Name: utilizing murine inducible telomerase alleles in the studies of tissue degeneration/regeneration and cancer
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Description: Watch this Scientific Journal Video about Utilizing Murine Inducible Telomerase Alleles in the Studies of Tissue Degeneration/Regeneration and Cancer at JoVE.com

J.H. is supported by NIH K99/R00 Pathway to Independence Award (5K99CA172700) and NCI Brain Cancer SPORE Career Development Award (2P50CA127001).

注:所有在这个协议的步骤已获得UT MD安德森癌症中心。 1.代mTERT-ER等位基因<ol><li>引入敲入靶向含有ERT2-LBD（配体结合域）的上游和在帧与mTERT基因组序列（外显子1至内含子2）和液氧矢量- PGK - 新- LOX片段（ 图1A）到小鼠ES细胞有电。 </li><li>培养胚胎干细胞的新霉素为6-10天。挑选新霉素抗性克隆并扩大在48孔板。提取使用商业试剂盒的基因组DNA并确认敲入等位基因通过Southern印迹。 </li><li>注入有超过95％的正常核型的ES 2行到C57BL / 6胚泡用显微操作盒倒置显微镜下。植入的胚泡到代孕母亲23的子宫。队友高档男性嵌合体（70-90％），以C57BL / 6 FE男性。 </li><li>确认杂mTERT-ERneo动物Southern杂交的基因分型。 </li><li>交配的杂合mTERT-ERneo动物和EIIa-Cre重组动物删除NeoR磁带。交配的杂合动物C57BL / 6动物为至少3倍，并进一步相互品种杂mTERT-ER动物，以产生纯合性。 </li></ol> 2.代LSL-mTERT等位基因<ol><li>引入敲入靶向载体含有LoxP位 - 三重塞 - 新 - LoxP位片段和mTERT基因组序列（外显子1和内含子2， 如图1B之间）导入小鼠ES细胞与电穿孔23。 </li><li>培养胚胎干细胞的新霉素为6-10天。挑选新霉素抗性克隆并扩大在48孔板。提取使用商业试剂盒的基因组DNA，并通过Southern印迹23确认敲入等位基因。 </li><li>仁济克拉2 ES细胞系以超过95％的正常核型到C57BL / 6胚泡用倒置显微镜下一个显微操作工具包。植入的胚泡到代孕母亲23的子宫。队友高档男性嵌合体（70-90％），以C57BL / 6雌性。 </li><li>确认杂LSL-mTERT动物Southern杂交的基因分型。 </li><li>交配的杂合动物C57BL / 6动物为至少3倍，并进一步相互品种杂LSL-mTERT动物，以产生纯合性。 </li></ol>在mTERT-ER和LSL-mTERT实验鼠体内 3.重新激活端粒酶对于mTERT-ER <ol><li>消毒所有的注射前的外科手术工具。 </li><li>麻醉小鼠用异氟烷腔（用于诱导4％，维持2％）在50％（体积/体积）氧气/ 50％（V / V）一氧化二氮的气体混合物。或者麻醉小鼠氯胺酮，甲苯噻嗪的混合物（100米克/千克体重+ 10毫克/公斤体重）。 </li><li>后达到深度麻醉，取出从感应腔麻醉动物，并保持其头部连接到所述腔室的异氟烷（2％），在管内。 </li><li>捏小鼠的脚垫，以确保动物深度麻醉。把软膏上双眼，以防止眼睛干燥。擦拭小鼠背部用聚维酮碘溶液。 </li><li>注入缓慢释放的4-OHT粒料用精密套管针（10 G）的背部皮肤下和一路推粒料向中线两肩之间。 </li><li>封切口的伤口夹器和监控小鼠从麻醉中恢复。 10天后取下夹子。