Name: screening for melanoma modifiers using a zebrafish autochthonous tumor model
Uploaded: 2012-11-13T13:00:00
Description: Watch this Scientific Journal Video about Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model at JoVE.com

Vi takker Dr. Leonard I. Zon i hvis laboratorium disse teknikker blev oprindeligt udviklet, Kristen Kwan og den sene Chi-Bin Chien for gaver af plasmider, der anvendes i dette arbejde, James Lister og David Raible for assistance med antistoffarvning og James Neiswender for Den mikroinjektion video. Dette arbejde blev finansieret af NIH tilskud R00AR056899-03 til CJC

1. Screening for Melanom Onset Modifiers <ol><li> Opret Gateway midterste entry kloner ved PCR amplifikation af fuldlængde åben læseramme på gener af interesse (GOI) og rekombinere til pDONR 221 ved anvendelse BP clonase II (Invitrogen). Brug Multisite Gateway-teknologi (Invitrogen) at rekombinere p5E_mitfa, pME_GOI, Tol2kit # 302 p3E_SV40polyA 6 og miniCoopR 5 for at gener af interesse under mitfa promotoren i miniCoopR vektoren (figur 1A). </li><li> Injice...

<ol>
	<li>Beroukhim, R., 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=The+landscape+of+somatic+copy-number+alteration+across+human+cancers.">The landscape of somatic copy-number alteration across human cancers.</a> Nature. 463, 899-905 (2010).</li><li>Curtin, J. 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=Distinct+sets+of+genetic+alterations+in+melanoma.">Distinct sets of genetic alterations in melanoma.</a> N. Engl. J. Med. 353, 2135-2147 (2005).</li><li>Lin, W. M., 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=Modeling+genomic+diversity+and+tumor+dependency+in+malignant+melanoma.">Modeling genomic diversity and tumor dependency in malignant melanoma.</a> Cancer Res. 68, 664-673 (2008).</li><li>Patton, E. E., 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=BRAF+mutations+are+sufficient+to+promote+nevi+formation+and+cooperate+with+p53+in+the+genesis+of+melanoma.">BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma.</a> Curr. Biol. 15, 249-254 (2005).</li><li>Ceol, C. J., 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=The+histone+methyltransferase+SETDB1+is+recurrently+amplified+in+melanoma+and+accelerates+its+onset.">The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset.</a> Nature. 471, 513-517 (2011).</li><li>Kwan, K. M., 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=The+Tol2kit:+a+multisite+gateway-based+construction+kit+for+Tol2+transposon+transgenesis+constructs.">The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.</a> Dev. Dyn. 236, 3088-3099 (2007).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function.">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> J. Vis. Exp. (25), e1115 (2009).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide of the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>White, R. M., 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=Transparent+adult+zebrafish+as+a+tool+for+in+vivo+transplantation+analysis.">Transparent adult zebrafish as a tool for in vivo transplantation analysis.</a> Cell Stem Cell. 2, 183-189 (2008).</li><li>Traver, 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=Transplantation+and+in+vivo+imaging+of+multilineage+engraftment+in+zebrafish+bloodless+mutants.">Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants.</a> Nat. Immunol. 4, 1238-1246 (2003).</li></ol>

The miniCoopR Fremgangsmåden muliggør ekspression af gener af interesse i zebrafisk melanocytter. Denne fremgangsmåde drager fordel af det faktum, at zebrafisk mitfa genet virker celle-uafhængigt. Af denne grund er melanocytter reddes af miniCoopR vektoren sikkert at indeholde minigenet og helst gen af ​​interesse, som det er fysisk koblet. Reddede melanocytter er klart synlige og kan fås i dyr der blev injiceret som en enkelt-celle embryoer. En specificeret grad af kimærisme er valgt, og dyr, overgå...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Gateway rekombination reagenser </td><td> Invitrogen </td><td></td><td></td></tr><tr><td> miniCoopR </td><td></td><td></td><td> Reference 5 </td></tr><tr><td> Mitfa antistof </td><td></td><td></td><td> Reference 5 </td></tr><tr><td> FITC gede anti-kanin IgG-antistof </td><td> Invitrogen </td><td></td><td></td></tr><tr><td> Vectashield </td><td> Vector Labs </td><td> H-1000 </td><td></td></tr><tr><td> casper zebrafisk </td><td></td><td></td><td> Henvisning 9 </td></tr><tr><td> 701N 10 pi Sprøjte </td><td> Hamilton / Fisher </td><td> 14-824 </td><td> &amp;nbsp; </td></tr><tr><td> 40 uM filter </td><td> BD Falcon / Fisher </td><td> 352.340 </td><td></td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 26140079 </td><td></td></tr></tbody></table>

