We thank the Bloomington Stock Center (Bloomington, USA), Dirk Bohmann, Katja Br&#252;ckner and Istvan Ando for fly stocks, and antibodies. We thank Marek Jindra and Colin Donohoe for comments on the manuscript. This work was supported by the Sofja Kovalevskaja Award to M.U. from the Alexander von Humboldt Foundation and DFG project UH243/1-1 to M.U. from the German Research Foundation.

注:该作品利用eyFLP1;法&gt; Y +&gt; Gal4的，UAS-GFP; FRT82B，浴缸，GAL80 MARCM 82B绿线测试仪11。穿越MARCM 82B绿测试处女的菌株如W的男性; UAS-A; UAS-B 的RNAi，FRT82BÇMUT基因型将产生在有丝分裂重组将第三同源染色体的右臂之间发生的后代。以这种方式，克隆对于位于远离FRT82B站点C基因纯合的突变体将在EAD和脑神经上皮内产生。这些克隆将表达绿色荧光蛋白，转基因的和双链RNA为基因B（ 图1A）。在X，2L，2R，3L和3R重组各种eyFLP-MARCM测试线路已建立并是飞社区内可用。 1.飞处理，十字架，和分期<ol><li>扩大在室温下适当MARCM测试仪的库存通过填充多个波ttles同时。翻转大人到新鲜瓶每2-3天，以便有足够的处女可以收集后续十字架。 注:当收集处女，避免长时间暴露于CO 2，因为这损害了女性的生育能力。 </li><li>根据实验的规模，成立了使用至少10 MARCM仪处女和4的男性（ 例如 ，荧光记者和免疫成像）或与至少30处女和10名男性（ 例如 ，瓶小瓶飞十字架RNA分离和侵袭的定量）。 </li><li>经过漫长的一天16小时/ 8小时的光暗周期下筹集到25℃后代。翻转父母到新的小瓶/瓶，每24小时，以方便幼虫可重复的分期，以防止过度拥挤。 注:在开始产蛋（AEL）后的第6天，晚三龄幼虫控制摄食停止，开始徘徊，进入pupariation。与此相反，幼虫-蛹过渡的开始可以被去奠定或不存在于幼虫EAD轴承增生或恶性克隆肿瘤。 </li></ol> 2.收集三龄幼虫<ol><li>对于免疫染色，收集有关20晚三龄幼虫（徘徊在墙上的那些可比的车身尺寸从食物）。使用镊子轻轻地从小瓶或瓶子挑幼虫，并将其转移到充满磷酸盐缓冲盐水（PBS）的胚胎的玻璃盘。 <ol><li>对于RNA分离和侵袭的定量，收集至少80晚三龄幼虫（徘徊在墙上的那些可比的车身尺寸从食物）。用喷瓶喷到PBS到含有幼虫飞瓶，直到表面被覆盖。 </li><li>软化食品的顶层用刮刀和倾含有幼虫食物醪到培养皿。使用镊子，轻轻一挑幼虫，并将它们转移到装有PBS胚胎玻璃盘。 </li></ol></li><li>幼虫洗净用PBS UNT金正日剩余的全部食品被删除。检查8荧光体视显微镜下的幼虫16X放大放弃所有的"豹仔"携带整个幼虫体内随机GFP阳性斑点（ 图2A，2B和见讨论部分 ）。 注:对于RNA分离包括在70％乙醇1分钟倒数第二个洗涤步骤。 </li><li>将含有幼虫选择在PBS冰上直到解剖菜。 30分钟内处理的幼虫，避免感冒和饥饿的不利影响。 </li></ol> 3.幼虫解剖<ol><li>解剖立体显微镜下的EAD，使用一对镊子轻轻按压在玻璃皿底幼虫的中间部分。随着第二个镊子，抢幼虫口钩，和他们拉从身体（ 图2C）的距离。 注:该拉力通常足以打破连接EAD PAI视神经秆R将脑叶。如果脑或其部分保持附着到EAD和它们的存在是不希望用于进一步的应用，削减光学秸秆与两对镊子的帮助。对于浸润性肿瘤（ 如 RAS V12 scrib 1）EAD和脑叶之间的连接变得越来越多地与时间由长满并迁移克隆细胞遮蔽。 <ol><li>以制备具有完整腹神经索（VNC）（ 图2D）的EAD /脑复杂，使用镊子切割幼虫在主体的中间。丢弃后半部。 </li><li>保持一个镊子的尖端之间的幼虫体的前半部分，通过在口钩与第2钳子的前端推压，并通过与所述第一钳对滚过它角质层翻转幼虫内向外。 </li></ol></li><li>使用镊子小心地取出所有多余的组织（如唾液腺，脂肪体，和内脏），同时使EAD对或连接口钩的EAD /大脑复杂。解开上覆角质层口钩。通过切断延伸到身体的肌肉和表皮（ 图1B，2C，2D）的轴突突起释放VNC。 注:口钩允许用钳子组织的安全操作和清洗过程中促进组织的更有效的下沉，易于识别。在PBS保持不固定的，当解剖EAD变化形态比较快的。限制清扫时间以20-30分钟。 </li><li>为了转移组织，涂层吹打其余主体上的P20的微量移液器尖端胴体几次上下。要传输更大的EAD /脑复合物，用剪刀剪的P20枪头。 注:涂布程序是至关重要的，以防止传输过程中粘附并解剖组织的损失。一个微管P20使用最小PBS转移到固定液，限制不必要的稀释。 <ol><li>对于免疫染色，转移解剖EAD到0.5毫升管装有400微升4％多聚甲醛（PFA）固定液。应用1.5毫升管1毫升PFA较大EAD /脑复合意味着侵袭的得分。 注意:PFA有剧毒。戴防护手套和防护服。避免皮肤，眼睛或粘膜接触。 </li><li>对于RNA分离转移解剖EAD到充满冷PBS一个新的玻璃盘。使用镊子从环和淋巴结清扫EAD，以尽量减少样品污染。 </li><li>通过切割使用镊子（ 图2E）的天线的光盘和口钩之间的连接分离从口挂钩EAD。为了避免RNA降解和基因表达的文物，限制了清扫时间为30分钟。继续执行步骤7。 注:一个有经验的研究者可以在需要的幼虫收集和洗涤30分钟时间除外解剖约60幼虫。总RNA的产量取决于EAD大小基因型和developmenta之间变化升阶段。从晚第三龄幼虫80-100控制EAD对解剖应该产生5-8微克总RNA。 </li></ol></li></ol> 4.固定，免疫染色，与安装<ol><li>固定在PFA固定剂的样品25分钟，同时在室温下章动。固定时间可以延长至60分钟，而不会改变组织形态和蛋白质的抗原性。 </li><li> （含有0.1％的Triton X-100的PBS）三次除去用PBST固定液和洗涤样品用于使用章动器10分钟。使用PBST的足够的体积为每个清洗步骤（400微升/ 0.5毫升管）。 注:固定EAD /脑复合意味着侵袭的得分不需要染色。 GFP信号保持固定后清晰可见。继续执行步骤4.4的最后清扫。 <ol><li>对于免疫染色，在200μl封闭液（PBST用0.3％BSA）中进行20分钟，温和搅拌在室温下的块组织。 <ol><li>与拘谨孵育进制抗体在4℃下稀释在100μl封闭溶液过夜，同时轻轻地摇动。请参阅第一抗体数据手册或发表的文献为推荐的抗体稀释。 