因为该过程完成后不久补充热量是不必要的。 </li><li>用4-OHT丸治疗期间应根据研究的目的进行优化。 </li></ol>对于LSL-mTERT &lt;OL&gt; <li>他莫昔芬（10毫克/毫升，溶解在玉米油中）（200微升/ 25克/天）的腹膜内注射连续两天。 </li></ol> 4.隔离神经干细胞和活化端粒酶体外 <ol><li>小鼠安乐死与二氧化碳和大脑被除去。按照市售的神经组织解离试剂盒根据制造商的协议。 </li><li>重悬在标解决方案4用1ml细胞培养基用于解4.小瓶冻干粉末不要旋涡。该溶液然后应等分并储存在-20℃以备后用。 </li><li>预加热，在37℃下在使用前15分钟，该混合物。 </li><li>使酶混合物1:方案1（50微升）和解决方案2（1900微升）。使酶混合物2:解决方案3（20微升）和解决方案4（10微升）。 </li><li>准备1,950微升酶混合物为1至400毫克的组织和漩涡。在37℃下预加热该混合物在使用前15分钟。 </li><li>取出1天小鼠的大脑，并保持前脑通过除去嗅球和小脑。保持前脑组织在1ml冷的培养基中，因为去除并存储在4℃。确保在1小时内处理的神经组织。 </li><li>确定组织的重量，以确保不超过每消化400毫克的限​​制。放置在35毫米直径的培养皿的盖子的大脑，并使用手术刀粉碎大脑。 </li><li>用1毫升移液管尖，加入1 ml的HBSS（W / O型的Ca / Mg）的和吸管件回入15ml管中。冲洗用HBSS（W / O型钙/镁）。离心机在300×g下在室温下2分钟，并小心地吸出上清液。 </li><li>添加1,950微升预先加热酶混合物1（溶液1和2）每达到400 mg组织。孵育在15ml试管15分钟，在37℃，通过反转或摇动该管每隔5分钟进行混合。 </li><li>每次准备组织样本30微升酶混合物2中加入20＃956，L的溶液3至10微升解决方案4.再放入样品。反转轻轻混合。不要旋涡。 </li><li>机械地使用宽嘴，火抛光的巴斯德吸移管通过移液器上下吹吸10次缓慢离解的组织。避免形成气泡。 </li><li>孵育在37℃进行10分钟，将试管倒数每3分钟。 </li><li>重复步骤4.11和4.12步骤，如果组织均大于200毫克。 </li><li>应用该细胞悬液，以70微米的细胞过滤网，放置在50毫升的试管中。通过细胞过滤应用10毫升PBS中。丢弃细胞过滤及离心分离细胞悬浮液，在300克20分钟，在室温下进行。吸上清彻底。 </li><li>重悬细胞与干细胞培养基，以供进一步的应用所需的体积。 </li><li>以激活端粒酶在LSL-mTERT神经干细胞，治疗的细胞与100μM的4-OHT两天。要激活端粒酶mTERT-ER的神经干细胞，保持细胞的培养液中有100μM4-OHT。 </li></ol> 5.端粒FISH <ol><li>准备从培养细胞的中期染色体。 </li><li>对FFPE（福尔马林固定石蜡包埋）组织切片，deparaffinize在二甲苯和再水合的乙醇系列，每次5分钟（100％，90％，70％，50％的乙醇）和PBS 5分钟。 </li><li>定位后，在甲醇:乙酸（3:1）为1-2小时。脱水在冷的乙醇系列，每次5分钟，（70％，90％，100％乙醇），并空气干燥。在1×PBS中，在37℃下进行5分钟，洗净。 </li><li>变性在4％甲醛的染色体，在37℃2分钟。脱水在冷的乙醇系列中各5分钟，（70％，90％，100％乙醇），并空气干燥。 </li><li>适用于12至25微升的PNA杂交混合，以每张幻灯片。 （杂交组合:70％甲酰胺，0.06X SSC，0.2％BSA，0.5纳克/微升的tRNA，0.5纳克/微升端粒或着丝粒探针） </li><li>密封盖玻片用橡皮泥。后变性染色体棉片和组织切片，在80℃下4米英寸杂交在室温或37℃下2-4小时，在潮湿的腔室中。 </li><li>在室温下，用洗涤缓冲液2×15分钟，洗净。 （洗涤液:70％甲酰胺，0.06X SSC，pH值7.2）。在室温下3×5分钟，用PBST洗。反染色幻灯片与DAPI或远红色荧光镜检。 </li></ol>

