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 tackar Harold Varmus, Brian C. Lewis, Douglas Hanahan, Danny Huang, Sharon Pang, Megan Wong och Manasi M. Godbole. Y.C.N.D. stöds av DOD grant W81XWH-16-1-0619 och NIH grant 1R01CA204916.

etik uttalande: djurförsök har utförts i enlighet med de riktlinjer och bestämmelser som anges av Institute for Animal Care och användning kommittén för Weill Cornell medicin.
 1. Val av aviär retrovirala vektorer (RCASBP A-baserad eller RCANBP(A)-based) <ul style="list-style: none;"> <li>vektorer härleddes från Rous sarkom virus 11. Aviär retrovirala vektorer kan leverera cDNAs (&#8804; 2,5 kb), kort hårnål RNAs (shRNAs), mikroRNA (Mirna) och andra ick...

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I denna studie beskrev vi en kraftfull musmodell, RIP-Tag; RIP-tva, att uppnå somatisk gen leverans via aviär retrovirus för identifiering och karakterisering av metastaserande faktorer. Även om RIP-Tag; RIP-tva möss utvecklar pankreas neuroendokrina tumörer, metastaserad faktorer som anges i denna musmodell kan även främja metastas av andra typer av cancer.
Vår metod har fördelen att införa somatiska genetiska förändringar specifikt i premaligna lesioner av pank...

De flesta cancerformer uppkommer somatiska mutationer 1. Konventionella genetiskt modifierade musmodeller (GEMM) har gett betydande insikter i bidrag till specifika genetiska förändringar till tumourigenesis 2. De har dock flera begränsningar. Den stora nackdelen med dessa modeller är att inte replikera de datatillämpningar tumörbildning hos människa, där endast vissa celler i en vävnad förvärva genetiska förändringar. Mutationerna i transgena och knockout möss är också könsceller med potential att påverka utvecklingen. Dessutom är genererar dessa musmodeller dyra och tidskrävande.
Metastaser är en viktig fråga i fältet av cancer. Modeling metastaser har varit svårt i GEMM. Spontan metastaser är sällsynt i musen. Penetrans är variabel och latens är lång i GEMM metastaser 3. Experimentell metastaser modeller sysselsätter direkt injektion av celler i cirkulationen av möss, så de tidiga stegen i metastaserande kaskad elimineras.
För att övervinna några av ovanstående begränsningar i att studera metastaserande faktorer i musmodeller, har vi utvecklat en bitransgenic musmodell, RIP-Tag; RIP-tva4. Strategin bygger på att kombinera ett högt-synkroniserad tumör progression musmodell, RIP-Tag 5, och receptorn för undergruppen-A aviär leukos virus, tva 6,7. Denna RIP-tagg; RIP-tvamusmodell tillåter gener införs somatically till en enda bitransgenic mus stam. Med SV40 T antigenet undertrycka tumör suppressiv funktioner Rb och p53, utvecklar möss pankreas neuroendokrina tumörer i ett liknande sätt som människans uppkomst, med stadier inklusive hyperplasi, angiogenes, adenom och invasiva carcinom. RIP-Tag modellen har varit mycket lärorikt för vår förståelse av kännetecknen för cancer, inte begränsat till bukspottskörteln neuroendokrina tumörer. Det har också använts i prekliniska studier 8.
Vi presenterar ett protokoll för somatiska genöverföring genom injektion av aviär retrovirus intracardiacally i RIP-Tag; RIP-tva möss. Framgångsrik infektion med RCASBP-derived aviär retrovirus kräver aktivt prolifererande målceller. Därför valde vi RIP-Tag; RIP-tva möss vid 7 veckors ålder, när hyperplasi utvecklar cirka 50% av Langerhanska. Vänstra ventrikulära intrakardiellt injektion av hög titer virus krävs för att uppnå en infektion verkningsgrad på 10-20% 4. Denna leveransmetod minskar betydande utspädning av viruspartiklar inom cirkulationen innan virus når Langerhanska.
Använder denna metod, har vi tidigare visat att Bcl-xL främjar cancer metastaser oberoende av dess anti-apoptotiska funktion 4,9. Denna anti-apoptotiska-oberoende metastaserande funktion observerades inte när Bcl-xL uttrycktes via en transgen i alla pankreas β-celler i hela tumörframkallande Ontogeni i RIP-Tag; RIP-Bcl-xL mus modell 10. Därför erbjuder våra musmodell en unik möjlighet att identifiera och karaktärisera gener funktioner när uttryckt i ett senare skede av tumourigenesis. Eftersom 2-4% av holmar utvecklas till tumörer i RIP-Tag; RIP-tva bitransgenic möss utan viral infektion och inte alla premaligna lesioner är infekterade med de RCASBP-derived aviär retrovirus, endast de faktorer som ger en selektiv fördel under naturliga uppkomst kan identifieras. I synnerhet känns metastaserande faktorer lättast igen av denna metod, eftersom metastaser bukspottskörteln lymfkörtlar eller andra organ inte förekommer normalt i RIP-Tag; RIP-tva möss.

