Name: identification and characterization of metastatic factors by gene transfer into the novel rip-tag; rip-tva murine model
Uploaded: 2017-10-16T12:30:00
Description: Watch this Scientific Journal Video about Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model at JoVE.com

Vi takker Harold Varmus, Brian C. Lewis, Douglas Hanahan, Danny Huang, Sharon Pang, Megan Wong og Manasi M. Godbole. Y.C.N.D. understøttes af DOD grant W81XWH-16-1-0619 og NIH grant 1R01CA204916.

etik erklæring: eksperimenter på dyr blev udført i overensstemmelse med de retningslinjer og forskrifter fastsat af Institute for Animal Care og brug Udvalget af Weill Cornell medicin.
 1. valg af aviær Retroviral vektorer (RCASBP A-baseret eller RCANBP(A)-based) <ul style="list-style: none;"> <li>vektorer var afledt af Rous sarkom virus 11. Aviær retroviral vektorer kan levere cDNAs (&#8804; 2,5 kb), korte hårnål RNA'er (shRNAs), MicroRNA (miRNAs) og andre n...

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I denne undersøgelse beskrev vi en kraftfuld musemodel, RIP-Tag; RIP-tva, at opnå somatiske gen levering via aviær retrovira til identifikation og karakterisering af metastatisk faktorer. Selv om RIP-Tag; RIP-tva mus udvikle pancreas Neuroendokrine tumorer, i denne musemodel metastatisk faktorer kan også fremme metastase af andre kræft-former.
Vores fremgangsmåde har fordelen af at indføre somatiske genetiske ændringer specielt i præmaligne læsioner i bugspytkirtlen...

De fleste kræftformer opstår fra somatiske mutationer 1. Konventionelle gensplejsede musemodeller (CLAUSS) har givet betydelig indsigt i specifikke genetiske ændringer tumordannelse 2bidrag. Men de har flere begrænsninger. Den store ulempe ved disse modeller er, at de ikke replikerer tumordannelse hos mennesker, hvor kun nogle celler i et væv erhverve genetiske ændringer sporadiske natur. Mutationer i transgene og knockout mus er også kønscelleoverførsel med potentiale til at påvirke udviklingen. Derudover er genererer disse musemodeller dyrt og tidskrævende.
Metastaser er et vigtigt spørgsmål inden for kræft. Modeling metastaser har været vanskeligt i CLAUSS. Spontan metastaser er sjældne i musen. Penetrans er variabel og latenstid er længe i CLAUSS af metastaser 3. Eksperimentelle metastase modeller beskæftiger direkte indsprøjtning af celler til omsætning af mus, så de tidlige trin i metastatisk cascade er elimineret.
For at overvinde nogle af de ovennævnte begrænsninger i at studere metastatisk faktorer i musemodeller, har vi udviklet en bitransgenic musemodel, RIP-Tag; RIP-tva4. Strategien er baseret på at kombinere brugen af en stærkt synkroniseret tumor progression musemodel, RIP-Tag 5, og receptoren for undergruppe-A aviær kvægleukose virus, tva 6,7. Denne RIP-Tag; RIP-tvamusemodel giver gener skal indføres somatically i en enkelt bitransgenic mus stamme. Med SV40 T antigen undertrykke tumor smittespredningshæmmende funktioner Rb og p53, udvikle mus i bugspytkirtlen Neuroendokrine tumorer i en lignende måde at menneskelige tumordannelse, med faser herunder hyperplasi, angiogenese, adenom og invasive karcinom. Denne RIP-Tag model har været meget lærerigt for vores forståelse af kendetegnende for kræft, ikke begrænset til pancreas Neuroendokrine tumorer. Det har også været brugt i prækliniske forsøg 8.
Vi præsenterer en protokol til overførsel af somatiske gener gennem indsprøjtning af aviær retrovira intracardiacally i RIP-Tag; RIP-tva mus. Vellykket infektion med RCASBP-afledte aviær retrovira kræver aktivt prolifererende målceller. Derfor valgte vi RIP-Tag; RIP-tva mus 7 uger gamle, hvornår hyperplasi udvikler i omkring 50% af pancreas holmene. Venstre ventrikel intracardiac injektion af høj titer vira er forpligtet til at opnå en infektion effektivitet på 10-20% 4. Reducerer denne leveringsmetode betydelig fortynding af virale partikler i omløb før virus når pancreas Holme.
Brug denne fremgangsmåde, har vi tidligere vist, at Bcl-xL fremmer kræft metastaser uafhængigt af dens anti-apoptotiske funktion 4,9. Denne anti-apoptotiske-uafhængig metastatisk funktion blev ikke observeret, når Bcl-xL blev udtrykt via en transgen pancreas β celler i hele tumorfremkaldende ontogeny i RIP-Tag; RIP-Bcl-xL mus model 10. Derfor tilbyder vores musemodel en unik mulighed for at identificere og karakterisere gener funktioner når udtrykt på et senere tidspunkt af tumordannelse. Fordi 2-4% af Holme udvikle sig til tumorer i RIP-Tag; RIP-tva bitransgenic mus uden viral infektion og ikke alle de præmaligne læsioner er inficeret med de RCASBP-afledte aviær retrovira, kun de faktorer, der giver en selektiv fordel i naturlige løbet af tumordannelse kan identificeres. Især genkendes metastatisk faktorer mest let af denne metode, fordi metastase i bugspytkirtlen lymfeknuder eller andre organer ikke normalt forekommer i RIP-Tag; RIP-tva mus.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">RCASBP-Y DV plasmid</td>
			<td style="width:123px;">Addgene</td>
			<td style="width:119px;">11478</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RCAS-RNAi plasmid</td>
			<td style="width:123px;">Addgene</td>
			<td style="width:119px;">15182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:123px;">Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fetal bovine serum</td>
			<td>Atlanta Biologicals</td>
			<td>25-005-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine, 100x</td>
			<td style="width:123px;">Corning</td>
			<td>25-005-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin solution, 100x</td>
			<td style="width:123px;">Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">PBS-/-, 1X</td>
			<td style="width:123px;">Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfect</td>
			<td>Qiagen</td>
			<td>301305</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyallomer centrifuge tube</td>
			<td>Beckman Coulter</td>
			<td>326823</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">0.45 mm Nalgene 
			Syringe Filters with PES Membrane</td>
			<td style="width:123px;">Thermo Scientific</td>
			<td style="width:119px;">194-2545</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Insulin Syringes&#160;</td>
			<td style="width:123px;">BD</td>
			<td>329461</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">synaptophysin</td>
			<td style="width:123px;">Vector Laboratories</td>
			<td>VP-S284</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">VECTASTAIN Elite ABC HRP Kit (Peroxidase, Rabbit IgG)&#160;</td>
			<td style="width:123px;">Vector Laboratories</td>
			<td>PK-6101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AmpliTaq DNA Polymerase with Buffer II</td>
			<td>Life Technologies</td>
			<td>N8080153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MyTaq DNA Polymerase</td>
			<td>Bioline</td>
			<td>BIO-21106</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification and characterization of metastatic factors by gene transfer into the novel rip-tag; rip-tva murine model