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The authors have nothing to disclose.

这里，我们报告两个诱导鼠端粒酶等位基因和如何重新激活端粒酶在体内和体外的产生。重新激活mTERT-ER活性的关键步骤是让他莫昔芬的连续浓度，所以我们选择使用由美国创新研究制造的4-OHT时间释放颗粒。粒料保持释放4OHT在1纳克/毫升的稳态血液水平长达60天。在先前的研究中，我们发现，重新激活端粒酶活性，这一战略能够改善退行性表型，如造血干细胞衰竭，端粒功能障碍G4 mTERT ER / ER诱导的组织损伤反应和器官功能衰竭多器官功能障碍老鼠20。 除了研究端粒酶在老化的功能，这两个可诱导的端粒酶等位基因也可用于研究癌症。以往的研究了利用这些等位基因探索在塑造基因组和冲击的T细胞胸腺淋巴瘤21和前列腺癌细胞22的生物学端粒耗损和端粒酶活化的作用。这两项研究表明，在后期一代G4 大气压端粒功能障碍- / - mTERT ER / ER和G4 PB-Cre4 PTEN的L / L p53的L / L LSL-mTERT L / L小鼠提供了一种机制，助长了收购早期突变事件肿瘤发生，但防止完全恶性肿瘤进展。当端粒酶被重新激活的端粒功能紊乱诱发的基因组不稳定性，DNA损伤检查点和猖獗染色体的不稳定的情况下被抑制，这使恶性肿瘤的新的生物特性，如前列腺癌21的骨转移和胸腺的脑浸润的充分进展Ç淋巴瘤21。类似的现象也观察到在其他类型的癌症，包括结肠癌和胰腺癌（含博士。豪强莹，亚当布廷和Ronald DePinho个人通信）。 因为叔ER蛋白可开和关状态来容易地切换这取决于是否将动物用他莫昔芬或载体处理，由mTERT-ER等位基因产生的肿瘤模型可以被用来测试抗端粒酶疗法。在先前的研究中，在G4 大气压产生的肿瘤- / - mTERT ER / ER动物从4-OHT释放;在端粒酶枯竭，肿瘤最终由于端粒功能障碍引起的检查站21复职萎缩。然而，一些针对获得性耐药的肿瘤通过激活端粒（ALT）机制的替代纤长端粒酶灭绝。这些ALT +抗肿瘤的进一步鉴定表明吨哎有参与线粒体生物学和氧化防御，包括线粒体合成和氧化防御PGC-1β的主控调节基因的异常转录。 PGC-1β或SOD2（氧化防御的另一个调节器）的击倒显著消除ALT +肿瘤细胞的同时保持端粒酶+肿瘤细胞保持相对完好21。 这两个敲入等位基因的一个限制是，它们只能承受端粒酶的活化在内源性水平，因为它们是在端粒酶基因的天然基因座。在大多数人类癌症，端粒酶实际上是过表达在高得多的水平。为了提高端粒酶的水平，我们可以考虑一个修饰是插入mTERT-ER构建体具有更强的启动子，如PGK（磷酸甘油酸激酶）启动子插入ROSA26基因座。 总之，这两个新的可诱导的鼠端粒酶等位基因提供了前所未有的遗传学IC工具来研究端粒酶的功能的老化和组织稳态以及肿瘤进展，尤其是在预采集端粒功能紊乱诱发的基因组不稳定性的背景。所述mTERT-ER等位基因也可用于通过杂交到其它肿瘤容易发生小鼠模型来研究抗端粒酶疗法。

