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, 其他需要直接从作者那里请求. </li>
	<li> 要过度感兴趣的基因 , 请使用下列向量之一, RCASBP (A) 12 ( 图 1A ), 会 x ( 图 1B ), 会-Y 13 ( 图 1C ), RCASBP-Y DV 14 , 或其他 ALV-一个向量 (https://home.ncifcrf.gov/hivdrp/RCAS/mice.html). </li>
	<li> 要通过 shRNA 来敲出感兴趣的基因 , 请使用下列矢量之一: 瑞 x DV 15 或会-rna 干扰 16 . </li>
	<li> 过度 rna , 生成会的向量, 如在 17 中所述。
	<li> 转换为 大肠杆菌 和 DNA 要求. </li>
	<li> 最好将 DNA 转化为 Stbl2 或 Stbl3 的合格细胞, 它们具有独特的基因型以稳定直接重复和反转录病毒序列。纯化质粒 dna 的方法, 将产生纯, 转染级 dna。5和 #181; 在最小浓度为0.1 和 #181 的情况下需要 DNA g; g/和 #181; i. </li>
</ul> 2。病毒在鸡成纤维细胞 DF1 细胞系中的传播 <ol> <li> 使用筛选过的吸管提示进行细胞培养, 以防止 cross-contaminating 病毒种群 18 。
	<li> 在 6 cm 盘中保持 DF1 细胞生长培养基 (DMEM 高糖, 10% 胎牛血清, 6 毫米 (最终浓度) l-谷氨酰胺, 10 单位/毫升青霉素, 10 和 #181; g/毫升链霉素) 在 37 o C 和 5% CO 2 。如果 DF1 细胞是从冷冻储存新鲜解冻, 培养他们至少3天前转染. </li>
	<li> 在转染前一天, 将 DF1 细胞传递到 6 cm 组织培养皿中, 使其在转染时为30-50% 汇合. </li>
	<li> 稀释5和 #181; DMEM (不含血清、青霉素或链霉素) 的纯病毒 DNA, 总体积为150和 #181; l 在组织培养罩内的离心管中. </li>
	<li> 添加30和 #181; L Superfect 直接进入稀释后的 DNA 管; 漩涡这种混合物十年代; 在室温下将混合物留在组织培养罩中5-10 分钟, 以允许转染复杂的形成. </li>
	<li> 虽然复杂的地层发生, 轻轻地从 DF1 细胞培养皿中吸取生长培养基, 并小心地用4毫升 PBS 洗涤细胞 -/-. </li>
	<li> 吸管5毫升生长培养基 (含血清和抗生素); 下一步, 将4毫升的猎鹰管保存起来; 并将其余的1毫升培养基添加到转染复合物中。由移混合两次, 并立即转移整个转染配合物 DF1 细胞。漩涡, 以确保细胞层覆盖;在 37 o C 上孵育2-3 小时. </li>
	<li> 吸入转染混合物; 用4毫升 PBS 洗涤细胞 --; 添加4毫升的新鲜生长培养基 (含血清和抗生素); 将单元格返回到 37 o C 孵化器. </li>
	<li> 当细胞到达 70-100, 传代细胞 (瞬态表达式可在转染后48到72小时). </li>
	<li> 继续传递单元格大约7天, 直到所有的细胞都被感染; 用 PCR 方法检测病毒 DNA 的整合, 用印迹法测定蛋白质表达; 用10% 亚砜和20% 胎牛血清 DMEM 培养基中的冷冻细胞. </li>
</ol> 3。 在体内 感染 RIP 标签;RIP-tva 鼠标 <ol> <li> 病毒收集和浓度 使用新的浓缩病毒进行 体内 感染。在一周内保存在 4 o C 中的病毒的效价不会显著降低。然而, 冷冻等分的病毒性股票显示10倍降低滴度后解冻。
	<ol> <li> 根据所需的病毒数量将病毒生产者 DF1 细胞扩展到 15 cm 的菜肴中。对于 在体内 鼠标感染, 请为每只鼠标准备一个平均盘. </li>
		<li> 预冷转子 (例如, SW28)、摆动桶和 ultracentrifuge 机到 4 o C. </li>
		<li> 从汇合的15厘米的菜肴中收集上清。用低速离心法在 1650 x g 处去除细胞碎片, 10 分钟, 在 4 o C. </li>
