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>
	<li>Vogelstein, B., Kinzler, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multistep+nature+of+cancer.">The multistep nature of cancer.</a> Trends Genet. 9 (4), 138-141 (1993).</li><li>Walrath, J. C., Hawes, J. J., Van Dyke, T., Reilly, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+engineered+mouse+models+in+cancer+research.">Genetically engineered mouse models in cancer research.</a> Adv Cancer Res. 106, 113-164 (2010).</li><li>Khanna, C., Hunter, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+metastasis+in+vivo.">Modeling metastasis in vivo.</a> Carcinogenesis. 26 (3), 513-523 (2005).</li><li>Du, Y. C., Lewis, B. C., Hanahan, D., Varmus, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+tumor+progression+factors+by+somatic+gene+transfer+into+a+mouse+model:+Bcl-xL+promotes+islet+tumor+cell+invasion.">Assessing tumor progression factors by somatic gene transfer into a mouse model: Bcl-xL promotes islet tumor cell invasion.</a> PLoS biology. 5 (10), e276 (2007).</li><li>Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heritable+formation+of+pancreatic+beta-cell+tumours+in+transgenic+mice+expressing+recombinant+insulin/simian+virus+40+oncogenes.">Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes.</a> Nature. 315 (6015), 115-122 (1985).</li><li>Fisher, G. H., 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=Development+of+a+flexible+and+specific+gene+delivery+system+for+production+of+murine+tumor+models.">Development of a flexible and specific gene delivery system for production of murine tumor models.</a> Oncogene. 18 (38), 5253-5260 (1999).</li><li>Orsulic, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+RCAS-TVA-based+approach+to+designer+mouse+models.">An RCAS-TVA-based approach to designer mouse models.</a> Mamm Genome. 13 (10), 543-547 (2002).</li><li>Tuveson, D., Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+medicine:+Cancer+lessons+from+mice+to+humans.">Translational medicine: Cancer lessons from mice to humans.</a> Nature. 471 (7338), 316-317 (2011).</li><li>Choi, S., 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=Bcl-xL+promotes+metastasis+independent+of+its+anti-apoptotic+activity.">Bcl-xL promotes metastasis independent of its anti-apoptotic activity.</a> Nat Commun. 7, 10384 (2016).</li><li>Naik, P., Karrim, J., Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rise+and+fall+of+apoptosis+during+multistage+tumorigenesis:+down-modulation+contributes+to+tumor+progression+from+angiogenic+progenitors.">The rise and fall of apoptosis during multistage tumorigenesis: down-modulation contributes to tumor progression from angiogenic progenitors.</a> Genes Dev. 10 (17), 2105-2116 (1996).</li><li>Hughes, S. H., Greenhouse, J. J., Petropoulos, C. J., Sutrave, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptor+plasmids+simplify+the+insertion+of+foreign+DNA+into+helper-independent+retroviral+vectors.">Adaptor plasmids simplify the insertion of foreign DNA into helper-independent retroviral vectors.</a> J Virol. 61 (10), 3004-3012 (1987).</li><li>Petropoulos, C. J., Payne, W., Salter, D. W., Hughes, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appropriate+in+vivo+expression+of+a+muscle-specific+promoter+by+using+avian+retroviral+vectors+for+gene+transfer+[corrected].">Appropriate in vivo expression of a muscle-specific promoter by using avian retroviral vectors for gene transfer [corrected].</a> J Virol. 66 (6), 3391-3397 (1992).</li><li>Dunn, K. J., Williams, B. O., Li, Y., Pavan, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest-directed+gene+transfer+demonstrates+Wnt1+role+in+melanocyte+expansion+and+differentiation+during+mouse+development.">Neural crest-directed gene transfer demonstrates Wnt1 role in melanocyte expansion and differentiation during mouse development.</a> Proc Natl Acad Sci USA. 97 (18), 10050-10055 (2000).</li><li>Loftus, S. K., Larson, D. M., Watkins-Chow, D., Church, D. M., Pavan, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+RCAS+vectors+useful+for+functional+genomic+analyses.">Generation of RCAS vectors useful for functional genomic analyses.</a> DNA Res. 8 (5), 221-226 (2001).</li><li>Bromberg-White, J. L., 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=Delivery+of+short+hairpin+RNA+sequences+by+using+a+replication-competent+avian+retroviral+vector.">Delivery of short hairpin RNA sequences by using a replication-competent avian retroviral vector.</a> J Virol. 78 (9), 4914-4916 (2004).</li><li>Harpavat, S., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RCAS-RNAi:+a+loss-of-function+method+for+the+developing+chick+retina.">RCAS-RNAi: a loss-of-function method for the developing chick retina.</a> BMC Dev Biol. 6 (2), (2006).</li><li>Huse, J. T., 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+PTEN-regulating+microRNA+miR-26a+is+amplified+in+high-grade+glioma+and+facilitates+gliomagenesis+in+vivo.">The PTEN-regulating microRNA miR-26a is amplified in high-grade glioma and facilitates gliomagenesis in vivo.</a> Genes Dev. 23 (11), 1327-1337 (2009).</li><li>Ahronian, L. G., Lewis, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+high-titer+RCAS+virus+from+DF1+chicken+fibroblasts.">Generation of high-titer RCAS virus from DF1 chicken fibroblasts.</a> Cold Spring Harb Protoc. 2014 (11), 1161-1166 (2014).</li><li>Green, M. R., Sambrook, J., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory manual. , (2012).</li><li>Du, Y. C., Lewis, B. C., Hanahan, D., Varmus, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+tumor+progression+factors+by+somatic+gene+transfer+into+a+mouse+model:+Bcl-xL+promotes+islet+tumor+cell+invasion.">Assessing tumor progression factors by somatic gene transfer into a mouse model: Bcl-xL promotes islet tumor cell invasion.</a> PLoS Biol. 5 (10), 2255-2269 (2007).</li><li>Du, Y. C., Chou, C. K., Klimstra, D. S., Varmus, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+for+hyaluronan-mediated+motility+isoform+B+promotes+liver+metastasis+in+a+mouse+model+of+multistep+tumorigenesis+and+a+tail+vein+assay+for+metastasis.">Receptor for hyaluronan-mediated motility isoform B promotes liver metastasis in a mouse model of multistep tumorigenesis and a tail vein assay for metastasis.</a> Proc Natl Acad Sci USA. 108 (40), 16753-16758 (2011).</li><li>Guernet, A., Grumolato, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9+editing+of+the+genome+for+cancer+modeling.">CRISPR/Cas9 editing of the genome for cancer modeling.</a> Methods. , (2017).</li></ol>

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 ...

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.

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