端粒酶是负责维持端粒的酶。这些都是在染色体的保护其完整性的目的重复序列。端粒酶全酶的核心部件是一个逆转录酶催化亚基（TERT）和RNA亚基（TERC）用作模板用于添加端粒重复1,2。而通常压制在分化的体细胞中，端粒酶表现出强大的活性和在生殖细胞，癌细胞中起重要作用和干细胞。 mTERC和mTERT敲除小鼠提供极大的体内模型系统以研究端粒酶的功能在两个干细胞和癌症。该mTERC和mTERT基因敲除小鼠（G1）没有表现出应有的端粒小鼠3长期储备的任何明显的表型。然而，连续互交mTERC和mTERT基因敲除小鼠晚代（G5-G6）导致端粒的逐步侵蚀，并最终引发了高度增生组织4严重退化。晚代（G5-G6）mTERC - / -小鼠是不育的，由于细胞凋亡的睾丸和生殖细胞耗竭的高比率。 G5-G6 mTERC - / -小鼠还表现出在高度增殖的组织包括骨髓，肠和皮肤退行性表型是由于细胞凋亡和有缺陷的自我更新能力的组织干细胞/祖细胞区室4高比率。造血干细胞在较晚代的骨髓mTERC - / -小鼠表现出损耗增殖潜能的，受损的自我更新能力，增强的细胞凋亡，并最终官能用尽5,6。同样地，逐步增加凋亡小体中肠腺，观察在每一代的G1-G6 mTERC - / -小鼠7,8。牛逼他功能失调端粒的有害作用似乎并不限于自成人神经干细胞的在体外的增殖，并在体内的神经发生高周转组织中严重受损晚代（G4-G5）mTERC - / -小鼠9 。端粒酶在干细胞中的作用是通过在一个罕见的人类遗传病症先天性角化不良（DKC），它在某些方面类似于过早老化10,11的结果进一步支持。 DKC由突变TERC，TERT，以及基因编码dyskerin，一个核心端粒酶亚基，和其它dyskerin相关蛋白12引起的。在平均，在DKC患者端粒是短于99％的年龄匹配的对照。该疾病的发病主要是由于再生障碍性贫血，这是造成有缺陷的维护的造血干细胞。该疾病的其他方面，例如口腔白斑，指甲营养不良，精神retardati上，睾丸萎缩和肺部并发症所有建议在晚代端粒酶基因敲除小鼠的受损的组织干细胞和祖细胞10，在与这些紧密协议的发现的功能。 DKC患者易发生骨髓增生异常综合征，并有恶性黏膜肿瘤的发生率增加。因此，端粒酶在人类患者中缺乏概括干细胞功能和肿瘤的倾向在后期产生端粒酶基因敲除小鼠中观察到的缺陷。 短端粒和高端粒酶活性的癌症标志。为正常或癌前细胞分裂时，低或无端粒酶活性导致端粒和激活蜂窝关卡类似于通过DNA双链-断裂（DNA双链断裂）13挑起的最终腐蚀。像古典双链断裂，端粒功能紊乱已显示诱导p53和相关的细胞反应，如衰老和/或凋亡作用Š14,15。当p53基因突变的失活，细胞周期和细胞存活是增强细胞的端粒功能障碍，它提供特点是易位和区域扩增和缺失16,17亲致癌突变的机制。与此同时，继续端粒功能障碍和相关猖獗的染色体不稳定性（即使在无p53细胞）似乎限制此类癌症的充分恶性进展。例如，晚代G4-G5 mTERC - / - INK4A / Arf的- / -小鼠显示出显著降低淋巴瘤的发病率与INK4A / Arf的无效小鼠由于端粒耗损进行比较。在另一项研究中，更先进的腺瘤性病变的G4 mTERT - / - APC 分钟小鼠大大由于显著生长停滞和凋亡抑制18,19与APC 分钟的小鼠的比较。该观察发现，端粒功能紊乱诱发的基因组不稳定性抑制肿瘤进展提示猜测端粒酶的活化可通过平息基因组不稳定性与癌症的细胞活力和/或中和p53依赖性或非依赖性的检查点机制兼容的电平使恶性进展中的一部分。 为了研究端粒酶活化是否可以停止甚至逆转干细胞的衰老，而端粒酶活化是否能促进对基因组不稳定的背景下，肿瘤发生，我们产生了两个诱导小鼠端粒酶等位基因。第一个是4-羟基（4-OHT）诱导性叔雌激素受体 ​​（mTERT-ER）的融合敲入等位基因。在不存在的4-OHT，小鼠纯合mTERT-ER（指定mTERT ER / ER）是端粒酶活性缺陷和维持相同的细胞遗传学和细胞表型的常规mTERT或米TERC基因敲除模式。当4-OHT处理，叔ER蛋白活性可以恢复到相媲美的天然TERT蛋白20，21的水平。第二 ​​等位基因是一种新颖的诱导TERT敲入含有一个内含子的loxP的阻流器- LoxP位盒等位基因（LSL- mTERT）;在Cre重组酶介导的LSL切除，mTERT重新表达下的内源性表达调控机制22。

<table><tbody><tr><td>Leica AM6000 Micromanipulation Kit</td><td>Leica</td><td>Per quote</td><td></td></tr><tr><td>Leica DMI6000 B inverted microscope</td><td>Leica</td><td>Per quote</td><td></td></tr><tr><td>Neomycin</td><td>Life technologies</td><td>21810031</td><td></td></tr><tr><td>isoflurane vaporizer</td><td>Veterinary Anesthesia Systems</td><td>911103</td><td></td></tr><tr><td>isoflurane</td><td>Henry Schein</td><td>1005796</td><td></td></tr><tr><td>ketamine-xylazine mixture</td><td>Sigma-Aldrich</td><td>K113-10ML</td><td></td></tr><tr><td>4-OHT powder</td><td>Sigma-Aldrich</td><td>H7904-5MG</td><td></td></tr><tr><td>Tamoxfien</td><td>Sigma-Aldrich</td><td>T5648-1G</td><td></td></tr><tr><td>4-OHT pellet</td><td>Innovative Research of America</td><td>Custermized</td><td></td></tr><tr><td>Precision Trochar (10 Gauge)</td><td>Innovative Research of America</td><td>MP-182</td><td></td></tr><tr><td>wound clip applier</td><td>Fischer Scientific</td><td>01-804</td><td></td></tr><tr><td>clip</td><td>Fischer Scientific</td><td>01-804-5</td><td></td></tr><tr><td>Neural Tissue Dissociation Kit</td><td>Miltenyi Biotec</td><td>130-092-628</td><td></td></tr><tr><td>HBSS</td><td>Life technologies</td><td>14025092</td><td></td></tr><tr><td>PBS</td><td>Life technologies</td><td>10010023</td><td></td></tr><tr><td>xylene</td><td>Fischer Scientific</td><td>X3P-1GAL</td><td></td></tr><tr><td>methanol</td><td>Fischer Scientific</td><td>A-433S4</td><td></td></tr><tr><td>acetic acid</td><td>Fischer Scientific</td><td>A38-500</td><td></td></tr><tr><td>ethanol</td><td>Fischer Scientific</td><td>22-032-104</td><td></td></tr><tr><td>formamide</td><td>Fischer Scientific</td><td>F84-1</td><td></td></tr><tr><td>PNA telomere probe</td><td>Panagene</td><td>F1001-5</td><td></td></tr><tr><td>PNA centromere probe</td><td>Panagene</td><td>F3003-5</td><td></td></tr><tr><td>20X SSC</td><td>Fischer Scientific</td><td>BP1325-4</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A2153-10G</td><td></td></tr><tr><td>tRNA</td><td>Sigma-Aldrich</td><td>R5636-1ML</td><td></td></tr><tr><td>Tween 20</td><td>Fischer Scientific</td><td>BP337-100</td><td></td></tr><tr><td>rubber cement</td><td>Walmart</td><td>Elmer&#039;s</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td></td></tr></tbody></table>