		<li> 将病毒上清液转移到 ultracentrifuge 管 (Polyallomer 离心管)。在 ultracentrifuge 中旋转1.5 小时, 95400 x g 4 o C。对于贝克曼 XL-100 ultracentrifuge 机, 使用加速: 1;Decel: 1 设置. </li>
		<li> 尽可能多地从 ultracentrifuge 管中去除上清。添加 pbs 没有钙和镁 (pbs --) 到 ultracentrifuge 管, 使最终100和 #181; l 病毒悬浮从一个 15 cm 菜。用膜盖住管子。重涡流的 ultracentrifuge 管, 在中速2分钟, 以不可见的病毒颗粒. </li>
		<li> 将 ultracentrifuge 管晃动 4 o C, 一小时至一夜. </li>
		<li> 将病毒悬浮液转移到离心管中。在注射到小鼠之前, 将病毒悬浮液加热到室温. </li>
	</ol> </li> <li> 病毒效价确定. 
	对于每一个新的病毒结构, 病毒滴度需要确定之前, 在体内 感染。
	<ol> <li> 种子 DF1 细胞在12孔组织培养板的所有井中 (建议: 2 x 10 4 细胞/井, 1 毫升/井), 使他们将是30% 汇合在感染时。一个 12-井板是需要一个病毒滴度测定. </li>
		<li> 在生长培养基上制作一系列10倍稀释的病毒上清液, 如: <ol> <li> 混合5和 #181; l 病毒上清与495和 #181; l 生长培养基为 10 2 稀释在离心管中。短暂的漩涡几秒钟. </li>
			<li> 40 和 #181; l 从 10 2 稀释为360和 #181; l 生长培养基在离心管为 10 3 稀释。短暂的漩涡几秒钟. </li>
			<li> 按照步骤 3.2.2.2 10 4 和 10 5 稀释. </li>
			<li> 设置5的6毫升聚苯乙烯管。分2.25 毫升培养基每管, 并标签他们 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 分别. </li>
			<li> 混合0.25 毫升从 10 5 稀释与2.25 毫升生长介质在6毫升聚苯乙烯管 10 6 稀释。短暂的漩涡几秒钟. </li>
			<li> 按照步骤 3.2.2.5 10 7 、10 8 、10 9 和 10 10 稀释。
			<li> 从 12-井板上的 DF1 细胞中删除原始培养基。将1毫升稀释病毒复制到每个井中 (未感染, 10 10 到 10 6 /毫升). </li>
		</ol> </li> <li> 允许单元格至少增长7天 (必要时执行 trypsinzation)。收集细胞, 以分离 DNA 和检查是否存在的病毒 dna PCR 的描述和 #34; 6。PCR 协议和 #34;如果这种病毒的效价每毫升有 10 8 感染单位 (毫升), 从 RCASBP 的阳性 398 bp PCR 产物将使用 10 8 到 10 6 的细胞 DNA, 而不是来自未感染的细胞, 或有 10 10 到 10 9 的细胞。病毒。#62 的效价; 1 x 10 8 每毫升的感染单位应用于 体内 感染. </li>
	本研究使用了 </ol> </li> <li> 心内注射 Hemizygous RIP 标记 小鼠, 而非纯合小鼠。Hemizygous RIP 标记 小鼠的胰腺神经内分泌肿瘤在10〜12周的年龄, 而纯合小鼠肿瘤潜伏期较短。因为细胞增殖是需要将病毒 cDNA 纳入宿主基因组, 7 周老 RIP 标签;tva 增生胰和 #946 小鼠; 使用细胞。请注意, 最正常的和 #946; 细胞没有增殖的成年小鼠。
	<ol> <li> 使用7周以前的 RIP 标记;RIP tva 鼠标在纯 C57BL/C 背景下进行实验. </li>
		<li> 麻醉鼠标使用氯胺酮/嗪组合 (150 毫克/千克; 15 毫克/千克)。使用热支持在整个麻醉期内保持鼠标体温. </li>
		<li> 用眼睛润滑剂来防止角膜干燥. </li>
		<li> 剃毛从胸腔的 RIP 标签;tva 鼠标。将鼠标放在背部, 胸部朝上。用脚趾夹紧观察动物的 non-responsiveness 以确认全身麻醉效果. </li>
		<li> 伸展前部肢体并用胶带固定它们。将鼠标和 #39 的标记用于注射。在胸骨切口和 xyphoid 过程的顶端之间标记一个位置, 并略微左 (解剖) 胸骨 ( 图 2 ). </li>
