Diese Arbeit wurde durch einen Zuschuss von der NIH Direktor Neue Innovator Award Program (ED) unterstützt. AA wurde zum Teil von Undergraduate-Auszeichnungen von der National Cancer Institute und der National Science Foundation unterstützt.

1. Die Herstellung der viralen Vektoren Die codierenden Sequenzen für das Gen von Interesse, sowie die positive und negative Kontrollen werden durch traditionelle Klonierungsverfahren (PCR-Amplifikation, Restriktionsspaltung und Ligation) in pBaBepuro eingefügt. Es gibt vier Restriktionsstellen auf dem Vektor, wobei die DNA eingefügt werden kann: BamHI, SnaBI, SalI und EcoRI. <ol><li> Bereiten Transfektion-grade Plasmid-DNA aus kompetenten Zellen mit dem QIAGEN Plasmid Midi Kit. </li><li> Messen DNA-Konzentration mit einem NanoDrop2000 Spektralphotometer. </li></ol> 2. Retrovirus Produktion Die Plat-E Verpackungszelllinie wird verwendet, um ecotrope Retrovirus, das die cDNA von Interesse für NIH 3T3-Zellen liefern produzieren wird. <ol><li> Gründung Plat-E Kulturen aus eingefrorenen Zellen <ol><li> Tauen schnell Plat-E-Zellen in einem 37 ° C Wasserbad und Transfer zu einem 15 ml konischen Röhrchen. Langsam 9 ml Plat-E-Medium (Dulbecco modifiziertes Eagle-Medium (DMEM) + 10% fötalem Rinderserum (FBS) + Penicillin und Streptomycin). </li><li> Zentrifugieren Sie die Röhrchen bei 180 x g für 5 min und den Überstand verwerfen. </li><li> Resuspendieren der Zellen mit 10 ml von PLAT-E-Medium, und in ein 10 cm Kulturschale. </li><li> Inkubieren der Zellen in einem 37 ° C, 5% CO 2 kultiviert, bis sie 80-90% Konfluenz (etwa 2 Tage). </li></ol></li><li> Zell Splitting <ol><li> Saugen Sie das Medium und waschen Sie die Zellen einmal mit PBS. </li><li> 2 ml 0,05% Trypsin-EDTA und Inkubation für 1 min bei RT. </li><li> Trennen Sie die Zellen, die durch Fingertippen, 10 ml Plat-E-Medium, und übertragen Sie die Zellsuspension in ein 15-ml-Tube. </li><li> Zentrifugieren Sie die Röhrchen bei 180 x g für 5 min und den Überstand verwerfen. </li><li> Die Zellen mit 10 ml Plat-E-Medium und Saatgut in neue Gerichte zu 1: 4-1: 6 Verdünnungen. </li></ol></li><li> Plat-E Seeding und Transfektion <ol><li> Seed 2 x 10 6Zellen pro 10 cm Kulturschale mit Plat-E-Medium ohne Antibiotika. </li><li> Die Zellen O / N Inkubieren bei 37 ° C, 5% CO 2 -Inkubator. </li><li> Am nächsten Tag, Transfer 300 ul Opti-MEM in 1,5 ml Mikrozentrifugenröhrchen. </li><li> Hinzufügen 27 ul Polyethylenimin (PEI), ein kostengünstiges Transfektionsreagenz 12, auf die vorbereitete Rohr mit Opti-MEM. Vorsichtig mischen Finger klopfen und Inkubation für 5 min bei RT. </li><li> In 9 ug Transfektion-Grade-Plasmid-DNA in der Opti-MEM / PEI Rohr, vorsichtig mischen durch Vortexen und Inkubation für 15 min bei RT. </li><li> Fügen Sie die DNA / PEI Komplex tropfenweise in die Plat-E Gericht, und Inkubation O / N bei 37 ° C, 5% CO 2. </li><li> Am nächsten Tag, saugen Sie den Medium, das das Transfektionsreagenz und 10 ml frisches Plat-E-Medium (ohne Antibiotika). </li><li> Rückkehr der