注:初级抗体的稀释可能需要进行优化。 </li><li>以下在PBST 5 10分钟洗涤后，用荧光二级抗体染色的组织在阻断溶液稀释，同时轻轻搅拌2小时，在室温下或在4℃下在黑暗中振摇过夜。 </li><li>洗样品三次10分钟在PBST中。 </li></ol></li></ol></li><li>用500微升的DAPI染色解决方案替换PBST。标记F-肌动蛋白，添加鬼笔环肽缀合至荧光染料DAPI染色溶液。 15分钟垂头在黑暗中后，10分钟用PBST洗涤组织一次。 注:F-肌动蛋白标记可以独立DAPI染色的条件下进行。 </li><li>对于最终夹层步骤中，使用1毫升圆周率转移组织放回PBS的填充的玻璃皿佩特。使用两对镊子夹断口钩。 <ol><li>分离，将通过切割视柄作为杂草丛生EAD可能妨碍分析用于侵袭性的量化从EAD的大脑。 </li></ol></li><li>将安装介质上的目标幻灯片的下降（15微升为22毫米×22毫米盖玻片）。用钳子提示的帮助下，介质分配到的薄层。 EAD转移，大脑或EAD /脑复合物与一个P20微量的安装介质。 </li><li>使用镊子尖端或钨​​棒分发组织拉直VNC上的幻灯片。 </li><li>以避免形成而安装的气泡，第一触摸盖玻片到安装介质的一个边缘，然后慢慢地降低到与钳的帮助下安装培养基。使用小片滤纸或组织的擦拭吸收周围盖玻片边缘多余的安装介质。 注:DABCO /聚乙烯醇（PVA）4-88安装介质变硬在一个小时内4℃。幻灯片可存放在4℃下个月。 </li></ol> 5.共焦成像<ol><li>获得使用配有20X，40X和60X的石油目标的共聚焦显微镜单焦部分和堆栈。 </li><li>防止像素的饱和度。使用相同的图像采集参数包括图像分辨率，激光输入功率，增益，偏移，帧平均，在一个Z系列的步长，并保持所有基因型这些设置以允许不同基因型21,22之间的像素强度的比较。 注:EAD /大脑复杂的可视化，产生最大的预测和各自的缝合，相邻的图像。对于最终图像的准备，后期使用图像处理软件面板组件，并调整亮度和对比度。 </li></ol> 6.定量肿瘤侵袭的<ol><li>定义一个评分系统，将表征不同层次的肿瘤invasivenes的Ş考虑入侵的空间格局和GFP阳性细胞扩散到大脑和VNC量。准备评价表的文档（ 图3A）。 </li><li>安装至少80固定在使用40微升每24毫米×50毫米的盖玻片安装介质的一个滑动每个基因型完好大脑。 <ol><li>为了让公正打分，问一个中立的一方匿名的幻灯片，让记分程序是公正的。评估由独立两名实验人员匿名幻灯片。公开点票完成后才会基因型。 </li></ol></li><li>评估配备了GFP过滤器设置荧光体视显微镜下恶性由安装大脑盲目得分的程度。使用标记标记已被视为已为避免重复计算（ 图3B）的大脑。 </li><li>计算大脑落入每个每基因型限定侵入类别的百分比。确定统计小号ignificance用卡方检验（ 图3C）。 </li></ol> 7. RNA提取，DNA酶处理和质量检查注意:在下面的步骤中使用的所有试剂（溶液，塑料器具）应无RNase活性。一定要穿戴手套和使用有机溶剂的工作化学罩。 <ol><li>用涂P20枪头转移干净EAD到1.5mL管中。 </li><li>让组织沉到试管底部，小心地取出PBS中，用100μl的RNA裂解试剂（足够用于多达140 EAD）代替它。 </li><li>裂解组织涡旋。此时，样品可以是深度冷冻在液氮中，并储存在-80℃或用标准的RNA分离协议直接处理。 注:从多个夹层轮类似基因型的样品可一旦RNA裂解试剂汇集。 </li><li>填写样本量0.9毫升RNA裂解试剂。增加±毫升氯仿和vigoro混合样品usly 15-20秒。 </li><li>以下2至3分钟，在室温下培养，离心样品以10,000 xg离心在4℃下15分钟。 </li><li>无色转移上含有RNA的水相到一个新的试管，而不会干扰相间。远离相间远，因为它含有蛋白质和基因组DNA。 注:将回收的体积应是RNA裂解试剂的用于均质化的体积的约60％。 </li><li>加入0.5ml异丙醇沉淀RNA。涡旋并在室温下孵育样品10分钟。 </li><li>离心样品以10,000 xg离心在4℃下15分钟。 </li><li>弃去上清液并用600μl的75％乙醇洗一次RNA沉淀。短涡和离心（在4℃万XG 7分钟）后丢弃所有的乙醇，并让RNA沉淀风干5-10分钟将开管放在一个干净的纸巾擦拭。 注:RNA颗粒可能是作为一个微小的不透明/白色ST可见熟在管或无形的底部。剩余的乙醇滴可与P10枪头仔细清除。 </li><li>通过一个枪头涡旋或通过溶液几次溶解在50μlDEPC处理的水的RNA。 </li><li>以最小化与基因组DNA污染，通过添加50微升含有DNA酶的1微升（2U /微升）和10微升10×DNA酶缓冲液中于DEPC处理过的水的混合物的治疗用DNase总RNA。涡和自旋向下。 </li><li>在水浴或30至40分钟的加热块在37℃下孵育样品。孵育后，加入100微升DEPC处理过的水到每个样品。 </li><li>从DNA酶失活酶的酶活性和清洁的RNA，加入200微升酚:以10,000 xg离心在4℃溶液中，涡旋1分钟并离心样品进行7分钟的氯仿:异戊醇（1 25:24）。 </li><li>仔细含RNA的上层水相转移到新管。添加等体积（200微升）氯仿，混合和从上面重复离心步骤。 </li><li>小心收集上层相并通过加入1/10体积的3M乙酸钠沉淀RNA，pH值5.2中的100％乙醇DEPC H 2 O和2.5体积制备。涡旋调匀。 注意:添加0.5微升糖原（20微克/微升），以沉淀组合有助于以可视化的RNA沉淀。为了尽量减少损失RNA，使用硅化，低结合的1.5 ml离心管中。 </li><li>在-20℃沉淀RNA为至少1小时。 注:潜伏期可能在一夜之间完成。样品可在-80℃放置为好。 </li><li>沉淀的RNA离心以10,000 xg离心在4℃下30分钟。洗净并按照7.9的说明干燥颗粒。 </li><li>在10-15微升DEPC处理过的水的溶解的RNA。在-80°C储存RNA。 </li><li>通过在230纳米，260纳米和利用分光光度计测量280nm处的OD确定RNA的数量和纯度。计算RNA合作通过应用该公约ncentration的1 OD在260纳米等于40微克/毫升RNA。 注意:"纯"RNA的一个260/280比等于2.0，而一个A 260/230比值应在2.0-2.2的范围内。与A 260/280比率1.7和2.0之间的RNA样品适用于下游应用，如cDNA合成。对于制备的mRNA-SEQ库RNA的质量和数量应使用自动电泳系统进行检查。的28S / 18S比率应大于1.8。 </li></ol>

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The authors declare that they have no conflict of interest.

