vi har i voresprotokol en detaljeret metode til kloning og vækst af plasmider indeholdende a-synuclein-promotoren, da vi fandt, at plasmider indeholdende denne region er ustabile, når de dyrkes med konventionelle procedurer (se Diskussion). Vi inkluderer også et billigt alternativ til kommercielt tilgængelige dual-luciferase-reagenser til high-throughput assays. Denne protokol kan let tilpasses til at målrette andre regulatoriske elementer af interesse eller til enhver proces, der kræver høj kapacitet forbigående transfektioner.

<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

Vi præsenterer en hurtig og billig high-throughput screening protokol til at identificere transskriptionsregulatorer af alfa-synuclein, et gen associeret med Parkinsons sygdom. 293T-celler transient transficeret med plasmider fra en opstillet ORF ekspressionsbibliotek sammen med luciferase reporter plasmider, i en en-gen-per-brønds mikroplade-format. Ildflueluciferaseaktivitet, analyseres efter 48 timer for at bestemme virkningerne af hvert bibliotek gen upon alfa-synuclein transskription, normaliseret til udtryk fra en intern kontrol konstruktion (en hCMV promotor dirigere Renilla luciferase). Denne protokol lettes af en bench-top robot indesluttet i et biosikkerhed kabinet, som udfører aseptisk væskehåndtering i 96-brønds format. Vores automatiske transfektionsprotokol kan let tilpasses til high-throughput lentiviral bibliotek produktion eller andre funktionelle screening protokoller kræver triple-transfektioner af et stort antal af unikke bibliotek plasmider i konjugeredenction med et fælles sæt af hjælper plasmider. Vi præsenterer også en billig og valideret alternativ til kommercielt tilgængelige, dual luciferase reagenser, som beskæftiger PTC124, EDTA og pyrofosfat at undertrykke ildflueluciferaseaktivitet, før måling af Renilla luciferase. Ved hjælp af disse metoder, vi screenet 7.670 humane gener og identificeret 68 regulatorer af alfa-synuclein. Denne protokol er nemt modificeres til at målrette andre gener af interesse.

Typiske luciferase værdier, F: R nøgletal og induktions-værdier for en enkelt halv plade er vist i figur 4. Bemærk hit i godt H2, en 32-fold inducer.. Gener, der forårsager overdreven toksicitet (for eksempel godt E3) eller brønde, der var dårligt transficerede, vil producere lave værdier for både ildflue og Renilla luciferaser, men et gennemsnit induktion værdi. Gener, der forårsager ikke-specifik induktion af luciferaseaktivitet, måske via interaktion med pGL4 rygrad, vil...

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

Vi præsenterer en hurtig og billig screeningsmetode til at identificere transskriptionsregulatorer ved hjælp af high-throughput robot transfektioner og en hjemmelavet dual-glød luciferaseassayet. Denne protokol hurtigt genererer direkte side-by-side funktionelle data for tusinder af gener og er let modificerbare at målrette ethvert gen af ​​interesse.

High-throughput Funktionel Screening ved hjælp af en hjemmelavet Dual-glød Luciferase Assay

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

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