Zellen in den Inkubator. </li></ol></li></ol> 3. NIH 3T3 Cells and Infection <ol><li> Gründung NIH3T3 Kulturen und Seeding <ol><li> Tauen schnell NIH 3T3-Zellen in einem 37 ° C Wasserbad und Transfer in ein 15-ml-Tube. Langsame Zugabe von 9 ml NIH 3T3-Medium (DMEM + 10% FBS). </li><li> Zentrifugieren Sie die Röhrchen bei 180 x g für 5 min und den Überstand verwerfen. </li><li> Resuspendieren der Zellen mit 10 ml von NIH 3T3-Medium und in ein 10 cm Kulturschale. </li><li> Inkubieren der Zellen in einem 37 ° C, 5% CO 2 -Inkubator. Es ist sehr wichtig, dass die NIH 3T3 Zellen werden stets underconfluent (50-60%), wenn die Frequenz der spontanen Transformation (und daher basale Foci-Bildung) erhöht, wenn eine Kultur Konfluenz erreicht. </li><li> Seed 3 x 10 5 Zellen pro 10 cm Schale und Inkubation O / N für die Infektion am nächsten Tag. </li></ol></li><li> Infektion <ol><li> Ernte der retroviralen Überstand vom PLAT-E-Schale unter Verwendung einer 10 ml Plastikspritze, die durch Filtrieren über eine 0,45 um Porengrße Nylonmembranfilter, und Überführung in eine 15 mlRohr. </li><li> 10 ml frisches Plat-E-Medium zu den Zellen (ohne Antibiotika), und bringt sie in den Inkubator. </li><li> Saugen Sie das Medium aus dem NIH 3T3 Zell Gericht angesteckt zu werden, und mit 5 ml regelmäßigen NIH 3T3 Medium und 5 ml virushaltige Überstand gefiltert. </li><li> Hinzufügen Polybren in die Schale in einer Konzentration von 6 ug / ml. </li><li> Die Zellen O / N bei 37 ° C, 5% CO 2. </li><li> Am nächsten Tag, wiederholen Sie die Schritte 3.2.1 - 3.2.5 für eine zweite Runde der Infektion. </li><li> Ersetzen Sie die virushaltige Medium mit regelmäßigen NIH 3T3 Medium. </li><li> Lassen Sie die NIH 3T3-Zellen zu 2-3 Wochen wachsen, Ersetzen des Mediums wie nötig. Überprüfen der Expression des Proteins von Interesse durch herkömmliche Protein Elektrophorese und Immunoblot auf ganzer Zellysate von Replika-Platten. </li></ol></li></ol> 4. Die Färbung und Quantifizierung <ol><li> Kristallviolett-Färbung <ol><li> Saugen Sie das NIH 3T3 Medium und Ort der Gerichteauf Eis. </li><li> Die Gerichte mit eiskaltem PBS zweimal waschen. </li><li> Befestigen Sie die Zellen mit eiskaltem Methanol für 10 min. </li><li> Entfernen Sie die Gerichte aus Eis, saugen Sie den Methanol und 3 ml 0,5% Kristallviolett-Lösung in 25% Methanol (RT) gemacht. </li><li> Das Geschirr Inkubation für 5 min bei RT. </li><li> Saugen Sie das Kristallviolett-Lösung und spülen Sie sorgfältig die Schale mit Milli-Q H 2 O, bis keine Farbe löst sich in der Spülung. </li><li> Lassen Sie die Gerichte nach O / N auf einem Labortisch trocknen (unbedeckt). </li></ol></li><li> Foci Quantifizierung <ol><li> Sobald die Gerichte sind trocken, mit einem Lineal, um die dunkel gefärbten Sammlungen von Zellen auf der Platte zu messen. Zählen nur jene, die größer als 5 mm im Durchmesser, wie die &quot;Foci&quot;. Nehmen Sie die Anzahl der Brennpunkte und berechnen Mittelwerte und die Bedeutung im Vergleich zu den Kontrollen. </li></ol></li></ol>