utilizing murine inducible telomerase alleles in the studies of tissue degeneration/regeneration and cancer

端粒功能障碍引起的基因组的完整性及其相关DNA损伤信号和检查点反应的损失设立了完善的驱动程序引起的老化过程中组织变性。癌症，与发病率随着年龄的增长大大增加了，其特点是短的端粒长度和高端粒酶活性。为了研究衰老和癌症的端粒功能障碍和重新激活端粒酶的作用，该协议显示了如何生成两个小鼠诱导端粒酶敲入等位基因4-羟基（4-OHT）诱导型叔雌激素受体 ​​（mTERT-ER）和液氧-Stopper-LOX TERT（LSL-mTERT）。本协议描述的过程诱导端粒功能障碍和重新激活mTERT-ER和LSL-mTERT小鼠端粒酶活性在体内 。代表性数据显示，端粒酶活性的再活化可以改善诱导端粒功能障碍的组织退行性表型。在奥德呃以确定肿瘤端粒酶活化的影响，我们产生前列腺肿瘤模型的G4 的PB-Cre4的Pten L / L p53的L / L LSL-mTERT L / L和胸腺T细胞淋巴瘤模型G4 大气压- / - mTERT ER / ER 。代表性的数据显示，基因组不稳定性通过端粒功能障碍引起的可以大大提高肿瘤发生的背景下，端粒酶激活。该协议还描述了用于分离的神经干细胞（NSCs）从mTERT-ER和LSL-mTERT小鼠和激活端粒酶活性的神经干细胞在体外的过程。代表性数据显示，端粒酶的活化可以提高在体外的自我更新能力和神经发生。最后，该协议描述的程序在两个鼠标FFPE进行端粒FISH（荧光原位杂交）（福尔马林固定石蜡和鳍嵌入式）脑组织和培养细胞的中期染色体。