<table border="0" cellpadding="0" cellspacing="0" width="747">
	<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

Metastaserande cancer står för 90 procent av dödsfallen hos patienter med solida tumörer. Det finns ett brådskande behov av att bättre förstå förare av cancer metastaser och identifiera nya terapeutiska mål. För att undersöka molekylära händelser som driver utvecklingen från primär cancer till metastas, har vi utvecklat en bitransgenic musmodell, RIP-Tag; RIP-tva. I denna musmodell driver råtta insulin arrangören (RIP) uttryck för SV40 T antigenet (Tag) och receptorn för undergruppen aviär leukos virus (tva) i pankreas β-celler. Möss utvecklar pankreas neuroendokrina tumörer med 100% penetrans genom väldefinierade steg som liknar människans uppkomst, med stadier inklusive hyperplasi, angiogenes, adenom och invasiva carcinom. Eftersom RIP-Tag; RIP-tva möss inte utvecklar metastaserande sjukdom, genetiska förändringar som främjar metastaser kan identifieras enkelt. Somatisk gen överför till tva-uttryckande, frodas pankreas β premaligna lesioner uppnås genom intrakardiellt injektion av aviär retrovirus härbärgerat den önskade genetiska förändringen. En titer av &#62; 1 x 108 infektiösa enheter per ml anses lämpligt för i vivo infektion. Dessutom kan aviär retrovirus infektera cellinjer som härstammar från tumörer i RIP-Tag; RIP-tva möss med hög verkningsgrad. Cellinjer kan också användas för att karaktärisera de metastaserande faktorerna. Här visar vi hur att utnyttja denna musmodell och cellinjer för att bedöma fungerar av kandidatgener i tumör metastasering.

Andelen i vivo och in vitro- infektion av RIP-Tag; RIP-tva tumörceller av RCASBP-baserade virus är ~ 20% ~ 80% respektive 20. I RIP-taggen. RIP-tva musmodell, cirka 4% av de 400 Langerhanska i varje mus kommer naturligt utvecklas till tumörer 20; Därför finns det tillräcklig mängd tumörceller i varje mus för histologiska och fenotypiska analys av den potentiella effekten av de gener som levereras ...

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 presenterar ett protokoll för att visa en del av ett system för överföring av somatisk gen utnyttja RIP-Tag; RIP-tva musmodell att studera funktionen av gener i metastaser. De aviär retrovirus levereras intracardiacally för att säkerställa genöverföring till pre-maligna, noninvasiv lesioner av pankreas β-celler i vuxna möss.

Identifiering och karakterisering av metastaserande faktorer av genöverföring i romanen RIP-taggen; RIP-tva murina modell

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 ...