<ol>
	<li>Clark, G. J., Cox, A. D., Graham, S. M., Der, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+assays+for+Ras+transformation.">Biological assays for Ras transformation.</a> Methods in enzymology. , 255-395 (1995).</li><li>Celis, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell biology : a laboratory handbook. , 345-352 (2006).</li><li>Raptis, L., Vultur, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neoplastic+transformation+assays.">Neoplastic transformation assays.</a> Methods in molecular biology. 165, 151-164 (2001).</li><li>Johnson, P. J., Coussens, P. M., Danko, A. V., Shalloway, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpressed+pp60c-src+can+induce+focus+formation+without+complete+transformation+of+NIH+3T3+cells.">Overexpressed pp60c-src can induce focus formation without complete transformation of NIH 3T3 cells.</a> Molecular and cellular biology. 5, 1073-1083 (1985).</li><li>Bonner, T. I., Kerby, S. B., Sutrave, P., Gunnell, M. A., Mark, G., Rapp, U. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+biological+activity+of+human+homologs+of+the+raf/mil+oncogene.">Structure and biological activity of human homologs of the raf/mil oncogene.</a> IMolecular and cellular biology. 5, 71400-71407 (1985).</li><li>Yancopoulos, G. D., 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=N-myc+can+cooperate+with+ras+to+transform+normal+cells+in+culture.">N-myc can cooperate with ras to transform normal cells in culture.</a> Proceedings of the National Academy of Sciences of the United States of America. 82, 5455-5459 (1985).</li><li>Fujiwara, S., 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=Transforming+activity+of+purinergic+receptor+P2Y,+G+protein+coupled,+8+revealed+by+retroviral+expression+screening.">Transforming activity of purinergic receptor P2Y, G protein coupled, 8 revealed by retroviral expression screening.</a> Leukemia &amp; lymphoma. 48, 978-986 (2007).</li><li>Morita, S., Kojima, T., Kitamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plat-E:+an+efficient+and+stable+system+for+transient+packaging+of+retroviruses.">Plat-E: an efficient and stable system for transient packaging of retroviruses.</a> Gene. 7, 1063-1066 (2000).</li><li>Morgenstern, J. P., Land, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+mammalian+gene+transfer:+high+titre+retroviral+vectors+with+multiple+drug+selection+markers+and+a+complementary+helper-free+packaging+cell+line.">Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line.</a> Nucleic acids research. 18, 3587-3596 (1990).</li><li>Kim, T. K., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cell+transfection:+the+present+and+the+future.">Mammalian cell transfection: the present and the future.</a> Analytical and bioanalytical chemistry. 397, 3173-3178 (2010).</li><li>Klaver, B., Berkhout, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+5'+and+3'+long+terminal+repeat+promoter+function+in+human+immunodeficiency+virus.">Comparison of 5' and 3' long terminal repeat promoter function in human immunodeficiency virus.</a> Journal of virology. 68, 3830-3840 (1994).</li><li>Fukumoto, 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=Cost-effective+gene+transfection+by+DNA+compaction+at+pH+4.0+using+acidified,+long+shelf-life+polyethylenimine.">Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified, long shelf-life polyethylenimine.</a> Cytotechnology. 62, 73-82 (2010).</li><li>Fox, E. J., Wright, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S-phase-specific+expression+of+the+Mad3+gene+in+proliferating+and+differentiating+cells.">S-phase-specific expression of the Mad3 gene in proliferating and differentiating cells.</a> The Biochemical journal. 359, 361-367 (2001).</li><li>Yun, J. S., Rust, J. M., Ishimaru, T., Diaz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+of+the+Mad+family+member+Mad3+in+cerebellar+granule+neuron+precursor+proliferation.">A novel role of the Mad family member Mad3 in cerebellar granule neuron precursor proliferation.</a> Molecular and cellular biology. 27, 8178-8189 (2007).</li><li>Gore, Y., Lantner, F., Hart, G., Shachar, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mad3+negatively+regulates+B+cell+differentiation+in+the+spleen+by+inducing+Id2+expression.">Mad3 negatively regulates B cell differentiation in the spleen by inducing Id2 expression.</a> Molecular biology of the cell. 21, 1864-1871 (2010).</li><li>Barisone, G. A., Yun, J. S., Diaz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+cerebellar+proliferation+to+tumorigenesis:+new+insights+into+the+role+of+Mad3.">From cerebellar proliferation to tumorigenesis: new insights into the role of Mad3.</a> Cell cycle. 7, 423-427 (2008).</li><li>Barisone, G. A., 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=Role+of+MXD3+in+proliferation+of+DAOY+human+medulloblastoma+cells.">Role of MXD3 in proliferation of DAOY human medulloblastoma cells.</a> PloS One. 7, e38508 (2012).</li><li>Nair, S. K., Burley, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+structures+of+Myc-Max+and+Mad-Max+recognizing+DNA.+Molecular+bases+of+regulation+by+proto-oncogenic+transcription+factors.">X-ray structures of Myc-Max and Mad-Max recognizing DNA. Molecular bases of regulation by proto-oncogenic transcription factors.</a> Cell. 112, 193-205 (2003).</li></ol>