		<li> 用聚维酮碘磨砂擦洗前胸壁, 或用70% 异丙醇或70% 乙醇浸泡过的纱布海绵擦洗. </li>
		<li> 绘制50和 #181; 空气进入无菌28克和 #189; 胰岛素注射器在500和 #181 的柱塞和半月板之间创造空间, 然后用注射器, 然后绘制100和 #181; l 病毒。在墙上没有任何液体的空气空间对于看到心脏脉搏是至关重要的. </li>
		<li> 保持针直立。用一只手握住鼠标绷紧的皮肤, 并将针插入到标记的位置。当一个鲜红的血脉冲出现在注射器中时, 停止推进针头, 用一只手把针的深度固定在鼠标上. </li>
		<li> 使用另一只手仔细和缓慢地推动柱塞提供病毒悬浮 (〜100和 #181; l 容量) 超过60秒的时间。提供10-20 和 #181; 我每次看到一个鲜红的血脉冲出现在注射器里。在透明的针毂上连续入口红色的含氧血, 表明针正确定位到左心室. </li>
		<li> 在最后一推后, 柱塞将病毒悬浮, 在看到下一个红色脉搏之前, 迅速将针头从胸腔中取出. </li>
		<li> 在注射部位施加轻柔压力, 以减少至少1分钟的出血量. </li>
		<li> 小心地将鼠标移到加热垫或热灯下直到完全清醒。小鼠将被观察直到麻醉消退, 这样他们可以离开刺激, 一个周期通常持续大约一个小时。每天都会对小鼠进行监测, 以得到均匀、完整的外衣和任何行为上的改变. </li>
	</ol> </li> </ol> 4。肿瘤的病理组织学分析 <ol> <li> 注射后九周, 安乐16周的 RIP 标记;tva 鼠标。记录大小和数量的原发性胰腺肿瘤和任何总转移. </li>
	<li> 在一个摇摆的平台上, 在室温下过夜, 用10% 的福尔马林固定胰腺、肝脏和/或其他器官. </li>
	<li> 第二天将固定组织转移到70% 乙醇。将组织加工成石蜡嵌段. </li>
	<li> 对突 (神经内分泌标记) 或胰岛素 (a 和 #946; 细胞特异标记) 进行免疫组织化学染色, 以确定胰腺和 #946 转移的指示; 胰腺淋巴结细胞或肝脏。然而, de-differentiated 肿瘤细胞或分化不良的肿瘤细胞可能会失去胰岛素的表达, 只能通过突免疫. </li>
</ol> 5。 体外 tva 表达细胞的感染 不需要使用浓缩病毒进行 体外 感染。下面描述的是两轮感染的协议。可以通过重复以下协议来进行进一步的感染. 
<ol> <li> 从汇合的 DF1 细胞中收集病毒上清液。在一周内将病毒上清液保存在 4 o C 中以进行感染。感染前, 通过0.45 和 #956; m 过滤器, 通过病毒上清, 以获得无细胞病毒. </li>
	<li> 简要冲洗 tva 表达的靶细胞 (即 N134 胰腺和 #946; 细胞瘤细胞系从 RIP 标签;tva 鼠标) 与 PBS -/- 删除所有的血清的痕迹, 其中含有胰蛋白酶抑制剂。用胰蛋白酶-edta (如 0.25% Trypsin-2.21 毫米 edta) 溶液简单冲洗 tva 表达靶细胞。加入胰蛋白酶-EDTA 溶液, 孵育2分钟。轻轻拍打盘子。在倒置显微镜下观察细胞。细胞通常在2到3分钟的种子靶细胞中分离成6厘米的板。使用 3 ~ 4 毫升过滤病毒上清作为生长培养基. 
	注意: 病毒上清液中的营养是由 DF1 细胞消耗的, 所以第二天改变培养基. </li>
	<li> 在第二天, 将足够多的病毒上清液加热到室温。从靶细胞中取出培养基, 用3-4 毫升病毒上清液取代. </li>
	<li> 在第三天, 根据需要通过细胞。如果细胞密度不够传代热身〜4毫升的常规生长培养基 (DMEM 与高糖, 10% 胎牛血清, 6 毫米 (最终浓度) l-谷氨酰胺, 10 单位/毫升青霉素, 10 和 #181; 链霉素), 以取代病毒上清. </li>
	<li> 扩展受感染的细胞并通过西方印迹检查候选蛋白表达式 19 。transwell 分析了候选基因对迁移和侵袭的影响。这些细胞系也可以测试其转移潜能的肝脏在尾静脉检测转移. </li>
</ol> 6。PCR 协议 解决方案应尽快在冰上组装。一个 mastermix 没有模板可以为大量的样品和 aliquoted 到 PCR 管。乘以样品数量所需的试剂数量。在服用一些添加到 mastermix 之前, 先用吸管尖端的前旋和旋流来混合试剂。在将每个试剂添加到 mastermix 管后, 吸管向上和向下, 以便在提示中提供任何残留物. 