该策略产生鼠mTERT-ER和LSL-mTERT敲入等位基因图1A和1B描述。具体来说，LoxP位 - 三重塞 - 新 - LoxP位片段插入外显子1和mTERT基因外显子2之间产生LSL-mTERT等位基因。以产生mTERT-ER等位基因，在帧与mTERT基因ERT2-LBD结构域插入外显子1的N-末端，我们表明，当端粒酶通过处理它们与4-OHT粒料4瞬时激活端粒障碍小鼠周，退行性表型可以改善在多器官如脑（ 图2A）和睾丸（ 图2B）。为了确定端粒酶活化对细胞在体外培养的影响，我们分离的神经干细胞（NSCs）从晚代G4 LSL-mTERT和G4 mTERT-ER小鼠。我们发现，当端粒酶被重新激活端粒功能失调的神经干细胞，神经干细胞的自我更新能力被大大增加（ 图3A），并在体外神经发生也显著增强（ 图3B）。为了确定端粒酶活化对肿瘤发生中的端粒功能紊乱的上下文的影响，我们产生的PB-Cre4的Pten L / L p53的L / L LSL-mTERT L / L（前列腺肿瘤模型） 和 ATM - / - mTERT ER / ER（胸腺T细胞淋巴瘤模型）晚代同伙。当端粒酶被重新激活通过他莫昔芬治疗在这些小鼠注射（ 图4A）或4-OHT粒料连续8周（ 图4B），所述肿瘤发生在两个前列腺肿瘤模型（ 图4A）和胸腺T细胞淋巴瘤模型大大增强（ <strong&gt;的图4B）。最后，我们报告了FFPE小鼠脑组织进行FISH端粒的协议（ 图5）和中期染色体（ 图5，小图）。 <img alt="图1" src="/files/ftp_upload/52599/52599fig1.jpg" /> 图1:（A）mTERT-ER敲入策略。电视，靶向载体; WT，野生型等位基因; KI，敲入等位基因; LA，左臂; RA，右臂; E1，外显子1; E2，外显​​子2;新，PGK启动子驱动的新霉素抗性基因; ER，改性的雌激素受体（ERT2）配体结合域; DT，dyphteria毒素基因。 （B）LSL-mTERT敲入策略。电视，靶向载体; WT，野生型等位基因; KI，敲入等位基因; LA，左臂; RA，右臂; E1，外显子1; E2，外显​​子2;新，PGK启动子驱动的新霉素抗性基因;塞，三重复转录终止序列; DT，dyphteria毒素基因。 <p类="jove_content"FO:保持together.within页="总是"&gt; <img alt="图2" src="/files/ftp_upload/52599/52599fig2.jpg" /> 图2:代表性数据显示端粒酶活化可改善器官的退行性表型的大脑的代表性图像（左）。 （右）与安慰剂或4-OHT 4周治疗的G0和G4 mTERT-ER小鼠的睾丸和。刻度条表示50μm左右。重新打印许可20。 <a href="https://www.jove.com/files/ftp_upload/52599/52599fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52599/52599fig3.jpg" /> 图3:代表性数据显示端粒酶活化增强自我更新和神经干细胞在体外的神经发生 （A） 的神经干细胞的代表性图像■从G4 LSL-mTERT隔离（上图） 和G4 mTERT-ER（下图）生长在用媒介物或4-OHT处理干细胞培养基。 （B）的神经干细胞，从G4 mTERT-ER在分化培养基中培养分离的体外神经发生（TUJ1 +）的代表性图像（有1％FBS）处理用媒介物或4-OHT。刻度条表示100μm左右。 <img alt="图4" src="/files/ftp_upload/52599/52599fig4.jpg" /> 图4:代表性数据显示端粒酶活化增强肿瘤对基因组不稳定性的背景下（A）中由G0 PB-Cre4的Pten L / L p53的L / L LSL-mTERT解剖前列腺肿瘤的代表性图像+/-，G4 PB- Cre4 PTEN的L / L p53的L / L LSL-mTERT- / - ，和G4 PB-Cre4 PTEN的L / L p53的L / L LSL-mTERT L / L他莫昔芬治疗。经许可后22重新打印。 （B）的由G0 大气压解剖胸腺T细胞淋巴瘤代表性图像- / - mTERT + / ER，G4大气压- / - mTERT ER / ER治疗用载体，和G4 大气压- / - mTERT ER / ER治疗4- OHT 8周。刻度条表示1厘米。 <img alt="图5" src="/files/ftp_upload/52599/52599fig5.jpg" /> 图5:端粒和着丝粒FISH对FFPE小鼠脑组织和中期（插图）代表图像 。红色表示DNA，绿色表示端粒染色，青色表示CEntromere染色。刻度条表示1毫米。

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Utilizing Murine Inducible Telomerase Alleles in the Studies of Tissue Degeneration/Regeneration and Cancer

Telomere and telomerase play essential roles in ageing and tumorigenesis. The goal of this protocol is to show how to generate two murine inducible telomerase knock-in alleles and how to utilize them in the studies of tissue degeneration/regeneration and cancer.

在组织变性/再生和癌症的研究利用小鼠诱导端粒酶等位基因

Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that ...

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10.2K 次观看.  UT MD Anderson Cancer Center. Telomere and telomerase play essential roles in ageing and tumorigenesis. The goal of this protocol is to show how to generate two murine inducible telomerase knock-in alleles and how to utilize them in the studies of tissue degeneration/regeneration and cancer. 

视频: Utilizing Murine Inducible Telomerase Alleles in the Studies of Tissue Degeneration/Regeneration and Cancer

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.

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.

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 ...

科研

癌症研究

Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. Recent findings revealed that TERRA molecules also interact with internal chromosomal regions to regulate gene expression in mouse embryonic stem (ES) cells. In line with this evidence, RNA fluorescence in situ hybridization (RNA-FISH) analyses have shown that only a subset of TERRA transcripts localize at chromosome ends. A better understanding of the dynamics of TERRA molecules will help define their function and mechanisms of action. Here, we describe a method to label and visualize single-telomere TERRA transcripts in cancer cells using the MS2-GFP system. To this aim, we present a protocol to generate stable clones, using the AGS human stomach cancer cell line, containing MS2 sequences integrated at a single subtelomere. Transcription of TERRA from the MS2-tagged telomere results in the expression of MS2-tagged TERRA molecules that are visualized by live-cell fluorescence microscopy upon co-expression of a MS2 RNA-binding protein fused to GFP (MS2-GFP). This approach enables researchers to study the dynamics of single-telomere TERRA molecules in cancer cells, and it can be applied to other cell lines.