Die Autoren haben nichts zu offenbaren.

Die FFA bietet eine schnelle und einfache Methode, um eine maligne Transformation in vitro zu bewerten. Es ist offen für das Screening einer relativ großen Anzahl von Kandidaten-Gene und ihrer bescheidenen technischen Anforderungen machen es kosteneffektiv. Ferner können zwei oder mehr Gene coexprimiert werden (manchmal als &quot;Kooperation&quot; assay), die tumorigene Potenzial der Kombination zu bewerten. Die Vorteile dieses Tests verlassen sich auf ihre einfache Technik, die einfache Quantifizierung und ...

Tumorzellen von ihren normalen Gegenstücken durch eine Vielzahl von Änderungen zu unterscheiden, von den Mustern der Genexpression Epigenomics morphologischen und proliferative Veränderungen. Unter den letzteren eine geringere Abhängigkeit von Serum, Kontaktverlust (Dichte) Inhibition des Erwerbs von verankerungsunabhängiges Proliferation und schließlich die Fähigkeit, Tumoren zu bilden, wenn sie in Tiere injiziert sind nützlich, messbare Indikatoren Entartungs 3. Mehrere in vitro- und in vivo-Assays für zelluläre Transformation entwickelt worden. In-vitro-Assays zielen auf die Identifizierung und Messung von Veränderungen der Kulturmorphologie (Fokus-Assay), Kultur Dynamik (Wachstumsrate, Sättigungsdichte) und Wachstumsfaktor (Wachstums verringerten Serum) oder Ankerplatz (Wachstum in Weichagar) Anforderungen. Der Goldstandard für die Bestimmung der bösartigen Natur eines Zelltyps bleibt der Tumorbildung (Xenografts) in Versuchstieren. Jedoch ter kostet und Länge der in vivo-Studien nicht immer machen sie zu rechtfertigen als ersten Validierungsschritt oder Screening von Kandidaten Onkogene. Obwohl keine in vitro-Test liefert eine definitive Beurteilung der onkogenen Potentials eines Gens, sie geben Einblick in onkogene Potenzial, das in vivo-Studien gezieltere Zukunft kann. Eine der am weitesten verbreiteten Systeme zur Bewertung der onkogenen Potential in vitro wird die Fokusbildungs-Assay 2. Dieser Ansatz beruht auf der Verwendung von NIH 3T3-Maus-Fibroblasten, einer nicht-transformierten Zelllinie, die starke Kontakthemmung zeigt. Überexpression eines Onkogens zu einem Verlust der Wachstumsdichte abhängig; transformierte Zellen können dann wachsen in mehreren Schichten bildet &quot;Foci&quot;, leicht gegen den Hintergrund Monoschicht von nicht-transformierten Zellen visualisiert. Die Fokusbildungs-Assay, dann misst die Fähigkeit eines Kandidaten-Onkogens zur malignen Transformation zu induzieren, wie durch den Verlust des Kontakts belegt INHIBition als messbare Phänotyp. Die FFA wurde zur Transformation durch die Überexpression von Proteinkinasen (zB Src 4, BRAF 5), Transkriptionsfaktoren (zB N-myc 6), G-Protein-gekoppelten Rezeptoren (zB P2RY8 7) und GTPasen (zB evaluieren Ras 1), unter anderem. Die relative Leichtigkeit dieses Assays ist es eine gute Wahl, die eine schnelle und optisch klare Antwort, ob die Überexpression des Gens ausreicht, um NIH 3T3-Maus-Fibroblasten-Zellen zu transformieren in vitro bereitstellt. Die in diesem Protokoll beschrieben FFA verwendet die Plat-E Verpackungszelllinie 8, die viralen Verpackungsproteine ​​enthält, und der retrovirale Vektor pBabePuro 9 (Addgene Plasmid 1764) zu Retrovirus zu produzieren. Nach Transfektion mit dem pBaBepuro Konstrukt das Gen von Interesse enthält, wird das PLAT-E-Zellinie ecotropen Retroviren, die verwendet werden können, um NIH 3T3 Zellen zu infizieren, zu erzeugen. Tseine Methode der viralen Gentransfer ist effizienter als herkömmliche chemische Transfektionsmethoden und bietet einen Weg, um nachhaltig das Gen 10 auszudrücken. Sobald sie in das Genom der NIH-3T3-Zellen eingebaut wird, wird die Überexpression des Gens von Interesse von der viralen langen terminalen Wiederholungen (LTR) -Promotor 11 angetrieben. Diese konstante Ausdruck kann verwendet werden, um festzustellen, ob das Gen von Interesse hat onkogene Aktivität, wie durch die Bildung von Foci auf den NIH 3T3-Zellen gemessen werden.