<ol> <li> 检测受感染或转染的单元格中是否存在 RCASBP. 
	以下协议要求11.5 和 #181; mastermix 和1和 #181 的 l; 每个样本的基因组 DNA 的 l。在添加每个试剂之前, 请务必先旋转。
	<ol> <li> 准备7.68 和 #181 的 mastermix; 核酸, 0.75 和 #181; 二甲基亚砜, 1.25 和 #181; l 10x 缓冲 II, 用于 AmpliTaq DNA 聚合酶, 1.375 和 #181; 25 mm 氯化镁 2 , 0.125 和 #181; 25 mm dNTP 的 l. </li>
		<li> 添加0.125 和 #181; 100 和 #181 的 l. RCAS5626F: (5 和 #39; ACCGGGGGATGCGTAGGCTTCA-3 和 #39;), 0.125 和 #181; 100 和 #181; m 反向底漆 RCAS6023R: (5 和 #39;-CCGCAACACCCACTGGCATTACC-3 和 #39;) mastermix. </li>
		<li> 添加0.075 和 #181; 我 AmpliTaq DNA 聚合酶到 mastermix. </li>
		<li> 通过用手指弹管来混合 mastermix。在离心机上旋转 matermix briefly 为 3 ~ 5 秒. </li>
		<li> 分11.5 和 #181; L 从 mastermix 到每个单独的 PCR 管。每次 aliquoting 前都要旋转到一个新的管子上. </li>
		<li> 旋流, 并添加1和 #181; L 基因组 DNA 模板到每个 PCR 管。准备基因组 DNA: <ol> <li> 200 和 #181 中的溶解细胞颗粒; 0.05 米氢氧化钠的 l。吸管向上和向下溶解颗粒. </li>
			<li> 在 PCR 机中加热98和 #176; c 为20分钟, 然后冷却到25和 #176; c. </li>
			<li> 添加20和 #181; L 1.0 M 三盐酸 (pH 7.5) 到管, 并存储在4和 #176 的 DNA 样本; C. </li>
		</ol> </li> <li> 通过轻弹和离心机将样品混合3-5 秒。将管子放入 PCR 机. </li>
		<li> PCR 条件设置为2分钟, 在92和 #176; c 为最初的变性, 其次40周期三十年代在94和 #176; c 为变性, 三十年代在67和 #176; c 为退火, 三十年代在72和 #176; c 为延长, 并且 10 m 在72和 #176; c 为最后的扩展. </li>
		<li> 在 PCR 完成后, 添加3和 #181; 对每个样品进行 6x DNA 加载染色。混合通过轻弹和离心机短暂3〜5秒. </li>
		<li> 2% (w/v) 琼脂糖凝胶电泳检测1x 泰缓冲液中的 PCR 产物。其 PCR 产物的大小为 398 bp. </li>
	</ol> </li> <li> 标记基因分型. 
	以下协议要求10.5 和 #181; mastermix 和2和 #181 的 l; 每个样本的基因组 DNA 的 l。在添加每个试剂之前, 请务必先旋转。
	<ol> <li> 准备 mastermix 4.5 和 #181; 核酸的水和4.00 和 #181; l 5x MyTaq 反应缓冲 MyTaq DNA 聚合酶. </li>
		<li> 添加0.4 和 #181; 100 和 #181 的 l 向前引 TagF2: (5 和 #39; GGACAAACCACAACTAGAATGCAGTG-3 和 #39;) 和0.4 和 #181; 100 和 #181; m 反向底漆 TagR2: (5 和 #39;-CAGAGCAGAATTGTGGAGTGG-3 和 #39;) 主组合. </li>
		<li> 添加0.8 和 #181; 10 和 #181 的 L beta-2-microglobulin 前体 (B2m) 底漆混合为内部控制。B2m 的 PCR 引物的内部控制, beta-2-microglobulin 前体 (B2m), 是 B2 F (5 和 #39; CACCGGAGAATGGGAAGCCGAA-3 和 #39;) 和 B2 R (5 和 #39; TCCACACAGATGGAGCGTCCAG-3 和 #39;). </li>
