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

We thank Harold Varmus, Brian C. Lewis, Douglas Hanahan, Danny Huang, Sharon Pang, Megan Wong, and Manasi M. Godbole. Y.C.N.D. is supported by DOD grant W81XWH-16-1-0619 and NIH grant 1R01CA204916.

Ethics Statement:Experiments on animals were performed in accordance with the guidelines and regulations set forth by the Institute for Animal Care and Use Committee of Weill Cornell Medicine.
1. Choice of Avian Retroviral Vectors (RCASBP(A)-based or RCANBP(A)-based)
<ul style="list-style: none;">
	<li>Vectors was derived from Rous sarcoma virus11. Avian retroviral vectors can deliver cDNAs (&#8804;2.5 kb), short hairpin RNAs (shRNAs), microRNAs (miRNAs), and other no...

<ol>
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In this study, we described a powerful mouse model, RIP-Tag; RIP-tva, to achieve somatic gene delivery via avian retroviruses for the identification and characterization of metastatic factors. Although RIP-Tag; RIP-tva mice develop pancreatic neuroendocrine tumors, metastatic factors identified in this mouse model may also promote metastasis of other cancer types.
Our approach has the advantage of introducing somatic genetic changes specifically into premalignant lesions of p...

Most cancers arise from somatic mutations 1. Conventional genetically engineered mouse models (GEMM) have provided significant insights into the contribution of specific genetic alterations to tumorigenesis 2. However, they have several limitations. The major drawback of these models is that they do not replicate the sporadic nature of tumor formation in humans, in which only some cells within a tissue acquire genetic alterations. The mutations in transgenic and knockout mice are also germline with potential to affect development. Moreover, generating these mouse models is expensive and time-consuming.
Metastasis is a significant issue in the field of cancer. Modeling metastasis has been difficult in GEMM. Spontaneous metastasis is rare in the mouse. Penetrance is variable and latency is long in GEMM of metastasis 3. Experimental metastasis models employ direct injection of cells into the circulation of mice, so the early steps in the metastatic cascade are eliminated.
To overcome some of the above limitations in studying metastatic factors in mouse models, we have developed a bitransgenic mouse model, RIP-Tag; RIP-tva4. The strategy is based on combining the use of a highly-synchronized tumor progression mouse model, RIP-Tag 5, and the receptor for subgroup-A avian leukosis virus, tva 6,7. This RIP-Tag; RIP-tvamouse model allows genes to be introduced somatically into a single bitransgenic mouse strain. With the SV40 T antigen suppressing the tumor suppressive functions of Rb and p53, mice develop pancreatic neuroendocrine tumors in a similar fashion to human tumorigenesis, with stages including hyperplasia, angiogenesis, adenoma, and invasive carcinoma.This RIP-Tag model has been very instructive for our understanding of hallmarks of cancer, not limited to pancreatic neuroendocrine tumors. It has also been used in preclinical trials 8.
We present a protocol for somatic gene transfer through injection of avian retroviruses intracardiacally into RIP-Tag; RIP-tva mice. Successful infection with RCASBP-derived avian retroviruses requires actively proliferating target cells. Therefore, we chose RIP-Tag; RIP-tva mice at 7 weeks of age, when hyperplasia develops in about 50% of the pancreatic islets. Left ventricular intracardiac injection of high titer viruses is required to achieve an infection efficiency of 10-20% 4. This delivery method reduces the significant dilution of viral particles within circulation before viruses reach pancreatic islets.
Using this approach, we have previously demonstrated that Bcl-xL promotes cancer metastasis independent of its anti-apoptotic function 4,9. This anti-apoptotic-independent metastatic function was not observed when Bcl-xL was expressed via a transgene in all pancreatic &#946; cells throughout tumorigenic ontogeny in the RIP-Tag; RIP-Bcl-xL mouse model 10. Therefore, our mouse model offers a unique opportunity to identify and characterize genes&#39; functions when expressed at a later stage of tumorigenesis. Because 2-4% of islets develop into tumors in the RIP-Tag; RIP-tva bitransgenic mice without viral infection and not all the premalignant lesions are infected with the RCASBP-derived avian retroviruses, only the factors that confer a selective advantage over the natural course of tumorigenesis can be identified. In particular, metastatic factors will be most easily recognized by this method, because metastasis to pancreatic lymph nodes or other organs does not normally occur in RIP-Tag; RIP-tva mice.

<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

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 and to identify novel therapeutic targets. To investigate molecular events that drive the progression from primary cancer to metastasis, we have developed a bitransgenic mouse model, RIP-Tag; RIP-tva. In this mouse model, the rat insulin promoter (RIP) drives the expression of the SV40 T antigen (Tag) and the receptor for subgroup A avian leukosis virus (tva) in pancreatic &#946; cells. The mice develop pancreatic neuroendocrine tumors with 100% penetrance through well-defined stages that are similar to human tumorigenesis, with stages including hyperplasia, angiogenesis, adenoma, and invasive carcinoma. Because RIP-Tag; RIP-tva mice do not develop metastatic disease, genetic alterations that promote metastasis can be identified easily. Somatic gene transfer into tva-expressing, proliferating pancreatic &#946; premalignant lesions is achieved through intracardiac injection of avian retroviruses harboring the desired genetic alteration. A titer of &#62;1 x 108 infectious units per ml is considered appropriate for in vivo infection. In addition, avian retroviruses can infect cell lines derived from tumors in RIP-Tag; RIP-tva mice with high efficiency. The cell lines can also be used to characterize the metastatic factors. Here we demonstrate how to utilize this mouse model and cell lines to assess the functions of candidate genes in tumor metastasis.

The in vivo and in vitro infection rate of RIP-Tag; RIP-tva tumor cells by RCASBP-based viruses are ~20% and ~80% respectively 20. In the RIP-Tag; RIP-tva mouse model, approximately 4% of the 400 pancreatic islets in each mouse will naturally develop into tumors 20; therefore there is sufficient amount of tumor cells in each mouse for histological and phenotypic analysis of the potential effect of the gene...

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

We present a protocol to demonstrate a novel somatic gene transfer system utilizing RIP-Tag; RIP-tva mouse model to study the function of genes in metastasis. The avian retroviruses are delivered intracardiacally to ensure gene transfer into pre-malignant, noninvasive lesions of pancreatic &#946; cells in adult mice.

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel 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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