Here, we present a protocol to generate cancer cell clones containing a MS2 sequence tag at a single subtelomere. This approach, relying on the MS2-GFP system, enables visualization of the endogenous transcripts of telomeric repeat-containing RNA (TERRA) expressed from a single telomere in living cells.

从活细胞中的单端粒表达的癌症细胞克隆的生成, 以可视化含有端粒重复的 rna terra

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in ...

遗传学

DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing1, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible2,3. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. 
We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. 
Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer4,5, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.

RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.

基于DNA载体的RNA干扰在癌症研究基因功能

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

医学

Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented with increasing depth, the incidence rate of cutaneous melanoma has shown a rapid and continuous increase during recent decades. In order to find effective preventative methods, it is important to understand the early steps of melanoma initiation in the skin. Previous data has demonstrated that follicular melanocyte stem cells (MCSCs) in the adult skin tissues can act as melanoma cells of origin when expressing oncogenic mutations and genetic alterations. Tumorigenesis arising from melanoma-prone MCSCs can be induced when MCSCs transition from a quiescent to active state. This transition in melanoma-prone MCSCs can be promoted by the modulation of either hair follicle stem cells&#39; activity state or through extrinsic environmental factors such as ultraviolet-B (UV-B). These factors can be artificially manipulated in the laboratory by chemical depilation, which causes transition of hair follicle stem cells and MCSCs from a quiescent to active state, and by UV-B exposure using a benchtop light. These methods provide successful spatial and temporal control of cutaneous melanoma initiation in the murine dorsal skin. Therefore, these in vivo model systems will be valuable to define the early steps of cutaneous melanoma initiation and could be used to test potential methods for tumor prevention.

The following procedure describes a method for spatial and temporal control of melanocytic tumor initiation in murine dorsal skin, using a genetically engineered mouse model. This protocol describes macroscopic as well as microscopic cutaneous melanoma initiation.

突变黑色素细胞干细胞引发小鼠黑色素瘤的时空控制

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented ...

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects (TEdeff) in Cancers

We have previously reported that a subset of cancers is defined by global transcriptional deregulations with widespread deficiencies in mRNA transcription elongation (TE)&#8212;we call such cancers as TEdeff. Notably, TEdeff cancers are characterized by spurious transcription and faulty mRNA processing in a large set of genes, such as interferon/JAK/STAT and TNF/NF-&#954;B pathways, leading to their suppression. The TEdeff subtype of tumors in renal cell carcinoma and metastatic melanoma patients significantly correlate with poor response and outcome in immunotherapy. Given the importance of investigating TEdeff cancers&#8212;as it portends a significant roadblock against immunotherapy&#8212;the goal of this protocol is to establish an in vitro TEdeff mouse model to study these widespread, non-genetic transcriptional abnormalities in cancers and gain new insights, novel uses for existing drugs, or find new strategies against such cancers. We detail the use of chronic flavopiridol mediated CDK9 inhibition to abrogate phosphorylation of serine 2 residue on the C-terminal repeat domain (CTD) of RNA polymerase II (RNA Pol II), suppressing the release of RNA Pol II into productive transcription elongation. Given that TEdeff cancers are not classified under any specific somatic mutation, a pharmacological model is advantageous, and best mimics the widespread transcriptional and epigenetic defects observed in them. The use of an optimized sublethal dose of flavopiridol is the only efficacious strategy in creating a generalizable model of non-genetic widespread disruption in transcription elongation and mRNA processing defects, closely mimicking the clinically observed TEdeff characteristics. Therefore, this model of TEdeff can be leveraged to dissect, cell-autonomous factors enabling them in resisting immune-mediated cell attack.

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects TEdeff in Cancers

The protocol details an in vitro murine carcinoma model of non-genetic defective transcription elongation. Here, chronic inhibition of CDK9 is used to repress productive elongation of RNA Pol II along pro-inflammatory response genes to mimic and study the clinically observed TEdeff phenomenon, present in about 20% of all cancer types.