screening for melanoma modifiers using a zebrafish autochthonous tumor model

Genomiske undersøgelser af humane cancere har givet et væld af oplysninger om gener, der er ændret i tumorer 1,2,3. En udfordring i forbindelse med disse undersøgelser er, at mange gener er ændret, og det kan være vanskeligt at skelne genetiske ændringer, der kørte tumorigenese fra at de opstod øvrigt under transformation. For at tegne denne skelnen er det fordelagtigt at have en assay, der kan kvantitativt måle effekten af ​​et ændret gen på tumor initiering og andre processer, der muliggør tumorer til at vare ved og formidle. Her præsenterer vi en hurtig metode til at screene et stort antal kandidat melanom modifikatorer i zebrafisk med en autokton tumormodel 4, som omfatter nødvendige trin for melanom initiering og vedligeholdelse. Et vigtigt reagens i dette assay er miniCoopR vektoren, som kobler en vildtype-kopi af mitfa melanocyt specifikation faktor til en Gateway rekombination kassette i hvilken kandidat melAnoma gener kan rekombineres 5. The miniCoopR vektor har en mitfa redning minigen, som indeholder promotoren, åbne læseramme og 3&#39;-utranslaterede region af det vildtype-mitfa genet. Det giver os mulighed for at lave konstruktioner under anvendelse fuld længde åbne læserammer af kandidat melanom modifikatorer. Disse individuelle kloner kan derefter injiceres i enkelt celle Tg (mitfa: BRAF V600E), p53 (lf), mitfa (lf) zebrafisk embryoner. The miniCoopR vektor bliver integreret ved Tol2-medieret transgenese 6 og redder melanocytter. Fordi de er fysisk koblet til mitfa redning minigenet, der kandidatgener udtrykt i reddede melanocytter, hvoraf nogle vil omdanne og udvikle sig til tumorer. Virkningen af ​​et kandidatgen for melanom initiering og melanomcellelinjer egenskaber kan måles med melanom-free overlevelseskurverne, invasion assays, antistof-farvning og transplantation assays.

Én-celle Tg (mitfa: BRAF V600E), p53 (lf), mitfa (lf) zebrafisk embryoner injiceret med miniCoopR vektor indeholdende melanom onkogen SETDB1 5 eller EGFP, hver især er under kontrol af mitfa promotoren. Embryoner med melanocyt redning blev udvalgt og fik lov til at modnes. Ved 2 måneders alderen dyr med melanocyt redning større end 4 mm 2 blev udvalgt. Dyrene blev screenet ugentligt for melanomer. Tumorforekomst kurverne for de voksne viste, at SETDB1</em...

Watch this Scientific Journal Video about Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model at JoVE.com

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

En hurtig måde at screene for melanom modifikatorer anvendelse af en zebrafisk autokton tumormodel er præsenteret. Den udnytter den miniCoopR vektor, som tillader ekspression af kandidat melanom gener i melanocytter. En fremgangsmåde til opnåelse af melanom-free overlevelseskurverne, en invasion assay, en protokol til antistoffarvning af skala melanocytter og en melanom transplantation assay er beskrevet.

Screening for Melanoma Modifiers anvendelse af et zebrafisk Autokton tumormodel

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

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