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

Biz Harold Varmus, Brian C. Lewis, Douglas Hanahan, Danny Huang, Sharon Pang, Megan Wong ve Manasi M. Godbole teşekkür ederim. Y.C.N.D. DOD grant W81XWH-16-1-0619 ve NIH grant 1R01CA204916 tarafından desteklenir.

etik beyanı: deneylerin hayvanlar üzerinde yönergeleri ve ileri hayvan bakım ve kullanım Komitesi Weill Cornell Tıp Enstitüsü tarafından belirlenen düzenlemelere uygun olarak gerçekleştirilen.
 1. seçim kuş Retroviral vektörler (RCASBP A-esaslı ya da RCANBP(A)-based) <ul style="list-style: none;"> <li>vektörler Rous sarkomu virüs 11 ' den elde edilen. Kuş retroviral vektörler cDNAs sunabilirsiniz (&#8804; 2,5 kb), kısa saç tokası RNA'ların (s...

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Bu çalışmada, bir güçlü fare modeli, RIP-Tag açıkladığımız; RIP-tva, yoluyla kimlik ve metastatik faktörler karakterizasyonu için kuş Retrovirüsler somatik gen teslim elde etmek için. Her ne kadar RIP-etiket; RIP-tva fareler geliştirmek pankreas nöroendokrin tümör, bu fare modelde tanımlanan metastatik faktörler de metastaz diğer kanser türleri teşvik.
Bizim yaklaşım somatik genetik değişiklikler özellikle premalign lezyonlar pankreas β hücrel...

En kanserleri somatik mutasyon 1ile ortaya çıkmaktadır. Geleneksel genetiği fare modelleri (et) tumorigenesis 2için belirli genetik değişiklik katkısını önemli anlayışlar hazırladık. Ancak, bazı sınırlamaları vardır. Bu modellerin en büyük dezavantajı sadece bir doku bazı hücrelerde genetik değişiklikler elde insanlarda tümör oluşumu sporadik doğası çoğaltılmaz var. Transgenik ve nakavt fareler mutasyonların da germline geliştirme etkiler için potansiyeli vardır. Ayrıca, bu fare modelleri üreten pahalı ve zaman alıcı olduğunu.
Metastaz kanser alanında önemli bir konudur. Metastaz modelleme et içinde zor olmuştur. Spontan metastaz fare nadirdir. Alellerle değişken ve gecikme süresi uzun metastaz 3et içinde. Metastatik cascade erken adımda ortadan kalkar böylece deneysel metastaz modelleri fareler, dolaşımı içine direkt enjeksiyon hücre istihdam.
Bazı fare modelleri metastatik faktörler eğitim yukarıdaki sınırlamaları aşmak için bir bitransgenic fare modeli, RIP-Tag geliştirdik; RIP-tva4. Strateji alt grubu-A kuş lökozu virüs, tva 6,7için bir son derece senkronize tümör ilerleme fare modeli, RIP-etiket 5ve reseptör kullanımını birleştirerek üzerinde temel alır. Bu RIP-etiket; RIP-tvafare modeli gen somatically bir tek bitransgenic fare yük sunulmasına olanak sağlar. Rb ve p53 Tümör baskılayıcı fonksiyonlarını baskılayarak SV40 T antijen ile fareler Hiperplazi, anjiogenez, adenoma ve invaziv karsinom gibi aşamaları ile pankreas nöroendokrin tümör insan tumorigenesis benzer bir şekilde geliştirin. Bu RIP-Tag model kanser, bunlarla sınırlı olmamak için pankreas nöroendokrin tümör işaretlerinden anlayışımız için çok öğretici oldu. Preklinik denemeler 8' de kullanılmaktadır.
Biz kuş Retrovirüsler intracardiacally enjeksiyonuyla RIP-etiket içine somatik gen transferi için bir iletişim kuralı mevcut; RIP-tva fareler. Kuş Retrovirüsler RCASBP elde edilen başarılı enfeksiyonu aktif Proliferasyona hedef hücrelerin gerekli. Bu nedenle, RIP-Tag seçti; RIP-tva fareler, 7 hafta-in yaş, ne zaman Hiperplazi pankreas adacıkları yaklaşık % 50 geliştirir. Sol ventrikül İntrakardiyak enjeksiyon yüksek titresi virüslerin % 10-20 4bir enfeksiyon verimlilik elde etmek için gereklidir. Virüs pankreas adacıkları ulaşmadan bu teslim yöntemi dolaşım içinde viral parçacıkların önemli seyreltme azaltır.
Bu yaklaşımı kullanarak, daha önce Bcl-xL kanser metastaz onun anti-apoptotik işlev 4,9/ bağımsız teşvik göstermiştir. BCL-xL RIP-etiket tumorigenic Ontojeni boyunca tüm pankreas β hücrelerindeki bir transgene ile ifade edildi zaman bu anti-apoptotik-bağımsız metastatik işlev gözlenen değil; RIP-Bcl-xL fare modeli 10. Bu nedenle, bizim fare modeli tanımlamak ve tumorigenesis daha sonraki bir aşamada ifade genler işlevleri tanımlamak için eşsiz bir fırsat sunmaktadır. Çünkü adacıkları % 2-4'ü geliştirmek tümörler RIP-Tag; içinde içine RIP-tva bitransgenic fareler viral enfeksiyon ve tüm premalign lezyonlar olmadan RCASBP elde edilen kuş Retrovirüsler ile enfekte, sadece tumorigenesis doğal seyri üzerinde seçici bir avantaj görüşmek faktörler belirlenebilir. Özellikle, pankreas lenf düğümleri veya diğer organlara metastaz normalde RIP-Tag oluşmaz çünkü metastatik faktörler en kolay bu yöntem tarafından tanınır; RIP-tva fareler.