慢性CDK9抑制的基于小鼠细胞系的模型，研究癌症中普遍存在的非遗传转录伸长缺陷（TE肯定）

We have previously reported that a subset of cancers is defined by global transcriptional deregulations with widespread deficiencies in mRNA transcription ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

延时成像的主要癌前乳腺上皮细胞来源于遗传工程小鼠乳腺癌模型

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Spontaneous Murine Model of Anaplastic Thyroid Cancer

Anaplastic thyroid cancer (ATC) is a rare but lethal malignancy with a dismal prognosis. There is an urgent need for more in-depth research on the carcinogenesis and development of ATC, as well as therapeutic methods, since standard treatments are essentially depleted in ATC patients. However, low prevalence has hampered thorough clinical studies and the collection of tissue samples, so little progress has been achieved in creating effective treatments. We used genetic engineering to create a conditionally inducible ATC murine model (mATC) in a C57BL/6 background. The ATC murine model was genotyped by TPO-cre/ERT2; BrafCA/wt; Trp53ex2-10/ex2-10 and induced by intraperitoneal injection with tamoxifen. With the murine model, we investigated the tumor dynamics (tumor size ranged from 12.4 mm2 to 32.5 mm2 after 4 months of induction), survival (the median survival period was 130 days), and metastasis (lung metastases occurred in 91.6% of mice) curves and pathological features (characterized by Cd8, Foxp3, F4/80, Cd206, Ki67, and Caspase-3 immunohistochemical staining). The results indicated that spontaneous mATC possesses highly similar tumor dynamics and immunological microenvironment to human ATC tumors. In conclusion, with high similarity in pathophysiological features and unified genotypes, the mATC model resolved the shortage of clinical ATC tissue and sample heterogeneity to some extent. Therefore, it would facilitate the mechanism and translational studies of ATC and provide an approach to investigate the treatment potential of small molecular drugs and immunotherapy agents for ATC.

Here, we present a standard pipeline to obtain murine ATC tumors by spontaneous genetically engineered mouse models. Further, we present tumor dynamics and pathological information about the primary and metastasized lesions. This model will help researchers to understand tumorigenesis and facilitate drug discoveries.

间变性甲状腺癌的自发性鼠模型

Anaplastic thyroid cancer (ATC) is a rare but lethal malignancy with a dismal prognosis. There is an urgent need for more in-depth research on the ...

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 ...

Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable model for tracing tumor progression and angiogenesis simultaneously in a visual and sensitive manner. Bioluminescence imaging displays its unique superiority in living imaging due to its advantages of high sensitivity, strong specificity, and accurate measurement. Presented here is a protocol to establish a tumor-bearing mouse model by injecting a Renilla luciferase-labeled murine breast cancer cell line 4T1 into the transgenic mouse with angiogenesis-induced Firefly luciferase expression. This mouse model provides a valuable tool to simultaneously monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging in a single mouse. This model may be widely applied in anti-tumor drug screening and oncology research.

This protocol describes the establishment of a tumor-bearing mouse model to monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging.

肿瘤进展和血管生成的双生物发光成像

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable ...

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by crossing the &#39;Tet-On&#39; system oTet-Cre mouse with a novel reverse tetracycline transactivator (rtTA) transgene linked to the Tert promoter, which we have demonstrated marks a novel population of adult brain stem cells. Here, administration of the tetracycline derivative doxycycline to mTert-rtTA::oTet-Cre mice will indelibly mark a population of cells that express a 4.4 kb fragment of the promoter region of the gene Tert. When combined the Rosa-mTmG reporter, mTert-rtTA::oTet-Cre::Rosa-mTmG mice will express membrane tdTomato (mTomato) until doxycycline treatment induces the replacement of mTomato expression with membrane EGFP (mGFP) in cells that also express Tert. Therefore, when these triple-transgenic lineage tracing mice receive doxycycline (the &#34;pulse&#34; period during which TERT expressing cells are marked), these cells will become indelibly marked mGFP+ cells, which can be tracked for any desirable amount of time after doxycycline removal (the &#34;chase&#34; period), even if Tert expression is subsequently lost. Brains are then perfusion-fixed and processed for immunofluorescence and other downstream applications in order to interpret changes to stem cell activation, proliferation, lineage commitment, migration to various brain niches, and differentiation to mature cell types. Using this system, any rtTA mouse can be mated to oTet-Cre and a Rosa reporter to conduct doxycycline-inducible &#34;pulse-chase&#34; lineage&#160;tracing experiments using markers of stem cells.

The ability to permanently mark stem cells and their progeny with a fluorophore using an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation in vivo. Lineage tracing can reveal novel information about lineage commitment, response to intervention(s), and multipotency.

成年小鼠大脑中诱导荧光标记干细胞的谱系示踪

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by ...