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

我们感谢哈罗德 Varmus, 布赖恩 c. 刘易斯, 道格拉斯 Hanahan, 丹尼黄, 沙龙庞, 梅根黄, 和玛纳斯河 m. 哥博利。y.c.n. D. 得到国防部 W81XWH-16-1-0619 和 NIH 补助金1R01CA204916 的支持。

道德声明: 动物实验是按照由威尔康奈尔医学研究所动物护理和使用委员会规定的准则和条例进行的. 
 1. 选择禽类逆转录病毒载体 (RCASBP (a) 基或 RCANBP (a)) <ul style="list-style: none;"> <li> 向量是从瓣肉瘤毒 11 派生的。禽逆转录病毒载体可以提供基因 (和 #8804; 2.5 kb), 短发夹 rna (shRNAs), rna (rna) 和其他编码 rna。在 材料 中列出了商业上可用的向量, ?...

<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>

在本研究中, 我们描述了一个强大的鼠标模型, RIP 标签;RIP-tva,通过禽病毒实现体细胞基因的传递, 以确定和鉴定转移因子。虽然RIP 标记;tva小鼠开发胰腺神经内分泌肿瘤, 在这种小鼠模型中发现的转移因子也可能促进其他癌症类型的转移。
我们的方法有优势, 引入体细胞遗传变化, 特别是前病变胰腺β细胞的控制方式, 从而更忠实地模仿偶发人类肿瘤的发展。这种方?...

大多数癌症都是由体细胞突变引起的1。传统的基因工程小鼠模型 (GEMM) 提供了重要的洞察力的贡献, 具体的基因改变的肿瘤发生的2。但是, 它们有几个限制。这些模型的主要缺点是, 它们不复制人类肿瘤形成的零星性质, 在这种情况下, 只有组织内的一些细胞才能获得基因改变。转基因和剔除小鼠的突变也系有可能影响发展。此外, 生成这些鼠标模型是昂贵和耗时的。
转移是一个重要的问题在癌症领域。模型转移在 GEMM 是困难的。小鼠自发转移是罕见的。显是可变的, 潜伏期很长, 在转移的 GEMM 3。实验性转移模型采用直接注射细胞进入小鼠的循环, 从而消除了转移级联的早期步骤。
为了克服在小鼠模型中研究转移因子的一些局限性, 我们开发了一个 bitransgenic 的鼠标模型, RIP 标签;RIP-tva4。该策略的基础是结合使用高度同步的肿瘤进展小鼠模型, RIP 标记5, 和受体的子群-禽白血病病毒, tva 6,7。此RIP 标记;RIP tva的鼠标模型允许基因被引入体细胞到一个单一的 bitransgenic 老鼠菌株。随着 SV40 T 抗原抑制 Rb 和 p53 的抑瘤作用, 小鼠的胰腺神经内分泌肿瘤与人的肿瘤形成相似, 包括增生、血管生成、腺瘤和浸润性癌。这个RIP 标签模型对我们了解癌症的特征非常有启发意义, 而不限于胰腺神经内分泌肿瘤。它也被用于临床前试验8。
我们提出了一个协议的体细胞基因转移通过注射禽病毒 intracardiacally 到RIP 标签;tva鼠标。成功的感染与 RCASBP 源性禽病毒需要积极增殖靶细胞。因此, 我们选择了RIP 标记;tva小鼠在7周的年龄, 当增生在约50% 的胰岛发育。需要高效价病毒的左心室心内注射, 以达到 10-20% 4的感染效率。这种传递方法减少病毒颗粒在传播到胰岛前的有效稀释。
使用这种方法, 我们以前已经证明, 亡促进肿瘤转移独立于其功能4,9。这种亡独立的转移功能没有观察到时, 通过转基因在所有的胰腺β细胞在整个瘤个体发育的RIP 标签;RIP-多 xL鼠标型号10。因此, 我们的鼠标模型提供了一个独特的机会, 以确定和表征基因的功能时, 表达的后期阶段的肿瘤。因为2-4% 的小岛发展成肿瘤在RIP 标签;tva bitransgenic 小鼠无病毒感染, 并非所有的前病灶都感染了 RCASBP 源性禽病毒, 只有具有选择性优势的因素才能确定肿瘤的自然病程。特别是, 转移因素将是最容易识别的这种方法, 因为转移到胰腺淋巴结或其他器官通常不会发生在RIP 标签;tva鼠标。

<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

转移性肿瘤占90% 的死亡患者的实体肿瘤。迫切需要更好地了解癌症转移的驱动因素, 并确定新的治疗靶点。为了研究推动从原发癌转移的分子事件, 我们开发了一个 bitransgenic 的小鼠模型, RIP 标签;RIP-tva。在这个老鼠模型中, 大鼠胰岛素启动子 (RIP) 驱动 SV40 T 抗原 (Tag) 和受体的表达的亚群 A 禽白血病病毒 (tva) 在胰腺β细胞。小鼠的胰腺神经内分泌肿瘤100% 显通过明确的阶段, 类似于人的肿瘤, 包括增生, 血管生成, 腺瘤, 和浸润性癌的阶段。因为RIP 标记;tva小鼠不发育转移性疾病, 促进转移的基因改变可以很容易地被识别出来。体细胞基因转移到 tva 表达, 增殖胰腺β前病变是通过心内注射的鸟类病毒窝藏期望的基因改变。#62 的效价; 1 x 108每毫升的感染单位被认为适合于体内感染。此外, 禽病毒可感染从肿瘤中提取的细胞系, 在RIP 标签;tva小鼠, 效率高。细胞系也可以用来表征转移的因素。在这里, 我们演示如何利用这种小鼠模型和细胞系来评估候选基因在肿瘤转移中的作用。

RIP 标记的体内和体外感染率;RIP tva肿瘤细胞由 RCASBP-based 病毒分别为 ~ 20% 和 ~ 80% 20。在RIP 标记中;RIP-tva鼠标模型, 大约4% 的400胰岛在每只老鼠将自然发展成肿瘤20;因此, 每只小鼠体内都有足够数量的肿瘤细胞进行组织学和表型分析, 以研究病毒所传递的基因的潜在作用。利用该系统, 确定了一种新的核功能--xL 在...

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

本文提出了一种利用 RIP 标记来演示一种新的体细胞基因转移系统的协议;tva小鼠模型研究基因在转移中的作用。禽病毒的 intracardiacally, 以确保基因转移到前, 无创性病变的胰β细胞在成年小鼠。

转移因子的鉴定和表征转移到新的RIP 标签;tva小鼠模型

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

肝大肠癌转移的高级动物模型：成像技术和转移性克隆性质

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

科研

癌症研究

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

转移性的型号同源小鼠肾细胞癌的临床前疗法的定量和纵向评估

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

疲劳的小鼠模型的外围照射引起

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

CRISPR 指南对小鼠前列腺病毒传递的基因改变

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

肿瘤免疫和非免疫微环境在小鼠皮下肿瘤中的富集与鉴定

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

骨肉瘤的三维骨细胞外基质模型

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

缺血再灌注损伤中转移性肝肿瘤的模样

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

分析肿瘤基因表达因子与CorExplorer门户网站

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

使用有机体片 3D 模型、机器学习和共声断层扫描量化脑转移肿瘤微环境

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

使用生物发光监测小鼠乳腺癌生长和转移集落形成

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