Metastatisk kræft tegner sig for 90% af dødsfald blandt patienter med solide tumorer. Der er et presserende behov for bedre at forstå driverne på kræft metastaser og til at identificere nye terapeutiske mål. For at undersøge molekylære begivenheder, der drev progressionen fra primær kræft til metastase, har vi udviklet en bitransgenic musemodel, RIP-Tag; RIP-tva. I denne musemodel drev rotte insulin promoter (RIP) udtryk for SV40 T antigen (Tag) og receptor for undergruppe en aviær kvægleukose virus (tva) i bugspytkirtlen β celler. Musene udvikle pancreas Neuroendokrine tumorer med 100% penetrans gennem veldefinerede trin, som ligner menneskets tumordannelse, med faser herunder hyperplasi, angiogenese, adenom og invasive karcinom. Fordi RIP-Tag; RIP-tva mus ikke udvikle metastatisk sygdom, genetiske ændringer, der fremmer metastaser kan identificeres nemt. Somatiske gen overføres til tva-udtrykker, prolifererende pancreas β præmaligne læsioner er opnået gennem intracardiac injektion af aviær retrovira husly det ønskede genmodifikation. En titer på &#62; 1 x 108 smitsomme enheder pr. ml er hensigtsmæssigt for i vivo infektion. Derudover kan aviær retrovira inficere cellelinjer afledt af tumorer i RIP-Tag; RIP-tva mus med høj effektivitet. Cellelinjer kan også bruges til at karakterisere de metastatisk faktorer. Her viser vi hvordan man kan udnytte denne musemodel og cellelinjer til at vurdere funktioner af kandidat gener i tumor metastaser.

I vivo og in vitro- infektion sats af RIP-Tag; RIP-tva tumorceller ved RCASBP-baseret virus er ~ 20% og ~ 80% henholdsvis 20. I RIP-Tag; RIP-tva musemodel, ca. 4% af de 400 i bugspytkirtlen Holme i hvert mus vil naturligt udvikle sig til tumorer 20; Derfor er der tilstrækkelig mængde af tumorceller i hvert mus for histologiske og fænotypiske analyse af den potentielle virkning af de gener, der er levere...

Watch this Scientific Journal Video about Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model at JoVE.com

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

Vi præsenterer en protokol for at demonstrere en roman somatiske gen overførselssystem udnytter RIP-Tag; RIP-tva musen model til at studere funktionen af gener i metastaser. De aviær retrovira leveres intracardiacally for at sikre overførsel af gener til præmaligne, noninvasive læsioner i bugspytkirtlen β celler i voksen mus.

Identifikation og karakterisering af metastatisk faktorer af genoverførsel i romanen RIP-Tag; RIP-tva Murine Model

Metastatic cancer accounts for 90% of deaths in patients with solid tumors. There is an urgent need to better understand the drivers of cancer metastasis ...

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Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

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