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 border="0" cellpadding="0" cellspacing="0">
	<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&#39;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。

Watch this Scientific Journal Video about Utilizing Murine Inducible Telomerase Alleles in the Studies of Tissue Degeneration/Regeneration and Cancer at JoVE.com

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 ...

utilizing-murine-inducible-telomerase-alleles-studies-tissue

Research

JoVE Journal

Medicine

10.1K 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

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

小鼠胰岛的分离和包膜下肾移植的方法

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

科研

医学

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

一个小鼠膀胱癌原位模型

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma (glioma) and medulloblastoma. The widely used stereotactic approach only allows for the injection of a single animal at a time, is labor intensive and requires highly specialized equipment. The guide screw method, initially developed by Lal et al.,1 was developed to eliminate cumbersome stereotactic procedures. We now describe a modified guide screw approach that is rapid and exceptionally safe; both of which are critical ethical considerations. Notably, our procedure now incorporates an infusion pump that allows up to 10 animals to be simultaneously injected with tumor cells.
To demonstrate the utility of this procedure, we established human U87MG glioma cells as intracranial xenografts in mice, which were then treated with AMG102; a fully human antibody directed to HGF/scatter factor currently undergoing clinical evaluation2-5. Systemic injection of AMG102 significantly prolonged the survival of all mice with intracranial U87MG xenografts and resulted in a number of complete cures. 
This study demonstrates that the guide screw method is an inexpensive, highly reproducible approach for establishing intracranial xenografts. Furthermore, it provides a relevant physiological model for validating novel therapeutic strategies for the treatment of brain cancers.

In order to evaluate novel therapeutic paradigms for the treatment of glioma, physiological relevant models are essential. We utilize an implantable guide screw procedure for establishment of intracranial xenograft models that is more rapid and safer than stereotactic approaches.

一个简单的指南螺杆颅内移植瘤小鼠的研究方法

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1/PD-L1, a member of the B7 family of immune-regulatory cell-surface proteins, plays an important role in the negative regulation of cell-mediated immune responses through its interaction with its receptor, programmed death-1 (PD-1) 1,2. Overexpression of B7-H1 by tumor cells has been noted in a number of human cancers, including melanoma, glioblastoma, and carcinomas of the lung, breast, colon, ovary, and renal cells, and has been shown to impair anti-tumor T-cell immunity3-8.
Recently, B7-H1 expression by pancreatic adenocarcinoma tissues has been identified as a potential prognostic marker9,10. Additionally, blockade of B7-H1 in a mouse model of pancreatic cancer has been shown to produce an anti-tumor response11. These data suggest the importance of B7-H1 as a potential therapeutic target. Anti-B7-H1 blockade antibodies are therefore being tested in clinical trials for multiple human solid tumors including melanoma and cancers of lung, colon, kidney, stomach and pancreas12.
In order to eventually be able to identify the patients who will benefit from B7-H1 targeting therapies, it is critical to investigate the correlation between expression and localization of B7-H1 and patient response to treatment with B7-H1 blockade antibodies. Examining the expression of B7-H1 in human pancreatic adenocarcinoma tissues through immunohistochemistry will give a better understanding of how this co-inhibitory signaling molecule contributes to the suppression of antitumor immunity in the tumor's microenvironment. The anti-B7-H1 monoclonal antibody (clone 5H1) developed by Chen and coworkers has been shown to produce reliable staining results in cryosections of multiple types of human neoplastic tissues4,8, but staining on paraffin-embedded slides had been a challenge until recently13-18. We have developed the B7-H1 staining protocol for paraffin-embedded slides of pancreatic adenocarcinoma tissues. The B7-H1 staining protocol described here produces consistent membranous and cytoplasmic staining of B7-H1 with little background.

Immunohistochemical Staining of B7-H1 PD-L1 on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1 (PD-L1) and its binding to PD-1 provide a major tumor-induced immunosuppressive signal in the tumor&#x2019;s microenvironment. An immunohistochemical staining technique to characterize the expression and localization of B7-H1 in pancreatic adenocarcinoma is described here.

B7-H1（PD-L1）免疫组化染色石蜡的嵌入式幻灯片的胰腺癌组织

B7-H1/PD-L1, a member  of the B7 family of immune-regulatory cell-surface proteins, plays an important  role in the negative regulation of cell-mediated ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

肝转移的临床前小鼠模型

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

一个<em&gt;在体外</em&gt;对雌激素敏感的乳腺癌休眠模式在骨髓中:分子机理研究与假设生成的一个工具

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible animal models mirror common components of human disease, including the bm12 transfer model. Upon transfer of bm12 splenocytes or purified CD4 T cells, C57BL/6 mice rapidly develop large frequencies of T follicular helper cells (Tfh), germinal center (GC) B cells, and plasma cells followed by high levels of circulating anti-nuclear antibodies. Since this model utilizes mice on a pure C57BL/6 background, researchers can quickly and easily study disease progression in transgenic or knockout mouse strains in a relatively short period of time. Here we describe protocols for the induction of the model and the quantitation Tfh, GC B cells, and plasma cells by multi-color flow cytometry. Importantly, these protocols can also be used to characterize disease in most mouse models of SLE and identify Tfh, GC B cells, and plasma cells in other disease models.

The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

系统性红斑狼疮的BM12诱导模型（SLE）的C57BL / 6小鼠

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible ...

Utilizing an Orally Dissolving Strip for Pharmacological and Toxicological Studies: A Simple and Humane Alternative to Oral Gavage for Animals

Prior to testing novel therapeutics in humans, short and long term preclinical (i.e., animal), repetitive pharmacological and toxicological testing is required. In most cases, the preferred route of administration is via oral delivery. At the present time, oral delivery is mostly accomplished using an oral gavage procedure, in part, because it can achieve consistent and precise dosing in the animal model. Although this method is well established it does have complications that can result in a high rate of animal attrition. To this end, the procedure introduced here describes an alternative to the oral gavage method in which the desired drug is incorporated into a tastant, orally dissolving strip (ODS) that can simply be presented to the test animal where it is then rapidly taken up with minimal manipulation of the test subject. Herein, we demonstrate that preclinical, oral drug delivery using the ODS method represents a safe, convenient, and humane alternative to oral gavage.

The following protocol describes a novel method for chronic oral drug administration using an orally dissolving strip (ODS) in lieu of the more commonly used oral gavage method. We demonstrate that preclinical, oral drug delivery using the ODS method represents a safe, convenient, and humane alternative to oral gavage.

利用一种口腔溶解地带的药理毒理研究:一个简单的和人道的替代口服灌胃动物

Prior to testing novel therapeutics in humans, short and long term preclinical (i.e., animal), repetitive pharmacological and toxicological testing is ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

人前列腺癌的临床前原位小鼠模型

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...