该技术产生在果蝇遗传马赛克是用于分析和操纵基因功能的33最复杂的工具之一。所述eyFLP-MARCM系统已被证明强大和健壮，因为它允许在视觉上标记，遗传背景明确克隆的诱导以空间受限的方式， 即 ，在组织中，其中无眼增强剂是活性9,18。当多种遗传病变在相同的细胞结合，这是特别重要的。虽然这些高度异常的修补程序时只限于EAD和脑部神经上皮使幼虫发育，分散在幼虫体内的热休克控制FLP（hsFLP）克隆的情况时，他们可能会导致杀伤力。显著，在飞EAD上皮细胞克隆的诱导激活其中致癌基因结合了肿瘤抑制基因的丧失概括在某些人类癌症的出现和实体上皮肿瘤进展。何wever，该系统也有其局限性，这将简要讨论。鉴于昆虫有一个开放的循环系统，不发生如在人类癌症中观察到的真实转移。因此，转移性传播的复杂的过程，不能如实地在果蝇模型。然而，EAD肿瘤如RAS V12 scrib 1个克隆做，因为他们打破围绕着EAD基底膜并侵入邻近脑11,12,31显示恶性属性。肿瘤侵袭性的程度可以定量并如本研究中所描述的不同基因型间比较。同样重要的是要注意，特定的遗传病变的镶嵌EAD存在可能会影响发育时间和幼虫生长的持续时间。幼虫分期将决定是否镶嵌组织的分析将使用相同的时间顺序或发育阶段的动物来完成。进行预试验以评估一个冲击上发育定时新EAD克隆基因型是可取的。 有技术局限性。首先，能够与eyFLP-MARCM工具建立不同克隆基因型的数目是很大的，但不是无限的。例如，驻留在不同的染色体上组合两个突变体等位基因是由两个染色体上的重组效率低同时阻碍，以及位于该小第四染色体上的基因通常是不可访问。基因转用RNAi抑制是一个替代的解决方案。直到GAL80阻遏被降解 - 然而，要认识到转基因时，不会有丝分裂重组立即表示是非常重要的。的Gal4的/ UAS表达系统的活性可以通过生长的幼虫在29℃得到增强。然而，重要的是要记住，升高的温度会影响各种发展，生理和分子参数，包括生长速率，发育时间，Metaboli游戏SM和基因表达的34-37。因此，适应于提供协议，如幼虫分期或用于转录分析解剖EAD的数目将是必要的。 该eyFLP-MARCM测试不是最健康的菌株中。在一个基因组折中的多个转基因飞健身和繁殖力。这些股票需要额外注意保持和处女女的大集合扩大。正如在这项研究中，MARCM 82B绿测试线11，虽然可行记载纯合子时，在遗传上不稳定的。这是从"豹仔"即显示各种多倍体组织中异位，GFP标记的克隆，包括肠，气管，脂肪体，和幼虫表皮的自发外观明显。这种现象发生在所有穿过独立于父亲的基因型。我们推测，异位GFP补丁从两间FR不受控制，随机重组导致T位在FLP出卡带，引起去除黄色+标记和"STOP卡带"的。在多倍体组织中， 微管蛋白启动子驱动的GAL80阻遏可能不足以阻止的Gal4的活性。遗传修饰的细胞中比EAD和脑等组织中的存在可能会引起可能影响了实验的克隆的行为全身信号。因此，这些"豹仔"应该被排除在分析。此外，我们经常重新建立由单一的男女杂交的MARCM 82B绿测试仪。 尽管上面提到的缺陷，在eyFLP-MARCM系统来研究基因功能的应用程序和克隆肿瘤及其环境之间的相互作用是多方面的。与GAL4 / UAS独立双元表达系统，如LexA的介绍/ LexOp 38或药物的可控QF / QUAS / QS 39,40将提供了转基因EXP精细控制ression和/或同时标记和不同的细胞群，包括克隆，非克隆上皮细胞和血细胞的操纵。这种镶嵌组织可根据此处所描述的协议来再次处理。 为从第三龄幼虫（协议第3节）的马赛克EAD和EAD /脑复合物夹层的方法，可以适用于早期发育阶段，以及。重要的是，EAD /脑配合的协议允许边路，haltere和腿部成虫盘的恢复。应这些用于RNA分离（协议部分3.3.2和7）中，感兴趣的圆盘应该从外部组织进行清洗和类似地处理到EAD。由于其较小的尺寸，应增加收集haltere和腿部盘的数量，以获得RNA的足够量。 用于从EAD（协议第7节）的总RNA提取协议是基于RNA的裂解试剂可得自不同的商业vendors（见材料/设备清单）。该过程的总体成功主要取决于:（i）具有足够数目的幼虫，（ⅱ）是快速，准确而解剖，和（iii）保持样品不含RNA酶。保持工作空间和工具的清洁，戴手套，使用无RNase的塑料制品和解决方案，并保持样品在冰上所有减少RNA降解。为了防止可能不是由DNA酶处理（协议第7.11节）将完全删除任何基因组DNA的RNA的污染，关键是要避免相间的第一次（协议第7.6节）收集的水相的时候。两个DNA污染和RNA退化可以通过自动电泳系统（ 图6B，6C）上运行样品来揭示。该RNA分离协议具有更广泛的应用。它已成功地用于从全幼虫在不同发育阶段的RNA提取，从成年人，和各种幼虫器官。将得到的RNA是同时适合定量RT-PCR和利用mRNA-SEQ 20,24,41,42全基因组转录组分析。相比于定量RT-PCR，在大多数量化几十选定的mRNA，mRNA的偏见使得测序全基因组表达分析。因此，可以比较在肿瘤24的不同的阶段诱导特异性遗传病变肿瘤整个转录组签名。重要的是要记住，RNA来自整个光盘，这样的结果，不仅反映在克隆而且在周围，非克隆组织变化是重要的。通过适应可用的协议执行上离解马赛克EAD荧光激活细胞分选（FACS）43,44可以适用于确定特定的细胞群， 例如 ，克隆（GFP +）和非克隆（GFP - ）的转录签名细胞。 这里描述的固定和染色协议是适合于同时联播用特异性抗体和转基因记者活动兴趣蛋白质taneous可视化。两个标记通过无论是在本研究中使用的两种转基因记者产生克隆细胞和DsRed的荧光的绿色荧光信号在整个固定和免疫染色被保留，并且可以通过共焦显微镜可以直接观察。然而，对荧光蛋白的抗体能够放大信号的强度。同样地，使用抗β半乳糖苷酶抗体将促进基于细菌lacZ基因的转基因记者检测。相对记者的活动可以通过测量克隆的相对像素强度与非克隆，利用组织图像分析软件（如斐济，ImageJ的）进行评估。当比较不同的克隆基因型报道分子活性，图像获取的设置必须保持恒定（协议第5和引用21,22）。虽然该协议与许多不同的一个运行良好ntibodies，可能有必要优化推荐的抗体浓度。改变也可能在固定的时间，类型和洗涤剂（曲通X-100，吐温-20，皂素）和固定剂（8％PFA中，4％的甲醛，乙醇）的浓度必需的。 不像上面讨论的协议，肿瘤侵袭的量化限制在三龄幼虫阶段和EAD /脑复杂。然而，它的总的理由可以适用于任何定量研究，其中认知偏差需要避免。该方法需要彻底性。如在时间肿瘤恶性的增加，有必要比较相同年龄的幼虫（ 例如，产卵后7或8天）。随着时间的克隆肿瘤可大规模长满，但肿瘤细胞可以保留在EAD之内。其中大脑从EAD（协议第4.4.1节）分离的步骤是至关重​​要的，因此为了避免假阳性侵袭计数。用所附EAD大脑的安装将弗拉特恩组织，导致杂草丛生EAD覆盖大脑结构，从而防止任何量化。理想情况下，一个想直接捕捉侵入过程中的生活幼虫。转基因记者可视化所涉及的细胞群体和组织可用，可以与eyFLP-MARCM系统相结合。不幸的是，侵入性过程需要几个小时到几天，一个时间帧，它与保持幼虫存活，同时仍然不相容。

癌代表了最遗传异质组疾病，其发病率和死亡率的显着提高，特别是在老年人全世界之一。癌症从逃脱固有的抑癌机制，并把失控的肿瘤启动细胞克隆起源。遗传病变共同促进生长，增殖和迁移，同时抑制死亡和分化的逐渐积累转化的初始良性增生为高度恶性，转移性和致命的肿瘤。它已成为明显的是，除了遗传改变，肿瘤进展需要在肿瘤和多种细胞类型（ 例如 ，成纤维细胞，免疫细胞和内皮细胞）在其微环境之间的周围的基质和串扰的改变。理解的分子原理恶变底层包括肿瘤 - 基质的相互作用是非常重要的发展防止措施灰和早期筛查策略，以及新的和有效的治疗方法，以打击癌症转移和耐药性。 果蝇果蝇已成为癌症研究1-4由于其快速产生时间一个有吸引力的系统，信令苍蝇和人类的促进几乎任何基因操纵在先进遗传工具有限遗传冗余和财富之间节点的显着的保护一个暂时和空间限制的方式。不同的恶性肿瘤的遗传背景明确肿瘤可在果蝇通过使用MARCM技术5在另外的野生型组织中祖细胞的一个子集引入GAIN-和丧失功能的突变再现地工程化。该MARCM工具结合FLP / FRT（FLP重组/ FLP识别目标）介导的有丝分裂重组6 FLP出7和Gal4的/ UAS（上游激活序列）8靶基因表达系统9。与任何基于UAS - 转基因的这种方法的表达，包括原癌基因或荧光蛋白的cDNA或倒置DNA重复对的dsRNA诱导的基因沉默，将被限制到细胞的克隆已经失去一个特定基因座和一个Gal4的阻遏由于重组（ 图1A）。标有绿色荧光（GFP）或红色荧光蛋白克隆补丁（ 例如，RFP，红色荧光蛋白，mCherry）可以很容易地在整个发展，孤立和分析跟踪。重要的是，它们的行为可以直接比较到相邻的野生型组织。因此，问题相关的所述细胞的遗传病变的自主性和非自主效应可以方便地研究。类似于哺乳动物，只有克隆，其中多个致癌病变被组合成为在果蝇恶性和概括哺乳动物癌细胞的关键特点。他们overproliferate，逃避凋亡，诱发炎症，成仙INVA西伯，最终杀死宿主10-17。 在这里，我们描述了一个协议，使用MARCM技术产生果蝇幼虫的眼睛/触角和脑组织基因定义克隆肿瘤。该方法依赖于一个MARCM仪股票其表达无眼增强剂（eyFLP）18,19的控制下的酵母FLP重组酶。以这种方式，在EAD的两个peripodial和柱状上皮和脑的整个胚胎和幼虫期（ 图1A，B和参考20）的神经上皮产生GFP标记的克隆。克隆可以轻松跟随直到成年的EAD发展到成人的眼睛，天线和头壳而神经上皮产生了产生差异化的视叶神经元的神经细胞。 为了方便马赛克组织，我们去的大量分子，功能和表型特征隶从三龄幼虫EAD和大脑的解剖协议，并概述了如何处理它们三个不同的应用程序:（一）检测转基因荧光记者和染色，（二）肿瘤侵袭和（三）分析量化基因表达的变化使用定量实时PCR（QRT-PCR）或高通量的mRNA测序（的mRNA-SEQ）（ 图1C）。 免疫染色协议可以用于可视化的任何感兴趣的蛋白质的特异性抗体。转基因荧光转录记者提供对特定信号传导途径的活性方便和精确的时空信息。细胞谱系特异性记者，另一方面，反映镶嵌组织内和不同的基因型的肿瘤中的细胞群体的定性和定量的变化。的入侵行为有利于量化基因型与肿瘤恶性的比较秒。最后，描述用于RNA分离镶嵌EAD的收集和处理的协议，适用于小型和大型的下游应用如逆转录随后分别定量RT-PCR和基因组范围内的mRNA-SEQ。从这些分析得到的定性和定量的数据提供新的见解克隆肿瘤的社会行为。此外，它们产生用于对各个基因，遗传网络和在不同阶段肿瘤微环境和肿瘤发生的各方面的作用，功能研究了坚实的基础。

<table border="0" cellpadding="0" cellspacing="0" width="1019">
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			<td height="84" style="height:84px;width:216px;">Agar</td>
			<td style="width:177px;">Gew&#252;rzm&#252;hle Brecht, Eggenstein, Germany</td>
			<td style="width:151px;">00262-0500</td>
			<td style="width:476px;">Fly food recipe: Prepare 20 L fly food with 160 g agar, 360 g yeast, 200 g soy flour, 1.6 kg yellow cornmeal, 1.2 L malt extract, 300 ml light corn syrup, 130 ml propionic acid and 200 ml 15% nipagin. Fly food should be cooked for 1 hour at 85&#176;C.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Corn syrup</td>
			<td style="width:177px;">Grafschafter Krautfabrik Josef Schmitz KG, Meckenheim, Germany</td>
			<td style="width:151px;">01939</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Propionic acid</td>
			<td style="width:177px;">Carl Roth GmbH, Karlsruhe, Germany</td>
			<td style="width:151px;">6026.1</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cornmeal</td>
			<td style="width:177px;">ReformKontor GmbH, Zarrentin, Germany</td>
			<td style="width:151px;">4010155063948</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Malt extract</td>
			<td style="width:177px;">CSM Deutschland GmbH, Bremen, Germany</td>
			<td style="width:151px;">728985</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Soy flour</td>
			<td style="width:177px;">Stockmeier Food GmbH, Herford, Germany</td>
			<td style="width:151px;">1000246441010</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Yeast</td>
			<td style="width:177px;">Werner Ramspeck GmbH, Schwabach, Germany</td>
			<td style="width:151px;">210099K</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Methyl-4-benzoate/ Nipagin</td>
			<td style="width:177px;">Sigma-Aldrich, Deisenhofen, Germany</td>
			<td style="width:151px;">H5501</td>
			<td style="width:476px;">Prepare a 15% stock solution with 70% EtOH</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Drosophila fly food vials</td>
			<td style="width:177px;">Kisker Biotech, Steinfurt, Germany</td>
			<td style="width:151px;">789008</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vial plugs</td>
			<td style="width:177px;">K-TK e.K., Retzstadt, Germany</td>
			<td style="width:151px;">1002</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Drosophila fly food bottles</td>
			<td style="width:177px;">Greiner Bio-One, Frickenhausen, Germany</td>
			<td style="width:151px;">960177</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bottle plugs</td>
			<td style="width:177px;">K-TK e.K., Retzstadt, Germany</td>
			<td style="width:151px;">1002 S</td>
			<td style="width:476px;"></td>
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		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Poly(vinyl alcohol) 4-88/[-CH2CHOH-]n</td>
			<td style="width:177px;">Sigma-Aldrich, Deisenhofen, Germany</td>
			<td style="width:151px;">81381</td>
			<td style="width:476px;">Mounting medium recipe: Dissolve 6 g Poly(vinyl alcohol) 4-88 in 39 ml Millipore H2O, 6 ml 1 M Tris (pH 8.5) and 12.5 ml glycerol. Stir overnight at 50&#176;C and centrifuge 20 min at 5000 rpm. Add DABCO to the supernatant to get a final concentration of 2.5%. Store aliquots at -20&#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1,4-Diazabicyclo[2.2.2]octane/ DABCO&#160;</td>
			<td style="width:177px;">Sigma-Aldrich, Deisenhofen, Germany</td>
			<td style="width:151px;">D2522</td>
			<td style="width:476px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Triton X-100</td>
			<td style="width:177px;">Sigma-Aldrich, Deisenhofen, Germany</td>
			<td style="width:151px;">9002-93-1</td>
			<td style="width:476px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphate-buffered saline/ PBS</td>
			<td style="width:177px;"></td>
			<td style="width:151px;"></td>
			<td style="width:476px;">137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4, pH 7.4 in Millipore H2O</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBST</td>
			<td style="width:177px;"></td>
			<td style="width:151px;"></td>
			<td style="width:476px;">0.1% Triton X-100 in PBS</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:216px;">Paraformaldehyde/ PFA</td>
			<td style="width:177px;">Sigma-Aldrich, Deisenhofen, Germany</td>
			<td style="width:151px;">158127</td>
			<td style="width:476px;">4% PFA fixative recipe: Dissolve 4 g PFA in 80 ml Millipore H2O on a magnetic stirrer plate heated to 55 &#176;C. Add 1M NaOH dropwise until all PFA particles are dissolved. Add 10 ml 10X PBS and adjust the pH to 7.4 with 1M HCl. Mix in 100 &#181;l Triton X-100 and fill up with Millipore H2O to 100 ml. Store aliquots at -20&#176;C. Avoid repeated thawing. (CAUTION: PFA is highly toxic. Prepare the PFA fixative in a fume hood. Wear a self-contained breathing apparatus, gloves and clothing. Avoid contact with skin, eyes or mucous membranes)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bovine Serum Albumin/ BSA</td>
			<td style="width:177px;">Sigma-Aldrich, Deisenhofen, Germany</td>
			<td style="width:151px;">A3059</td>
			<td style="width:476px;">Blocking solution recipe: Dissolve 0.3% BSA in PBST</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">4&#8217;,6-Diamidino-2-phenylindol Dihydrochlorid/ DAPI</td>
			<td style="width:177px;">Carl Roth GmbH, Karlsruhe, Germany</td>
			<td style="width:151px;">6335</td>
			<td style="width:476px;">DAPI staining solution: Prepare a stock solution of 5 mg/ml in Millipore H2O and store aliquots at 4&#176;C. Used in dilution 1:1000 in PBST.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alexa Flour 546 Phalloidin</td>
			<td style="width:177px;">Invitrogen, Karlsruhe, Germany</td>
			<td style="width:151px;">A22283</td>
			<td style="width:476px;">Used in dilution 1:500 in PBST.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mouse anti-H2 antibody</td>
			<td style="width:177px;">Kurucz et al., 2003</td>
			<td style="width:151px;"></td>
			<td style="width:476px;">Used in dilution 1:500 in blocking solution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Cy5 AffiniPure Donkey Anti-Mouse IgG (H+L)</td>
			<td style="width:177px;">Jackson ImmunoResearch, Suffolk, UK</td>
			<td style="width:151px;">715-175-151</td>
			<td style="width:476px;">Used in dilution 1:500 in blocking solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dumont #5 forceps</td>
			<td style="width:177px;">Fine Science Tools, Heidelberg, Germany</td>
			<td style="width:151px;">11295-10</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass embryo dish (30 mm)</td>
			<td style="width:177px;">Thermo Scientific, Schwerte, Germany</td>
			<td style="width:151px;">E90</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tungsten needles</td>
			<td style="width:177px;">Fine Science Tools, Heidelberg, Germany</td>
			<td style="width:151px;">10130-20</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nickel Plated Pin Holder</td>
			<td style="width:177px;">Fine Science Tools, Heidelberg, Germany</td>
			<td style="width:151px;">26018-17</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microscope slides</td>
			<td style="width:177px;">VWR, Darmstadt, Germany</td>
			<td style="width:151px;">631-1553</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Coverslips 22 x 22 mm (#1 Menzel-Gl&#228;ser)</td>
			<td style="width:177px;">VWR, Darmstadt, Germany</td>
			<td style="width:151px;">631-1336</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Coverslips 24 x 50 mm (0.13-0.16 mm)</td>
			<td style="width:177px;">Thermo Scientific, Schwerte, Germany</td>
			<td style="width:151px;">1076371</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Kimtech Science Precision Wipes</td>
			<td style="width:177px;">Thermo Scientific, Schwerte, Germany</td>
			<td style="width:151px;">06-677-70</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissecting stereomicroscope</td>
			<td style="width:177px;">Olympus, Hamburg, Germany</td>
			<td style="width:151px;">SZX7 (DF PLAPO 1X-4 Japan)</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Fluorescent stereomicroscope</td>
			<td style="width:177px;">Olympus, Hamburg, Germany</td>
			<td style="width:151px;">SZX16 (SDF PLAPO 0.8X Japan) with DP72 CCD camera</td>
			<td style="width:476px;">Equipped with the narrow blue bandpass filter set for excitation of GFP other blue excitable fluorochromes (excitation filter BP460-480 nm, barrier filter BA495-540 nm) and narrow green excitation and longpass barrier filter for RFP and other green excitable fluorochromes (excitation filter BP530-550, barrier filter BA575IF). Software: Olympus cellSens Standard 1.11.</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Confocal microscope</td>
			<td style="width:177px;">Olympus, Hamburg, Germany</td>
			<td style="width:151px;">FV1000</td>
			<td style="width:476px;">Equipped with inverted IX81 microscope. Objectives: 20&#215; UPlan S-Apo (NA 0.85), 40&#215; UPlanFL (NA 1.30) and 60&#215; UPlanApo (NA 1.35). Lasers: UV laser Diode LD405 (50mW), Argon laser, multi-line 457/(476)/ 488/515 (40mW), Yellow/Green laser diode LD560 (15mW) and Red Laser diode 635 (20mW). Software: Fluoview 2.1c Software</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Drosophila cooled incubator</td>
			<td style="width:177px;">Ewald Innovationstechnik GmbH, Bad Nenndorf, Germany</td>
			<td style="width:151px;">&#160;Sanyo MIR553</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Squirt bottle</td>
			<td style="width:177px;">VWR, Darmstadt, Germany</td>
			<td style="width:151px;">215-8105</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BD Clay Adams Nutator Mixer</td>
			<td style="width:177px;">VWR, Darmstadt, Germany</td>
			<td style="width:151px;">15172-203</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ELMI Digital Rocking Shaker</td>
			<td style="width:177px;">VWR, Darmstadt, Germany</td>
			<td style="width:151px;">DRS-12</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">5 PRIME Isol-RNA Lysis Reagent</td>
			<td style="width:177px;">VWR, Darmstadt, Germany</td>
			<td style="width:151px;">2302700</td>
			<td style="width:476px;">The protocol is compatible with other TRIzol-based reagents e.g. TRIreagent from Sigma (Cat. Nr.T9424), TRIzol reagent from Thermofisher (Cat. Nr. 15596).</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Diethyl pyrocarbonate/ DEPC</td>
			<td style="width:177px;">Sigma-Aldrich, Deisenhofen, Germany</td>
			<td style="width:151px;">D5758</td>
			<td style="width:476px;">Dilute DEPC 1:1000 in Millipore H2O, stir overnight and autoclave.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">UltraPure Phenol:Chloroform:Isoamyl Alcohol (25:24:1)</td>
			<td style="width:177px;">ThermoFischer Scientific, Invitrogen, Karlsruhe, Germany</td>
			<td style="width:151px;">15593-031</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Chloroform</td>
			<td style="width:177px;">Merck, Darmstadt, Germany</td>
			<td style="width:151px;">102445</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TURBO DNase (2 U/&#181;l)</td>
			<td style="width:177px;">Thermo Scientific, Schwerte, Germany</td>
			<td style="width:151px;">AM2238</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Invitrogen UltraPure Glycogen</td>
			<td style="width:177px;">ThermoFischer Scientific, Invitrogen, Karlsruhe, Germany</td>
			<td>10-814-010</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Sodium acetate trihydrate</td>
			<td style="width:177px;">VWR, Darmstadt, Germany</td>
			<td style="width:151px;">27652.232</td>
			<td style="width:476px;">Prepare a 3M Sodium acetate solution with DEPC-H2O and adjust the pH to 5.2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2-Propanol</td>
			<td style="width:177px;">Merck, Darmstadt, Germany</td>
			<td style="width:151px;">109634</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethanol</td>
			<td style="width:177px;">Merck, Darmstadt, Germany</td>
			<td style="width:151px;">100983</td>
			<td style="width:476px;">Prepare a 75% EtOH dilution with DEPC-H2O.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">UV/Vis-Spectrophotometre NanoDrop ND-8000</td>
			<td style="width:177px;">Thermo Scientific, Schwerte, Germany</td>
			<td style="width:151px;">ND-8000</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Eppendorf Microcentrifuge (Refrigerated)</td>
			<td style="width:177px;">Thermo Scientific, Schwerte, Germany</td>
			<td style="width:151px;">5417R</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Experion RNA StdSens Analysis Kit</td>
			<td style="width:177px;">Bio-Rad Laboratories GmbH, M&#252;nchen, Germany</td>
			<td style="width:151px;">7007103</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Experion Automated Electrophoresis Station</td>
			<td style="width:177px;">Bio-Rad Laboratories GmbH, M&#252;nchen, Germany</td>
			<td style="width:151px;">7007010</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SuperScript III Reverse Transcriptase</td>
			<td style="width:177px;">Thermo Scientific, Schwerte, Germany</td>
			<td style="width:151px;">18080044</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Oligo d(T) Primer</td>
			<td style="width:177px;">Integrated DNA Technologies, Leuven, Belgium</td>
			<td style="width:151px;"></td>
			<td style="width:476px;">Prepare 100 &#181;M stock solutions in DEPC-H20 and store at -20&#176;C</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">dNTP Mixture</td>
			<td style="width:177px;">Takara Bio Europe/Clonetech, Saint-Germain-en-Laye, France</td>
			<td style="width:151px;">4030</td>
			<td style="width:476px;">Store aliquots of 25 &#181;l at -20&#176;C</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">CFX96 Touch Real-Time PCR Detection System</td>
			<td style="width:177px;">Bio-Rad Laboratories GmbH, M&#252;nchen, Germany</td>
			<td style="width:151px;">1855195</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">iQ SYBR Green Supermix</td>
			<td style="width:177px;">Bio-Rad Laboratories GmbH, M&#252;nchen, Germany</td>
			<td style="width:151px;">170-8882</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Hard-Shell PCR Plates 96-well, thin wall</td>
			<td style="width:177px;">Bio-Rad Laboratories GmbH, M&#252;nchen, Germany</td>
			<td style="width:151px;">HSP9601</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Microseal &#39;B&#39; Film</td>
			<td style="width:177px;">Bio-Rad Laboratories GmbH, M&#252;nchen, Germany</td>
			<td style="width:151px;">MSB1001</td>
			<td style="width:476px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">rp49 Forward primer</td>
			<td style="width:177px;">Integrated DNA Technologies, Leuven, Belgium</td>
			<td style="width:151px;"></td>
			<td>5&#39; TCCTACCAGCTTCAAGATGAC 3&#39;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">rp49 Reverse primer</td>
			<td style="width:177px;">Integrated DNA Technologies, Leuven, Belgium</td>
			<td style="width:151px;"></td>
			<td>5&#39; CACGTTGTGCACCAGGAACT 3&#39;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">mmp1 Forward primer</td>
			<td style="width:177px;">Integrated DNA Technologies, Leuven, Belgium</td>
			<td style="width:151px;"></td>
			<td>5&#39; AGGGCGACAAGTACTACAAGCTGA 3&#39;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">mmp1 Reverse primer</td>
			<td style="width:177px;">Integrated DNA Technologies, Leuven, Belgium</td>
			<td style="width:151px;"></td>
			<td>5&#39; ACGTCTTGCCGTTCTTGTAGGTGA 3&#39;</td>
		</tr>
	</tbody>
</table>

the drosophila imaginal disc tumor model: visualization and quantification of gene expression and tumor invasiveness using genetic mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

为了证明eyFLP-MARCM技术产生在果蝇 EAD定义基因型的GFP标记的补丁的潜力，三种克隆的诱导:（1）控制只表达GFP的，（2）恶性肿瘤表达小的致癌形式在肿瘤抑制基因乱画的纯合损耗（scrib 1）的背景G蛋白的Ras（RAS V12），和（3）过度增长，但非侵入ras基因的V12 scrib 1 JNK DN克隆，其中该君N-末端激酶（JNK）是由它的显性负形式（ 图4， 表1基因型）的表达失活。它已经显示，ras基因V12 scrib 1克隆的肿瘤的侵袭性要求JNK信号的异常活化及其下游转录因子10,12,23,24。此外，RAS V12 scrib 1 </s向上&gt;肿瘤促进在果蝇幼虫强烈的炎症反应，导致免疫细胞（称为血细胞）的浸润到EAD 14,25。 为了监测水平和镶嵌组织内JNK的活性空间分布和不同的基因型的肿瘤之一，已建立的转基因，JNK响应TRE-红色荧光蛋白记者采用26。解剖，固定EAD和EAD的共聚焦显微镜/脑络合物揭示了在组织轴承恶性ras基因的V12 scrib 1肿瘤（ 图4B）的TRE-DsRed的报道基因活性的显着上调。的红色荧光蛋白信号标记的ras基因的V12 scrib 1克隆在EAD（ 图4B）以及肿瘤细胞散布在所述脑叶和侵入的VNC（ 图4E，4F）。与此相反，TRE-DsRed的记者活性状况TY从触角延伸至控制盘（ 图4A&#39;）的眼部分单元串仍然受限。阻断JNK信号导致TRE-红色荧光蛋白信号的克隆RAS V12 scrib 1 JNK DN肿瘤（ 图4C，4D）显着降低。因此，这些数据为JNK信号以激活在恶性ras基因V12 scrib 1肿瘤TRE依赖性转录反应的要求提供功能的证据。谱系特异性hmlΔ-DsRed的记者，设计用于跟踪果蝇血细胞27,28表明免疫细胞在组织中的轴承恶性ras基因的V12 scrib 1肿瘤（ 图5B）的积累。与此相反，在对照和ras V12 scrib 1 JNK DN镶嵌EAD和脑组织中检测到仅单个血细胞或几个局部血球补丁（ 图5A，5C）。与泛血球抗Hemese抗体的免疫染色（H2）29证实hmlΔ，红色荧光蛋白阳性细胞（ 图5D，5E）的免疫细胞识别。与公开的报道12,23,24一致，肿瘤侵袭的偏见量化证实JNK的核心作用，促进肿瘤的侵袭到邻近脑叶和VNC（ 图3C）的信号。最后，总RNA从马赛克EAD隔离。对样品进行要么定量RT-PCR或分析质量和数量的自动化电泳系统。 图6A显示基质金属蛋白酶显著JNK依赖上调1（MMP1）成绩单EAD轴承恶性RAS V12 scrib 1克隆肿瘤，证实了先前公布调查结果12,24,30,31。使用自动化电泳系统的总RNA评估表现为T帽子三个独立的EAD样品具有28S / 18S比上述1.8（ 图6B）。然而，EAD 2的RNA是部分降解，由凝胶（ 图6C）上的涂抹反射。与此相反，EAD 3样品具有低的浓度。凝胶图像（ 图6B）和小峰上电泳（ 图6C）的较高分子量的多条带，还建议DNA污染。因此，仅EAD 1样品将是适合于制备的mRNA-SEQ库。 <img alt="图1" src="/files/ftp_upload/54585/54585fig1.jpg" /> 图1: 用于产生在果蝇 EAD和下游应用 遗传马赛克的MARCM系统的原理图 （A）中的MARCM系统允许产生具有定义的遗传病变克隆斑块的在另外的野生型（杂合子）。上下文。 FLP的组织特异性启动子（ 例如， 无眼 ）的控制下表达催化两个FRT元件侧翼STOP盒之间的重组标有黄色（y）的基因（ 动作&gt; Y +&gt; Gal4的 ）。在除去停止盒时，普遍表达Gal4的转录激活因子（ACT-GAL4）可以驱动任何基于无人机系统，转基因，如UAS-GFP（也UAS-ras基因V12）的表达。在一个亲代细胞，但是，Gal4的活性是通过一个GAL80阻抑其表达由微管蛋白启动子（ 桶-GAL80）控制的阻止。在细胞周期的G2期，FLP介导远端的FRT位点的同源染色体之间的非姐妹染色单体的交换。重组的染色体的有丝分裂过程中的偏析会产生两个子细胞，对于特定的基因组位点之一是纯合的突变体（ 例如 ，scrib <s<html>达&gt; 1），而其他将携带两个野生型等位基因。在纯合突变细胞GAL80的损失将允许的GFP表达。相反，野生型姐妹保持GFP阴性。 （ 二 ）第三龄幼虫的示意图描述了前方连接口钩和向后通过光秆大脑中的EAD对。该eyFLP-MARCM系统诱导EAD和脑叶的神经上皮内GFP标记的克隆。 （C）的解剖EAD和大脑可以进行多样的下游应用，包括肿瘤侵袭的免疫化学和转基因报告基因活性（ⅰ）的检测，定量（ii）和转录分析用的候选（QRT-PCR）或无偏的方法（表达-seq）（ 三 ）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54585/54585fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <html> "FO:保together.within页="1"&gt; <img alt="图2" src="/files/ftp_upload/54585/54585fig2.jpg" /> 图2: 三龄幼虫和EAD和EAD的典型例子排序/用脑配合不同的下游应用 （AB）与eyFLP-MARCM 82B青仪引起的第三龄幼虫轴承控制GFP标记克隆的荧光显微照片。 （A）代表控制幼虫与限制在EAD和脑叶克隆。 （B）一种"豹幼虫"搭载在各种组织中的GFP阳性克隆整个身体的一个例子。解剖EAD并附着在口钩（D）的EAD /脑络合物，和（E）EAD（C）的明视场图像从所有外部组织，包括口钩清洗。比例尺= 2毫米（AB）和500微米（CE）。fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/54585/54585fig3.jpg" /> 图 3: 肿瘤的侵袭性的定量 （A）大脑的荧光共焦图片来自RAS V12 scrib 1幼虫解剖（7天AEL）代表了四种不同等级的肿瘤侵袭范围从非侵入性的真实案例（0分） 1脑叶侵入（分数1），侵入二者脑叶（分数2），以一个很强的肿瘤浸润与克隆组织涵盖脑叶和进入的VNC（分数3）。图像是对得分2的多个共焦部分和实施例凸起和3是从2×2共焦图象拼接。比例尺= 100微米。（B）用于偏见的固定大脑的匿名显微镜载玻片的例子荧光立体显微镜下得分。拿下大脑标记笔防止重复注册。比例尺= 500微米（C）相对于高度侵袭性RAS V12 scrib 1肿瘤，抑制JNK信号（RAS V12 scrib 1 JNK DN）克隆细胞的侵袭淘汰。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54585/54585fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54585/54585fig4.jpg" /> 图4: 转录 TRE-DsRed的 记者在恶性 ras基因 "V12"scrib&#39;1 克隆肿瘤的 激活 JNK依赖（A。 </STRONG&gt;）在控制EAD（6天AEL）中，JNK敏感TRE-红色荧光蛋白标记记者从触角眼睛部分（箭头）运行细胞的窄条纹。 （B）中的TRE-DsRed的报道的活性ras基因的V12 scrib显着增强1 GFP阳性克隆比周围非克隆EAD上皮。 （C）JNK活性的抑制导致RAS V12 scrib 1 JNK DN克隆明显减少红色荧光信号强度。 （ 四 ）红色荧光蛋白信号的进一步加强揭示了围绕RAS V12 scrib 1 JNK DN克隆细胞信号传导JNK的非自治激活。 （E）在每天8 AEL，RAS V12 scrib 1细胞茂密生长的整个EAD和分布在脑叶的VNC（箭头）。 （F）的克隆的细胞侵入的区域M的VNC（特写）在E中的箭头arked富集的红色荧光蛋白信号。的多个共焦切片（AC）显示突起，（E）是由3×3共焦图象拼接和（D，F）的代表单个部分。比例尺= 50微米。 EAD，眼睛/触角光盘; BL，脑叶; VNC，腹神经索。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54585/54585fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54585/54585fig5.jpg" /> 图 5: 天堂特定 HMLΔ-DsRed 转基因记者透露 RAS V12 scrib 富集 1 肿瘤相关血细胞 （A）在控制EAD，hmlΔ-红色荧光蛋白记者标记的血细胞CLU<html>讲演者被困在眼睛和触角上皮细胞的压痕。 （ 二 ）由RAS V12 scrib的EAD和脑组织1 GFP标记的肿瘤呈散在各地的克隆组织血细胞数量的增加。 （ 三 ）有关血细胞的数量大大抑制后JNK信号在RAS V12 scrib 1 JNK DN镶嵌组织减少。 （DE）HmlΔ-红色荧光蛋白阳性血细胞也标有H2-抗体检测泛血球标记Hemese。 （AC）的多个共焦截面中，（A）是从2×2和（BC）缝合显示突起从3×3共焦图象拼接。 （DE）代表一个部分。比例尺= 100微米（AC）和10微米（DE）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54585/54585fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> e_content"FO: - together.within页保留="1"&gt; <img alt="图6" src="/files/ftp_upload/54585/54585fig6.jpg" /> 图6:。 数量和EAD解剖分离的总RNA的质量评价 （ 一 ）使用各基因型MMP1转录水平的4种生物实时定量重复-PCR结果的代表性的例子正常化RP49。在基因表达的倍数变化用的相对标准曲线法32计算。数据是平均值±标准差; *** P &lt;0.001使用学生配对双尾t检验与方差不等。 （B，C）使用自动化电泳系统总RNA的质量和数量的评价。虚拟凝胶图像（B）表示对应于上电泳的良好定义的峰（℃）的18S和28S个rRNA的两个突出的频带。 DNA污染和RNA的降解是由反映MEAR（箭头）和extranumerary带和峰（箭头）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54585/54585fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 飞株 </td><td> 来源/评论 </td></tr><tr><td> eyFLP1;法&gt; Y +&gt; Gal4的，UAS-GFP; FRT82B，浴缸，GAL80 </td><td> eyFLP-82B MARCM绿色测试11 </td></tr><tr><td> 瓦; UAS-ras基因V12; FRT82B scrib 1 / TM6B </td><td> 12 </td></tr><tr><td> 瓦; UAS-ras基因V12; UAS-JNK DN FRT82B scrib 1 / TM6B </td><td> 12 </td></tr><tr><td> 瓦; hmlΔ，红色荧光蛋白; FRT82B </td><td>这项研究中， 瓦; hmlΔ -红色荧光蛋白从卡佳布鲁克纳28 </td></tr><tr><td> W¯¯; UAS-ras基因V12hmlΔ，红色荧光蛋白; FRT82B scrib 1 / TM6B </td><td>这项研究</td></tr><tr><td> 瓦; UAS-ras基因V12hmlΔ，红色荧光蛋白; UAS-JNK DN FRT82B scrib 1 / TM6B </td><td>这项研究</td></tr><tr><td> 瓦; TRE-红色荧光蛋白; FRT82B </td><td>这项研究中， 瓦; TRE-红色荧光蛋白从德克Bohmann 26 </td></tr><tr><td> 瓦; UAS-ras基因V12 TRE-红色荧光蛋白; FRT82B scrib 1 / TM6B </td><td>这项研究</td></tr><tr><td> 瓦; UAS-ras基因V12 TRE-红色荧光蛋白; UAS-JNK DN FRT82B scrib 1 / TM6B </td><td>这项研究</td></tr></tbody></table> 表1: 本研究中使用 果蝇 线 综述 。

Watch this Scientific Journal Video about The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics at JoVE.com

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

该协议将演示如何生成的果蝇眼睛/触角成虫盘（EAD）荧光标记，基因定义克隆肿瘤。它介绍了如何从第三龄幼虫，以及如何处理它们的可视化和量化基因表达的变化和肿瘤的侵袭性解剖EAD和大脑。

该<em&gt;果蝇</em&gt;成虫盘瘤模型:基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

the-drosophila-imaginal-disc-tumor-model-visualization-quantification

Nanyang Technological University

Research

JoVE Journal

Cancer Research

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

11.0K 次观看.  University of Cologne. 该协议将演示如何生成的果蝇眼睛/触角成虫盘（EAD）荧光标记，基因定义克隆肿瘤。它介绍了如何从第三龄幼虫，以及如何处理它们的可视化和量化基因表达的变化和肿瘤的侵袭性解剖EAD和大脑。 

视频: The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

小鼠原位膀胱肿瘤模型和肿瘤检测系统

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

科研

癌症研究

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

In the early stages of cancer, transformed mutant cells show cytological abnormalities, begin uncontrolled overgrowth, and progressively disrupt tissue organization. Drosophila melanogaster has emerged as a popular experimental model system in cancer biology to study the genetic and cellular mechanisms of tumorigenesis. In particular, genetic tools for Drosophila imaginal discs (developing epithelia in larvae) enable the creation of transformed pro-tumor cells within a normal epithelial tissue, a situation similar to the initial stages of human cancer. A recent study of tumorigenesis in Drosophila wing imaginal discs, however, showed that tumor initiation depends on the tissue-intrinsic cytoarchitecture and the local microenvironment, suggesting that it is important to consider the region-specific susceptibility to tumorigenic stimuli in evaluating tumor phenotypes in imaginal discs. To facilitate phenotypic analysis of tumor progression in imaginal discs, here we describe a protocol for genetic experiments using the GAL4-UAS system to induce neoplastic tumors in wing imaginal discs. We further introduce a diagnosis method to classify the phenotypes of clonal lesions induced in imaginal epithelia, as a clear classification method to discriminate various stages of tumor progression (such as hyperplasia, dysplasia, or neoplasia) had not been described before. These methods might be broadly applicable to the clonal analysis of tumor phenotypes in various organs in Drosophila.

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

Mosaic clone analysis in Drosophila imaginal disc epithelia is a powerful model system to study the genetic and cellular mechanisms of tumorigenesis. Here we describe a protocol to induce tumors in Drosophila wing imaginal discs using the GAL4-UAS system, and introduce a diagnosis method to classify the tumor phenotypes.

诱导和诊断肿瘤<em&gt;果蝇</em&gt; Imaginal Disc Epithelia

In the early stages of cancer, transformed mutant cells show cytological abnormalities, begin uncontrolled overgrowth, and progressively disrupt tissue ...

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

肿瘤细胞系基因表达的条件性击倒研究单核/巨噬细胞在微环境中的招募

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

肺定植法在肺部组织循环肿瘤细胞可视化中的建立

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

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 ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

脱细胞和细胞肺模型研究肿瘤转移

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene disruption, many of these studies have made use of complex gene knockout models. While these studies with knockout mice offer an elegant and time-tested system for investigating genotype-to-phenotype relationships, a rapid and scalable method for assessing candidate genes that play a role in AML cell proliferation or survival in AML models will help accelerate the parallel interrogation of multiple candidate genes. Recent advances in genome-editing technologies have dramatically enhanced our ability to perform genetic perturbations at an unprecedented scale. One such system of genome editing is the CRISPR-Cas9-based method that can be used to make rapid and efficacious alterations in the target cell genome. The ease and scalability of CRISPR/Cas9-mediated gene-deletion makes it one of the most attractive techniques for the interrogation of a large number of genes in phenotypic assays. Here, we present a simple assay using CRISPR/Cas9 mediated gene-disruption combined with high-throughput flow-cytometry-based competition assays to investigate the role of genes that may play an important role in the proliferation or survival of human and murine AML cell lines.

This manuscript describes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR-Cas9-based method for simple and expeditious investigation of the role of multiple candidate genes in Acute Myeloid Leukemia (AML) cell proliferation in parallel. This technique is scalable and can be applied in other cancer cell lines as well.

基于 crispr-cas9 的竞争检测对遗传依赖性的调查

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene ...

The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% with current standard of care. Tumors often recur within 9 months following initial surgery, radiation and chemotherapy, at which point treatment options become limited. This highlights the pressing need for the development of better therapeutics to prolong survival and increase the quality of life for these patients.
Tumor Treating Fields (TTFields) therapy was developed to take advantage of the effect of low frequency alternating electrical fields on cells for cancer therapy. TTFields have been demonstrated to disrupt cells during mitosis and slow tumor growth. There is also growing evidence that they act through stimulating immune responses within exposed tumors. The advantages of TTFields therapy include its noninvasive approach and increased quality of life compared to other treatment modalities such as cytotoxic chemotherapies. The Food and Drug Administration approved TTFields therapy for the treatment of recurrent glioblastoma in 2011 and for newly diagnosed glioblastoma in 2015. We report on the effects of TTFields during mitosis, the results of electric fields modeling, and proper transducer array placement. Our protocol outlines the clinical application of TTFields on a patient post-surgery, using the second-generation device.

Glioblastoma is the most common and aggressive primary brain malignancy in adults, with most tumors recurring after initial treatment. Tumor Treating Fields (TTFields) therapy is the newest treatment modality for glioblastoma. Here, we describe the proper application of TTFields-transducer arrays on patients and discuss theory and aspects of treatment.

肿瘤治疗胶质母细胞瘤的临床应用

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.