<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

Metastatik kanser ölümleri solid tümör olan hastalarda yüzde 90'ını hesapları. Kanser metastaz sürücüleri daha iyi anlamak için ve yeni tedavi hedefleri belirlemek için acil bir ihtiyaç vardır. İlerleme birincil kanser metastaz için sürücü moleküler olayları araştırmak için bir bitransgenic fare modeli, RIP-Tag geliştirdik; RIP-tva. Bu fare modelinde, sıçan insülin organizatörü (RIP) pankreas beta hücrelerinde SV40 T antijen (etiket) ve reseptör alt grubu bir kuş lökozu virüs (tva) için ifade kullanıyor. Fareler pankreas nöroendokrin tümörlerin % 100 alellerle Hiperplazi, anjiogenez, adenoma ve invaziv karsinom gibi aşamaları ile insan tumorigenesis için benzer iyi tanımlanmış aşamalarla ile geliştirmek. Çünkü RIP-etiket; RIP-tva fareler metastatik hastalık geliştirmek değil, metastaz teşvik genetik değişikliklere kolayca belirlenebilir. Somatik gen aktarım tva ifade, pankreas β Proliferasyona premalign lezyonlar elde edilir kuş Retrovirüsler istenen genetik değişikliği barındıran İntrakardiyak sızdırma yoluyla. Bir titresi &#62; ml başına 1 x 108 bulaşıcı birim vivo içinde enfeksiyon için uygun kabul edilir. Buna ek olarak, kuş Retrovirüsler tümörler RIP-etiket içinde türetilmiş hücre hatları bulaşabilir; RIP-tva fareler yüksek verimlilik ile. Hücre hatları metastatik faktörler karakterize etmek için de kullanılabilir. Burada nasıl bu fare modeli kullanmak ve tümör metastazı aday genler işlevleri değerlendirmek için satırları hücre göstermektedir.

Vivo ve in vitro enfeksiyon oranı RIP-etiket; RIP-tva tümör hücreleri RCASBP tabanlı virüs tarafından 20~ % 20 ve % 80 ~ sırasıyla vardır. RIP-etiket; RIP-tva fare modeli, her fare 400 pankreas adacıkları yaklaşık % 4'ü doğal olarak geliştirmek tümörler 20; Bu nedenle tümör hücreleri her fare virüsler tarafından teslim genlerin potansiyel etkisinin histolojik ve fenotipik analiz iç...

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

Biz bir roman somatik gen aktarım sistem RIP-etiket kullanmak göstermek için bir iletişim kuralı mevcut; RIP-tva metastaz genlerinde fonksiyonu çalışmaya fare modeli. Kuş Retrovirüsler gen transferi önceden Malign, invaziv olmayan lezyonlar yetişkin farelerde pankreas β hücrelerinin içine sağlamak için intracardiacally teslim edilir.

Kimlik ve metastatik faktörler gen transferi ile karakterizasyonu roman RIP-etiketinin içine; RIP-tva fare modeli

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

İleri Hayvan Karaciğer Kolorektal Metastaz Model: Görüntüleme Teknikleri ve Metastatik Klonların Özellikleri

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

Kanser Araştırmaları

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

Preklinik Tedaviler Nicel ve Boyuna Değerlendirme A sinjeneik Fare Modeli Yapmış Bir Renal Hücreli Karsinom

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

Periferik Radyasyonla Bağlı Yorgunluk A Fare Modeli

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

Virüs gen değiştirme için fare prostat CRISPR kılavuzları teslimini

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

Zenginleştirme ve kurulan subkutan fare tümör tümör bağışıklık ve Non-immün Microenvironments karakterizasyonu

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

Üç boyutlu kemik hücre dışı matriks modeli osteosarkom için

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

İskemi Reperfüzyon Hasarı Ayarında Metastatik Karaciğer Tümörlerinin Murine Modeli

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

CorExplorer Web Portalı ile Tümör Gen Ekspresyonu Faktörlerinin Analizi

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

Organ-On-A Chip 3D Modeli, Makine Öğrenimi ve Konfokal Tomografi Kullanarak Beyin Metastatik Tümör Mikro-Ortamının Ölçülmesi

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

Biyolüminesans Kullanarak Farelerde Meme Kanseri Büyümesi ve Metastatik Koloni Oluşumunun İzlenmesi

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