Name: focus formation: a cell-based assay to determine the oncogenic potential of a gene
Uploaded: 2014-12-31T15:00:00
Description: Watch this Scientific Journal Video about Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene at JoVE.com

Dette arbeidet ble støttet av et stipend fra NIH direktørens New Innovator Award Program (ED). AA ble støttet delvis av lavere priser fra National Cancer Institute og National Science Foundation.

1. Å gjøre de virale vektorer De kodende sekvenser for genet av interesse, så vel som positive og negative kontroller, blir satt inn i pBABEpuro av tradisjonelle kloningsmetoder (PCR amplifikasjon, restriksjonsspalting og ligering). Det er fire restriksjonsseter på vektor hvor DNA kan settes inn: BamHI, SnaBI, EcoRI og Sali. <ol><li> Forbered transfeksjon-grade plasmid DNA fra kompetente celler ved hjelp av Qiagen plasmid midi kit. </li><li> Måle DNA-konsentrasjon ved hjelp av en Nano...

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FFA gir en rask og enkel metode for å evaluere ondartet transformasjon in vitro. Det er mottagelig for screening av et forholdsvis stort antall kandidatgener, og de beskjedne tekniske krav at det er kostnadseffektivt. Videre kan to eller flere gener bli koekspresjon (noen ganger referert til som en &quot;samarbeid&quot; assay) for å evaluere karsinogent potensial av kombinasjonen. Fordelene med denne analysen er avhengig av sin enkle teknikk, sin enkle kvantifisering og dens relativt korte omløpstiden. Det m...

Tumorceller kan skilles fra deres normale motstykker av en lang rekke modifikasjoner, fra genekspresjonsmønster til Epigenomics til morfologiske og proliferative endringer. Blant de sistnevnte, redusert avhengighet av serum, tap av kontakt (tetthet) hemming, oppkjøpet av ankeruavhengig spredning og til slutt evnen til å danne svulster når injisert i dyr er nyttige, målbare indikatorer for malign transformasjon tre. Flere in vitro og in vivo er blitt utviklet for cellulær transformasjon. In vitro assays sikte på identifisering og måling av endringer i kultur morfologi (fokusdannelse assay), dynamikk kultur (veksthastighet, metningstetthet) og vekstfaktor (vekst i redusert serum) eller anker (vekst i myke agar) krav. Den gullstandard for bestemmelse av ondartet karakter av en celletype forblir tumordannelse (xenotransplantater) i forsøksdyr. Imidlertid than koste og lengden på in vivo studier ikke alltid gjør dem forsvarlig som et første valideringstrinnet eller screening av kandidat onkogener. Selv om ingen in vitro-analysen gir en klar vurdering av onkogene potensialet av et gen, de gi innsikt i onkogene potensial som kan innskrenke fremtid in vivo studier. En av de mest brukte systemer for evaluering av onkogene potensial in vitro er Focus Formation Assay 2. Denne metoden baserer seg på bruk av NIH 3T3 mus fibroblast, en ikke-transformert cellelinje som viser sterk inhiberende kontakt. Overekspresjon av et oncogen som resulterer i tap av tetthetsavhengig vekst; transformerte cellene kan deretter vokse i flere lag, som danner &quot;foci&quot;, lett visualisert på bakgrunn monolag av ikke-transformerte celler. Focus Formation analysen, da, måler evnen til en kandidat onkogen å indusere ondartet transformasjon, som dokumentert av tap av kontakt spesition som en målbar fenotype. FFA er blitt brukt til å evaluere transformasjon ved overekspresjon av protein-kinaser (for eksempel Src 4, BRAF 5), transkripsjonsfaktorer (for eksempel, N-myc-6), G-protein-koblede reseptorer (f.eks P2RY8 7) og GTPases (f.eks Ras 1), blant andre. Den relative letthet med denne analysen gjør det til et godt valg som vil tilveiebringe en rask og visuelt klart svar på hvorvidt overekspresjon av genet som er tilstrekkelig til å transformere NIH 3T3-muse-fibroblast-celler in vitro. FFA er beskrevet i denne protokollen bruker Plat-E pakkingscellelinje 8, som gir virale pakkeproteinene, og den retrovirale vektor pBABEpuro 9 (Addgene plasmid 1764) for å produsere retrovirus. Etter transfeksjon med pBABEpuro konstruksjon inneholdende genet av interesse, vil Plat-E-cellelinjen produserer ekotropisk retrovirus som kan bli anvendt for å infisere NIH 3T3-celler. Thans viral metode for genet levering er mer effektiv enn tradisjonelle kjemiske transfeksjonsmetoder og det gir en måte å bærekraftig uttrykke genet 10. Når inkorporert i genomet av NIH 3T3-celler, er overekspresjon av genet av interesse som drives av virale lange terminale gjentagelser (LTR) promoter 11. Denne konstante uttrykk kan benyttes for å bestemme om genet av interesse har onkogen aktivitet, som målt ved dannelse av foci, på NIH 3T3-celler.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">pBABE-puro vector</td>
			<td style="width:127px;">Addgene</td>
			<td style="width:123px;">Plasmid 1764</td>
			<td style="width:171px;">cloning vector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Platinum-E Retroviral Packaging Cell Line, Ecotropic</td>
			<td style="width:127px;">Cell Biolabs, Inc.</td>
			<td style="width:123px;">RV-101</td>
			<td>cell line for viral production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">NIH 3T3 Cell Line murine</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:123px;">93061524</td>
			<td>cell line for focus formation assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">10 mL BD Luer-Lok tip syringe</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:123px;">309604</td>
			<td>viral production reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">0.45 &#956;m Puradisc Syringe Filter</td>
			<td style="width:127px;">Whatman</td>
			<td style="width:123px;">6750-2504</td>
			<td>viral production reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Polyethylenimine (PEI)</td>
			<td style="width:127px;">Polysciences, Inc.</td>
			<td style="width:123px;">23966-2</td>
			<td>cell transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Polybrene Infection / Transfection Reagent</td>
			<td style="width:127px;">EMD Millipore</td>
			<td style="width:123px;">TR-1003-G</td>
			<td>cell transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Crystal Violet</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:123px;">C581-25</td>
			<td>cell stain reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Plasmid Plus Midi Kit</td>
			<td style="width:127px;">QIAGEN</td>
			<td style="width:123px;">12945</td>
			<td>plasmid purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">BD Falcon Tissue Culture Dishes</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:123px;">353003</td>
			<td>cell culture supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">11995-065</td>
			<td>cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">0.05% Trypsin-EDTA</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">25300-054</td>
			<td>cell culture supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Opti-MEM I Reduced Serum Medium</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">31985-062</td>
			<td>cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">16000-044</td>
			<td>cell culture media</td>
		</tr>
	</tbody>
</table>

focus formation: a cell-based assay to determine the oncogenic potential of a gene

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.

MXD3 er en grunnleggende helix-sløyfe-helix leucine glidelås (bHLHZ) transkripsjonsfaktor som er medlem av MYC / MAX / MAD nettverk. Det er et atypisk medlem av familien MAD 13-15, og det har blitt rapportert å være involvert i kreftutvikling 16,17. Sammenlignet med pBABEpuro (negativ kontroll) og MYC (positiv kontroll), de NIH 3T3 retter der MXD3 ble overuttrykt hadde signifikant mindre foci (Figur 1a). Dataene i figur 1B ble samlet fra flere eksperimenter for...

Watch this Scientific Journal Video about Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene at JoVE.com

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

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