Identifikation af Transskription Factor Regulators ved hjælp af Medium-Throughput Screening af Arrayed Biblioteker og en Dual-Luciferase-baserede Reporter
Summary
For at identificere nye regulatorer af transskription faktorer, vi udviklet en tilgang til at screene arrayed lentiviral eller retroviral RNAi biblioteker ved hjælp af en dual-luciferase-baserede transskriptions-reporter assay. Denne tilgang tilbyder en hurtig og relativt billig måde at screene hundredvis af kandidater i et enkelt eksperiment.
Abstract
Transskription faktorer kan ændre udtrykket af mange mål gener, der påvirker en række downstream processer gør dem gode mål for anti-cancer behandlinger. Men, direkte målretning transskription faktorer er ofte vanskeligt og kan forårsage bivirkninger, hvis transskription faktor er nødvendig i en eller flere voksne væv. Identifikation upstream regulatorer, der afvigende aktivere transskription faktorer i kræftceller tilbyder et mere realistisk alternativ, især hvis disse proteiner er nemme at narkotika. Her beskriver vi en protokol, der kan bruges til at kombinere arrayed medium-skala lentiviral biblioteker og en dual-luciferase-baserede transskriptions-reporter assay at identificere nye regulatorer af transskription faktorer i kræftceller. Vores tilgang tilbyder en hurtig, nem og billig måde at teste hundredvis af gener på i et enkelt eksperiment. For at demonstrere brugen af denne tilgang, udførte vi en skærm af en arrayed lentiviral RNAi bibliotek, der indeholder flere regulatorer af Ja-associeret protein (YAP) og transskriptionelle co-aktivator med PDZ-bindende motiv (TAZ), to transskriptionelle co-aktivatorer, der er downstream effektorer af Hippo pathway. Denne fremgangsmåde kan dog ændres til at screene for regulatorer af stort set alle transskriptionsfaktorer eller co-faktor og kan også bruges til at screene CRISPR/CAS9-, cDNA- eller ORF-biblioteker.
Introduction
Formålet med denne analyse er at bruge viralbiblioteker til at identificere regulatorer af transskriptionsfaktorer på en relativt hurtig og billig måde. Afvigende transskriptionsaktivitet er forbundet med kræft og metastase1,,2,3,4,5,6, så målretning transskription faktorer i kræftceller er en lovende terapeutisk tilgang. Transskriptionsfaktorer er imidlertid ofte vanskelige at målrette farmakologisk7 , og mange er nødvendige for normal cellefunktion i voksent væv8,9,10. Målretning af kræft-associerede veje, der afvigende aktivere transskription faktorer til at drive sygdom er en mere realistisk tilgang med potentiale til at have mindre alvorlige bivirkninger. Den kommercielle tilgængelighed af arrayed lentivirale og retrovirale RNAi, CRISPR/CAS9, cDNA eller ORF biblioteker giver forskerne mulighed for at teste betydningen af mange gener i et enkelt eksperiment. Der kræves dog en pålidelig udlæsning for ændret transskriptionsaktivitet.
Her beskriver vi brugen af en dobbelt-luciferase-baserede transskriptionel reporter assay og arrayed lentiviral biblioteker til at identificere proteiner, der regulerer transskription faktorer i kræftceller. I denne analyse, shRNAs at målrette kræft-associerede gener leveres til pattedyr kræftceller via lentiviral transduktion og celler er udvalgt til stabil integration ved hjælp af puromycin. Cellerne er næste transfected med en reporter konstruktion, der udtrykker firefly luciferase drevet af en promotor specifikt til transskription faktor, der er ved at blive undersøgt, og en kontrol konstruktion, der udtrykker Renilla luciferase fra en konstituerende aktiv promotor, der ikke er lydhør ekonnert over for transskription faktor, der undersøges. Vi demonstrerer denne tilgang med en proof-of-concept skærm for lovgivere af YAP og TAZ, de kritiske downstream effektorer af Hippo pathway8,10,11. Unormal aktivitet af YAP og TAZ fremmer flere trin i den metastatiske kaskade11 og er observeret i mange kræftformer11,12,13. Men, hvordan YAP og TAZ bliver afvigende aktiveret i nogle kræftceller er endnu ikke fuldt forstået. YAP og TAZ binder ikke DNA, men rekrutteres i stedet til initiativtagere af andre transskriptionsfaktorer. Medlemmer af TEA domæne (TEAD) familie af transskription faktorer er de vigtigste bindende partnere for YAP og TAZ, og er afgørende for de fleste YAP og TAZ-afhængige funktioner. Vores reporter konstruere udtrykker firefly luciferase fra en YAP / TAZ-TEAD-lydhør promotor og tidligere undersøgelser har vist, at det trofast registrerer ændringer i YAP-TEAD og TAZ-TEAD transskriptionelle aktivitet2,14,15.
Vores tilgang er hurtig, medium-throughput, og kræver ikke screening faciliteter, automatiserede robotter, eller dyb sekventering af pooled biblioteker. Omkostningerne er relativt lave, og der er mange kommercielt tilgængelige biblioteker at vælge imellem. Det nødvendige udstyr og de nødvendige reagenser er også relativt standard i de fleste laboratorier. Det kan bruges til at screene for lovgivere af stort set enhver transskription faktor, hvis en luciferase-baserede reporter eksisterer eller genereres. Vi bruger denne tilgang til at screene shRNAs i kræftceller, men enhver celle linje, der kan transfected med rimelig effektivitet kunne bruges med enhver form for arrayed bibliotek.
Protocol
Representative Results
Discussion
I denne undersøgelse demonstrerer vi en tilgang til medium-gennemløb screening af arrayed viral biblioteker i kombination med en dual-luciferase-baserede transskriptionsreporter assay, der kan bruges til at identificere og teste nye regulatorer af transskription faktorer. Det er vigtigt at karakterisere og optimere reportersystemet for hver cellelinje før enhver skærm. Der bør gøres forsøg for at bekræfte, at journalisten er lydhør over for ændret aktivitet af den transskriptionsfaktor, der undersøges, og omfa…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Vi vil gerne takke Emily Norton og Mikaelan Cucciarre-Stuligross for at hjælpe med udarbejdelsen af shRNA vektorer. Dette arbejde blev delvist støttet af en Susan G. Komen Career Catalyst Grant, der tildeles J.M.L. (#CCR17477184).
Materials
| 2.0 ml 96-well deep well polypropylene plate | USA Scientific | 1896-2000 | For bacterial mini-prep |
| Trypsin – 2.50% | Gibco | 15090-046 | Component of trypsin-EDTA |
| 96 well flat bottom white assay plate | Corning | 3922 | For dual-luciferase assay |
| Ampicillin – 100 mg/ml | Sigma-Aldrich | 45-10835242001-EA | For bacterial mini-prep |
| Bacto-tryptone – powder | Sigma-Aldrich | 95039 | Component of LB broth |
| Dual-luciferase reporter assay system, which include LAR II reagent (reagent A), Stop & Glo substrate (reagent B substrate) and Stop & Glo buffer (reagent B buffer) – Kit | Promega | E1960 | For dual-luciferase assay |
| Dulbecco's phosphate buffered saline w/o calcium, magnesium and phenol red – 9.6 g/L | Himedia | TS1006 | For PBS |
| EDTA – 0.5 M | VWR | 97061-406 | Component of trypsin-EDTA |
| Ethanol – 100% | Pharmco-AAPER | 111000200 | For bacterial mini-prep |
| Foetal Bovine Serum – 100% | VWR | 97068-085 | Component of complete growth media |
| Hexadimethrine bromide (Polybrene) – 8 mg/ml | Sigma-Aldrich | 45-H9268 | For virus infection |
| HyClone DMEM/High glucose – 4 mM L-Glutamine; 4500 mg/L glucose; sodium pyruvate | GE Healthcare life sciences | SH30243.01 | Component of complete growth media |
| I3-P/i3 Multi-Mode Microplate/EA | Molecular devices | For dual-luciferase assay | |
| L-Glutamine – 200 mM | Gibco | 25030-081 | Component of complete growth media |
| Lipofectamine 3000 (Transfection Reagent 2) – 100% | Life technologies | L3000008 | For transfections |
| Molecular Biology Water – 100% | VWR | 02-0201-0500 | For dilution of shRNA vector for virus packaging |
| NaCl – powder | BDH | BDH9286 | Component of LB broth |
| NanoDrop One Microvolume UV-Vis Spectrophotometer | Thermo scientific | For measuring vector DNA concentration | |
| Opti-MEM (Transfection Buffer) – 100% | Gibco | 31985-062 | For transfections |
| Penicillin Streptomycin – 10,000 Unit/ml (Penicillin); 10,000 µg/ml (Streptomycin) | Gibco | 15140-122 | Component of complete growth media |
| PureLink Quick Plasmid Miniprep Kit – Kit | Thermo Fisher Scientific | K210010 | For bacterial mini-prep |
| Puromycin – 2.5 mg/ml | Sigma-Aldrich | 45-P7255 | For antibiotic selection after infection |
| TC20 automated cell counter | Bio-Rad | For cell counting | |
| X-tremeGENE 9 DNA transfection reagent (Transfection Reagent 1) – 100% | Roche | 6365787001 | For virus packaging |
| Yeast extract – powder | VWR | J850 | Component of LB broth |
| P3000 (Transfection Reagent 3) – 100% | Life technologies | L3000008 | For transfections |
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