Fokus Formation: En cellebaseret assay til bestemmelse af onkogent potentiale af et gen
Summary
Focus Formation-analysen giver en enkel metode til at vurdere transformerende potentiale en kandidat onkogen.
Abstract
Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.
Introduction
Tumorceller kan skelnes fra deres normale modparter med en bred vifte af ændringer, fra mønstre af genekspression til Epigenomics til morfologiske og proliferative ændringer. Blandt sidstnævnte mindre afhængighed af serum, tab af kontakt (tæthed) hæmning, erhvervelse af forankringsuafhængig spredning og, i sidste ende, evnen til at danne tumorer ved injektion i dyr er nyttige, målbare indikatorer for malign transformation 3. Adskillige in vitro og in vivo assays er blevet udviklet til cellulær transformation. In vitro assays sigte på at identificere og måle ændringer i kultur morfologi (fokus dannelse assay), kultur dynamik (vækstrate, mætning tæthed) og vækstfaktor (vækst i reduceret serum) eller ankerplads (vækst i blød agar) krav. Den gyldne standard til bestemmelse af ondartet karakter af en celletype forbliver tumordannelse (xenografter) i forsøgsdyr. Men than koster og længden af in vivo studier ikke altid gøre dem berettiget som et første skridt validering eller screening af kandidat onkogener. Selv om ingen in vitro assay tilvejebringer en konkret vurdering af den onkogene potentiale af et gen, de giver indsigt i onkogent potentiale, der kan indsnævre fremtid in vivo-undersøgelser. En af de mest anvendte systemer til evaluering onkogent potentiale in vitro er i fokus Formation Assay 2. Denne tilgang er baseret på anvendelsen af NIH 3T3 muse-fibroblast, en ikke-transformeret cellelinie, der viser stærk kontakt inhibering. Overekspression af et onkogen resulterer i tab af tæthed-afhængig vækst; transformerede celler kan derefter vokse i flere lag, der udgør "foci" let visualiseres på baggrund monolag af ikke-transformerede celler. Focus Formation Assay, derefter måler evnen af en kandidat onkogen, at inducere malign transformation, som det fremgår af tab af kontakt Inhibition som en målelig fænotype. FFA er blevet anvendt til at evaluere transformation ved overekspression af proteinkinaser (f.eks Src 4, BRAF 5), transkriptionsfaktorer (fx N-myc 6), G-protein-koblede receptorer (f.eks P2RY8 7) og GTPaser (f.eks Ras 1), blandt andre. Den relative lethed af dette assay gør det til et godt valg, der vil give en hurtig og visuelt klart svar på, hvorvidt overekspression af genet er tilstrækkelig til at omdanne NIH 3T3 muse fibroblastceller in vitro.
Beskrevet i denne protokol FFA anvender Plat-E pakkecellelinie 8, som giver virale emballage proteiner, og den retrovirale vektor pBABEpuro 9 (Addgene plasmid 1764) til fremstilling af retrovirus. Efter transfektion med pBABEpuro konstruktion indeholdende genet af interesse, vil Plat-E cellelinje producerer ecotropisk retrovirus, der kan anvendes til at inficere NIH 3T3-celler. Thans viral metode til genlevering er mere effektiv end traditionelle kemiske transfektionsfremgangsmåder og det giver en måde at bæredygtigt udtrykke gen 10. Når inkorporeres i genomet af NIH 3T3-celler, er overekspression af genet af interesse drives af virale lange terminale gentagelser (LTR) promotoren 11. Denne konstante ekspression kan anvendes til at bestemme, hvorvidt genet af interesse har onkogen aktivitet, målt ved dannelsen af foci, på NIH 3T3-celler.
Protocol
Representative Results
Discussion
Den FFA giver en hurtig og nem metode til at evaluere malign transformation in vitro. Det er muligt at foretage en screening af et relativt stort antal kandidatgener, og dens beskedne tekniske krav gør det omkostningseffektivt. Endvidere kan to eller flere gener co-udtrykt (undertiden benævnt "samarbejde" assay) at evaluere tumorigene potentiale kombination. Fordelene ved denne analyse påberåbe sig sin ligetil teknik, dens lette kvantificering og dens relativt korte turn-around tid. Det skal dog u…
Declarações
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af en bevilling fra NIH direktørs New Innovator Award Program (ED). AA blev delvist understøttet af bachelor priser fra National Cancer Institute og National Science Foundation.
Materials
| pBABE-puro vector | Addgene | Plasmid 1764 | cloning vector |
| Platinum-E Retroviral Packaging Cell Line, Ecotropic | Cell Biolabs, Inc. | RV-101 | cell line for viral production |
| NIH 3T3 Cell Line murine | Sigma-Aldrich | 93061524 | cell line for focus formation assay |
| 10 mL BD Luer-Lok tip syringe | BD Biosciences | 309604 | viral production reagent |
| 0.45 μm Puradisc Syringe Filter | Whatman | 6750-2504 | viral production reagent |
| Polyethylenimine (PEI) | Polysciences, Inc. | 23966-2 | cell transfection reagent |
| Polybrene Infection / Transfection Reagent | EMD Millipore | TR-1003-G | cell transfection reagent |
| Crystal Violet | Fisher Scientific | C581-25 | cell stain reagent |
| Plasmid Plus Midi Kit | QIAGEN | 12945 | plasmid purification |
| BD Falcon Tissue Culture Dishes | BD Biosciences | 353003 | cell culture supplies |
| Dulbecco's Modified Eagle Medium (DMEM) | Gibco | 11995-065 | cell culture media |
| 0.05% Trypsin-EDTA | Gibco | 25300-054 | cell culture supplies |
| Opti-MEM I Reduced Serum Medium | Gibco | 31985-062 | cell culture media |
| Fetal Bovine Serum (FBS) | Gibco | 16000-044 | cell culture media |
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