		<li> 添加0.4 和 #181; 我 MyTaq DNA 聚合酶到 mastermix. </li>
		<li> 通过用手指弹管来混合 mastermix。在离心机上旋转 mastermix 3 ~ 5 秒. </li>
		<li> 分10.5 和 #181; L 从 mastermix 到每个单独的 PCR 管。每次 aliquoting 前都要旋转到一个新的管子上. </li>
		<li> 旋流, 并添加2和 #181; L 基因组 DNA 模板到每个 PCR 管。基因组 DNA 的制备如上所述. </li>
		<li> 通过轻弹和离心机混合解决方案3-5 秒。将管子放入 PCR 机. </li>
		<li> PCR 条件设置为3分钟, 在95和 #176; c 为最初的变性, 其次35周期十五年代在95和 #176; c 为变性, 十五年代在65和 #176; c 为退火, 三十年代在72和 #176; c 为延长, 并且 10 min 在72和 #176; c 为最后的扩展. </li>
		<li> 在 PCR 完成后, 将3和 #181 添加到每个样品的6x 加载染料中。混合通过轻弹和离心机短暂3〜5秒. </li>
		<li> 2% (w/v) 琼脂糖凝胶在1x 泰缓冲液中检测 PCR 产物。标签和 B2m PCR 产品的大小分别为 450 bp 和 300 bp. </li>
	</ol> </li> <li> tva 基因分型. 
	下面的协议要求10.5 和 #181, 每个样本的试剂混合的 L。在添加每个试剂之前, 请务必先旋转。
	<ol> <li> 准备5.6 和 #181 的 mastermix; 核酸免费水, 0.5 和 #181; 二甲基亚砜, 1.25 和 #181; l 10x 缓冲 II 为 AmpliTaq DNA 聚合酶, 和1.375 和 #181; l 25 毫米氯化镁 2 , 0.125 和 #181; 25 mm dNTP. </li>
		<li> 添加0.125 和 #181; 100 和 #181 的 l tva-3 (5 和 #39; GCCCTGGGGAAGGTCCTGCCC-3 和 #39;), 0.125 和 #181; 100 和 #181; m tva-5 (5 和 #39; CTGCTGCCCGGTAACGTGACCGG-3 和 #39;) mastermix. </li>
		<li> 添加1.275 和 #181; L 10 和 #181; M B2m 底漆混合为内部控制对 mastermix. </li>
		<li> 添加0.125 和 #181; 我 AmpliTaq DNA 聚合酶到 mastermix. </li>
		<li> 通过用手指弹管来混合 mastermix。在离心机中旋转 mastermix 3-5 秒. </li>
		<li> 分10.5 和 #181; L 从 mastermix 到每个单独的 PCR 管。每次 aliquoting 前都要旋转到一个新的管子上. </li>
		<li> 旋流, 并添加2和 #181; L 基因组 DNA 模板到每个 PCR 管。基因组 DNA 的制备如上所述. </li>
		<li> 将解决方案通过轻弹和离心机混合使用3-5 秒将管子放入 PCR 机. </li>
		<li> PCR 条件设置为2分钟, 在92和 #176; c 为最初的变性, 跟随35周期三十年代在94和 #176; c 为变性, 三十年代在60和 #176; c 为退火, 30 秒在72和 #176; c 为延长, 10 分钟在72和 #176; c 为最后的扩展. </li>
		<li> 在 PCR 完成后, 将3和 #181 添加到每个样品的6x 加载染料中。混合通过轻弹和离心机简要地为 3-5 s. </li>
		<li> 2% (w/v) 琼脂糖凝胶电泳检测1x 泰缓冲液中的 PCR 产物。tva 和 B2m PCR 产物的大小分别为 500 bp 和 300 bp. </li>
	</ol>
	</li>
</ol>