<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 eine basische Helix-Loop-Helix-Leucin-Zipper (bHLHZ) Transkriptionsfaktor, der ein Mitglied der MYC / MAX / MAD-Netzwerk ist. Es ist ein atypisches Mitglied der Familie MAD 13-15, und es wurde berichtet, dass bei der Karzinogenese 16,17 beteiligt sein. Im Vergleich zu pBaBepuro (negative Kontrolle) und MYC (positive Kontrolle), die NIH 3T3 Geschirr wo MXD3 überexprimiert wurde, hatten signifikant weniger Foci (1A). Die Daten in Abbildung 1B wurden aus verschi...

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

Der Fokus Formation Assay bietet eine einfache Methode, um die transformierende Potential eines Kandidaten Onkogen zu bewerten.

Fokus Formation: Ein Assay auf Zellbasis, um das onkogene Potential eines Gens zu bestimmen

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

focus-formation-cell-based-assay-to-determine-oncogenic-potential

Research

JoVE Journal

Medicine

27.7K Aufrufe.  University of California, Davis.  Der Fokus Formation Assay bietet eine einfache Methode, um die transformierende Potential eines Kandidaten Onkogen zu bewerten. 

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

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarker in einem Tiermodell für die Aufdeckung Neural, hämatologischen und Behavioral Korrelate der PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Forschung

Medizin

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

A Novel Surgical Ansatz intratracheale Verabreichung von Wirkstoffen in einem Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Hochdurchsatz-Durchflusszytometrie zellbasierten Assay auf Antikörper gegen N-Methyl-D-Aspartat-Rezeptor oder Dopamin-2-Rezeptor im menschlichen Serum Detect

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

Eine modifizierte<em&gt; In-vitro-</em&gt; Invasion Assay, um die mögliche Rolle von Hormonen, Cytokinen und / oder Wachstumsfaktoren bei der Vermittlung der Invasion von Krebszellen fest,

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

A Novel<em&gt; In vivo</em&gt; Gentransfer-Technik und<em&gt; In-vitro-</em&gt; Zellbasierte Assays für das Studium der Knochenverlust in Skeletterkrankungen

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Dreidimensionale (3D) Tumor Spheroid Invasionsassay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

Nutzung der Koloniebildung in Weichagar-Assay auf Inhibitoren der Tumorigenität in Brustkrebs-Zellen zu identifizieren

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes an artificial small guide RNA (sgRNA) to guide tRNA 3&#8242; processing endoribonuclease, tRNase ZL, to recognize a target RNA. sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells. We have screened an sgRNA library containing 156 heptamer-type sgRNAs for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines. Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers. The protocol includes how to construct the sgRNA library, how to assess the effect of each sgRNA on cell viability, and how to further evaluate the effective sgRNAs by flow cytometry. Around 2,000 hits would be expected to be obtained by screening the full-scale sgRNA library composed of 16,384 heptamers.

Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers.

TRUE Gene Silencing: Screening eines Heptamer-Typ Kleiner Leitfaden RNA-Bibliothek für potentielle Krebs Therapeutika

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes ...

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

Die Injektion von syngenen murinen Melanomzellen ihre metastatische Potential in der Lunge zu bestimmen

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB functions in separating the cerebrospinal fluid (CSF) from the blood and restricting the molecular exchange to a minimum extent. An in vitro model of the BCSFB is based on cells derived from a human choroid plexus papilloma (HIBCPP). HIBCPP cells display typical barrier functions including formation of tight junctions (TJs), development of a transepithelial electrical resistance (TEER), as well as minor permeabilities for macromolecules. There are several pathogens that can enter the central nervous system (CNS) via the BCSFB and subsequently cause severe disease like meningitis. One of these pathogens is Neisseria meningitidis (N. meningitidis), a human-specific bacterium. Employing the HIBCPP cells in an inverted cell culture filter insert system enables to study interactions of pathogens with cells of the BCSFB from the basolateral cell side, which is relevant in vivo. In this article, we describe seeding and culturing of HIBCPP cells on cell culture inserts. Further, infection of the cells with N. meningitidis along with analysis of invaded and adhered bacteria via double immunofluorescence is demonstrated. As the cells of the CP are also involved in other diseases, including neurodegenerative disorders like Alzheimer`s disease and Multiple Sclerosis, as well as during the brain metastasis of tumor cells, the model system can also be applied in other fields of research. It provides the potential to decipher molecular mechanisms and to identify novel therapeutic targets.

The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

Ein Choroidplexus Epithelial Zell-basierte Modell des menschlichen Blut-Liquor-Schranke Bakterielle Infektion von der basolateralen Seite zu studieren

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The ...