The overall goal of this procedure is to identify and characterize metastatic factors by gene transfer into the Novel RIP-Tag;RIP-tva mouse model. This method can help answer key questions in the cancer metastasis field such as identifying the drivers and the Novel therapeutic targets. The main advantage of this mouse model of cancer is to identify metastatic factors by somatic gene transfer into pancreatic beta cell precursor legions.Begin by harvesting one 15 centimeter dish of virus producing DF1 cells per mouse to be infected. First, collect the medium from the confluent 15 centimeter dishes. Then remove cell debris by low speed centrifugation in a pre-chilled centrifuge for 10 minutes at 1650 times G and four degrees celsius.Next, transfer the viral supernatant to polyallomer ultracentrifuge tubes. Spin in an ultracentrifuge for 1.5 hours at 9540 times G and four degrees celsius. Remove as much supernatant from the ultracentrifuge tubes as possible.Then add PBS to the ultracentrifuge tube to make the final 100 microliter viral suspension per 15 centimeter dish. Cover the tubes with laboratory film and re-suspend the invisible viral pellet by vortexing for two minutes at medium speed. Then rock the ultracentrifuge tubes at four degrees celsius for one hour or overnight.After the incubation, transfer the viral suspension to a microcentrifuge tube and proceed to titer the virus. Begin the titering procedure by ensuring the DF1 cells seated into 12 well tissue culture plates have reached 30%confluency. One twelve well plate is needed for each viral titer determination.Begin a tenfold serial dilution of the viral supernatants by first mixing five microliters of viral supernatant with 495 microliters of growth medium in a microcentrifuge tube to make a ten to the second dilution. Vortex briefly for a few seconds. Then transfer 40 microliters of the ten to the second dilution to 360 microliters of growth medium in a microcentrifuge tube for the ten to the third dilution.Vortex briefly for a few seconds. Continue this process to dilute to ten to the fourth and ten to the fifth. Next aliquot 2.25 milliliters of growth medium into each of five six milliliter polystyrene tubes.And label them ten to the sixth, ten to the seventh, ten to the eighth, ten to the ninth, and ten to the tenth respectively. Mix 0.25 milliliters of the ten to the fifth dilution with 2.25 milliliters of growth medium in the six milliliter polystyrene tube for the ten to the sixth dilution. Vortex briefly for a few seconds.Repeat the procedure for ten to the seventh, ten to the eighth, ten to the ninth, and ten to the tenth dilutions. Next remove the original culture medium from the DF1 cells on the 12 well plates. And add one milliliter of diluted virus into each well in duplicate.Allow the cells to grow at 37 degrees celsius and 5 CO2 for at least seven days. Begin the infection procedure by first anesthetizing a seven week old RIP-Tag RIP-tva mouse. Apply eye lubricant to both eyes to prevent corneal drying.Shave the hair from the chest cavity of the RIP-Tag RIP-tva mouse, position the mouse on its back with the chest facing up on an absorbent lab bench paper on top of a snuggle safe warmer. Perform a toe pinch to check the animal for nonresponsiveness and to confirm full anesthesia effect. Extend the front limbs and secure them with tape.Mark the chest of the mouse for injection. Mark the location midway between the sternal notch and the top of the xiphoid process, and slightly left anatomically of the sternum. Then scrub the anterior chest wall with either a povidone iodine scrub or a chlorhexidine scrub, followed by a 70%isopropyl alcohol or a 70%ethanol soaked gauze sponge.Draw 50 microliters of air into a sterile 28.5 gauge insulin syringe to create space between the plunger and the meniscus of a 500 microliter syringe. Draw up 100 microliters of virus. The airspace without any liquid on the wall is crucial for seeing the cardiac pulse.Keep the needle upright, hold the skin of mouse taut with one hand and insert the needle into the marked location. When a bright red pulse of blood appears in the syringe, stop advancing the needle and fix the depth of the needle in the mouse with one hand. Use the other hand to carefully and slowly push the plunger to deliver the viral suspension over about a 60 second period.Deliver 10-20 microliters whenever a bright red pulse of blood appears in the syringe. The continuous entrance of red oxygenated blood into the transparent needle hub indicates the proper positioning of the needle into the left ventricle. After the last push of the plunder to deliver viral suspension and before seeing the next red pulse of blood, quickly retract the needle out of the chest cavity.Apply gentle pressure over the injection site for at least one minute to reduce the bleeding. Then carefully move the mouse onto a heating pad or under a heat lamp until it is fully conscious. RIP-Tag RIP-tva mice were infected with the indicated RCASBP-Bcl-xL retro virus at seven weeks of age and euthanized at 16 weeks of age.This photograph shows representative synaptophysin staining of metastatic pancreatic neuroendocrine tumors in pancreatic lymph nodes. Here the RIP-Tag RIP-tva mice were infected with the indicated RCASBP-RHAM B.This photograph shows the representative synaptophysin staining of metastatic pancreatic neuroendocrine tumors in pancreatic lymph nodes. Here the synaptophysin staining is seen in the liver after injection of the RCASBP-RHAMM B virus.It is important to remember the title of greater than ten to the eight infectious units per milliliter is required for in vivo infection of proliferating TVA cells in RIP-Tag RIP-tva mouse and RCASBP retro viral rector can deliver CDNA up to 2.5 kilobases. After the development of this mouse model, it paved the way to a paradigm shifting finding that BCL-xL actually promotes cancer metastasis independent of it's antiapoptotic function.

Research

JoVE Journal

Cancer Research

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

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

Advanced djurmodell av Colorectal metastas i Lever: avbildningsmetoder och egenskaper av metastaserande kloner

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Ett syngent musmodell av metastaserad njurcellscancer för kvantitativ och Longitudinal Bedömning av Prekliniska terapier

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

En musmodell av trötthet inducerad av perifera bestrålning

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration

Virus leverans av CRISPR guider till murina prostatan för gen ändring

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) ...

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

Anrikning och karakterisering av de tumör immun och icke-immun mikromiljö i etablerade subkutan murina tumörer

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Tredimensionella ben extracellulärmatrix modell för osteosarkom

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Murin modell av metastaserande levertumörer i inställningen av ischemia reperfusion skada

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

Analyzing Tumor Gene Expression Factors with the CorExplorer Web Portal

Analysera tumör gen uttrycks faktorer med CorExplorer webb Portal

Differential gene expression analysis is an important technique for understanding disease states. The machine learning algorithm CorEx has shown utility ...

Quantifying the Brain Metastatic Tumor Micro-Environment using an Organ-On-A Chip 3D Model, Machine Learning, and Confocal Tomography

Kvantifiera hjärnan metastaserande tumör mikromiljö med hjälp av en organ-on-A Chip 3D-modell, machine learning, och confocal tomografi

Brain metastases are the most lethal cancer lesions; 10-30% of all cancers metastasize to the brain, with a median survival of only ~5-20 months, ...

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

Övervakning av bröstcancertillväxt och metastatisk kolonibildning hos möss med bioluminescens

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several ...