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作者没有什么可透露的。

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><tbody><tr><td>RCASBP-Y DV plasmid</td><td>Addgene</td><td>11478</td><td></td></tr><tr><td>RCAS-RNAi plasmid</td><td>Addgene</td><td>15182</td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>fetal bovine serum</td><td>Atlanta Biologicals</td><td>25-005-CI</td><td></td></tr><tr><td>L-glutamine, 100x</td><td>Corning</td><td>25-005-CI</td><td></td></tr><tr><td>Penicillin-Streptomycin solution, 100x</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>PBS&lt;sup&gt;-/-&lt;/sup&gt;, 1x</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>Superfect</td><td>Qiagen</td><td>301305</td><td></td></tr><tr><td>Polyallomer centrifuge tube</td><td>Beckman Coulter</td><td>326823</td><td></td></tr><tr><td>0.45 mm Nalgene&lt;br/&gt; Syringe Filters with PES Membrane</td><td>Thermo Scientific</td><td>194-2545</td><td></td></tr><tr><td>Insulin Syringes</td><td>BD</td><td>329461</td><td></td></tr><tr><td>synaptophysin</td><td>Vector Laboratories</td><td>VP-S284</td><td></td></tr><tr><td>VECTASTAIN Elite ABC HRP Kit (Peroxidase, Rabbit IgG)</td><td>Vector Laboratories</td><td>PK-6101</td><td></td></tr><tr><td>AmpliTaq DNA Polymerase with Buffer II</td><td>Life Technologies</td><td>N8080153</td><td></td></tr><tr><td>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 ...

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

8.7K 次观看.  Weill Cornell Medicine. 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。

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

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

核素荧光报告基因成像在啮齿动物肿瘤模型中跟踪肿瘤进展

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

科研

癌症研究

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

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

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 ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

使用快速和定量的细胞内胆碱膜模型发现转移调节器

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

人类前列腺癌转移的原位小鼠模型

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

医学

A Robust Discovery Platform for the Identification of Novel Mediators of Melanoma Metastasis

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was established using robust, reproducible in vivo models and standardized methods to identify novel players in melanoma metastasis. This approach allows for data inference at specific experimental stages to precisely characterize a gene's role in metastasis. Models are established by introducing genetically modified melanoma cells via intracardiac, intradermal, or subcutaneous injections into mice, followed by monitoring with serial in vivo imaging. Once preestablished endpoints are reached, primary tumors and/or metastases-bearing organs are harvested and processed for various analyses. Tumor cells can be sorted and subjected to any of several 'omics' platforms, including single-cell RNA sequencing. Organs undergo imaging and immunohistopathological analyses to quantify the overall burden of metastases and map their specific anatomic location. This optimized pipeline, including standardized protocols for engraftment, monitoring, tissue harvesting, processing, and analysis, can be adopted for patient-derived, short-term cultures and established human and murine cell lines of various solid cancer types.

This article describes a workflow of techniques employed for testing novel candidate mediators of melanoma metastasis and their mechanism(s) of action.

用于鉴定黑色素瘤转移新介质的强大发现平台

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

乳腺癌的一种新的转基因小鼠模型：转移性乳腺癌通过瞄准导管上皮与腺病毒Cre重组酶的启动

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

体外肺转移及微环境的实时成像

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer metastasis. The 4T1 cells expressing GFP in combination with imaging and confocal microscopy are used to monitor tumor growth, track metastasizing tumor cells, and quantify the metastatic burden. These approaches are supplemented by digital histopathology that allows the automated and unbiased quantification of metastases. In this method the routinely prepared histological lung sections, which are stained with hematoxylin and eosin, are scanned and converted to the digital slides that are then analyzed by the self-trained pattern recognition software. In addition, we describe the flow cytometry approaches with the use of multiple cell surface markers to identify alveolar macrophages in the lungs. To determine impact of alveolar macrophages on metastases and antitumor immunity these cells are depleted with the clodronate-containing liposomes administrated intranasally to tumor-bearing mice. This approach leads to the specific and efficient depletion of this cell population as confirmed by flow cytometry. Tumor volumes and lung metastases are evaluated in mice depleted of alveolar macrophages, to determine the role of these cells in the metastatic progression of breast cancer.

Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used.

研究肺泡巨噬细胞的作用乳腺癌转移

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer ...