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?...

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><tbody><tr><td>Agar</td><td>Gew&amp;uuml;rzm&amp;uuml;hle Brecht, Eggenstein, Germany</td><td>00262-0500</td><td>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&amp;nbsp;&amp;deg;C.</td></tr><tr><td>Corn syrup</td><td>Grafschafter Krautfabrik Josef Schmitz KG, Meckenheim, Germany</td><td>01939</td><td></td></tr><tr><td>Propionic acid</td><td>Carl Roth GmbH, Karlsruhe, Germany</td><td>6026.1</td><td></td></tr><tr><td>Cornmeal</td><td>ReformKontor GmbH, Zarrentin, Germany</td><td>4010155063948</td><td></td></tr><tr><td>Malt extract</td><td>CSM Deutschland GmbH, Bremen, Germany</td><td>728985</td><td></td></tr><tr><td>Soy flour</td><td>Stockmeier Food GmbH, Herford, Germany</td><td>1000246441010</td><td></td></tr><tr><td>Yeast</td><td>Werner Ramspeck GmbH, Schwabach, Germany</td><td>210099K</td><td></td></tr><tr><td>Methyl-4-benzoate/ Nipagin</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>H5501</td><td>Prepare a 15% stock solution with 70% EtOH</td></tr><tr><td>Drosophila fly food vials</td><td>Kisker Biotech, Steinfurt, Germany</td><td>789008</td><td></td></tr><tr><td>Vial plugs</td><td>K-TK e.K., Retzstadt, Germany</td><td>1002</td><td></td></tr><tr><td>Drosophila fly food bottles</td><td>Greiner Bio-One, Frickenhausen, Germany</td><td>960177</td><td></td></tr><tr><td>Bottle plugs</td><td>K-TK e.K., Retzstadt, Germany</td><td>1002 S</td><td></td></tr><tr><td>Poly(vinyl alcohol) 4-88/[-CH&lt;sub&gt;2&lt;/sub&gt;CHOH-]&lt;sub&gt;n&lt;/sub&gt;</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>81381</td><td>Mounting medium recipe: Dissolve 6 g Poly(vinyl alcohol) 4-88 in 39&amp;nbsp;ml Millipore H&lt;sub&gt;2&lt;/sub&gt;O, 6 ml 1 M Tris (pH 8.5) and 12.5 ml glycerol. Stir overnight at 50&amp;nbsp;&amp;deg;C and centrifuge 20&amp;nbsp;min at 5,000 rpm. Add DABCO to the supernatant to get a final concentration of 2.5%. Store aliquots at -20&amp;nbsp;&amp;deg;C.</td></tr><tr><td>1,4-Diazabicyclo[2.2.2]octane/ DABCO&amp;nbsp;</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>D2522</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>9002-93-1</td><td></td></tr><tr><td>Phosphate-buffered saline/ PBS</td><td></td><td></td><td>137 mM NaCl, 2.7 mM KCl, 10 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; and 1.8 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, pH 7.4 in Millipore H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>PBST</td><td></td><td></td><td>0.1% Triton X-100 in PBS</td></tr><tr><td>Paraformaldehyde/ PFA</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>158127</td><td>4% PFA fixative recipe: Dissolve 4&amp;nbsp;g PFA in 80 ml Millipore H&lt;sub&gt;2&lt;/sub&gt;O on a magnetic stirrer plate heated to 55&amp;nbsp;&amp;deg;C. Add 1 M NaOH dropwise until all PFA particles are dissolved. Add 10 ml 10x PBS and adjust the pH to 7.4 with 1 M HCl. Mix in 100 &amp;micro;l Triton X-100 and fill up with Millipore H&lt;sub&gt;2&lt;/sub&gt;O to 100 ml. Store aliquots at -20 &amp;deg;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><td>Bovine Serum Albumin/ BSA</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>A3059</td><td>Blocking solution recipe: Dissolve 0.3% BSA in PBST</td></tr><tr><td>Fluorescently labeled phalloidin</td><td>Invitrogen, Karlsruhe, Germany, Molecular Probes</td><td>A12379, A22283, A22284</td><td>Phalloidin stock solution: Dissolve powder (300 U) in 1.5 ml Methanol. Store at -20 &amp;deg;C. For staining, use in dilution 1:250 (A12379, A22283) and 1:100 (A22284) in PBST.</td></tr><tr><td>4&amp;rsquo;,6-Diamidino-2-phenylindol Dihydrochlorid/ DAPI</td><td>Carl Roth GmbH, Karlsruhe, Germany</td><td>6335</td><td>DAPI staining solution: Prepare a stock solution of 5 mg/ml in Millipore H&lt;sub&gt;2&lt;/sub&gt;O and store aliquots at 4 &amp;deg;C. Used in dilution 1:1,000 in PBST.</td></tr><tr><td>Mouse anti-H2 antibody</td><td>Kurucz et al., 2003</td><td></td><td>Used in dilution 1:500 in blocking solution</td></tr><tr><td>Cy5 AffiniPure Donkey Anti-Mouse IgG (H+L)</td><td>Jackson ImmunoResearch, Suffolk, UK</td><td>715-175-151</td><td>Used in dilution 1:500 in blocking solution</td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools, Heidelberg, Germany</td><td>11295-10</td><td></td></tr><tr><td>Glass embryo dish (30&amp;nbsp;mm)</td><td>Thermo Scientific, Schwerte, Germany</td><td>E90</td><td></td></tr><tr><td>Tungsten needles</td><td>Fine Science Tools, Heidelberg, Germany</td><td>10130-20</td><td></td></tr><tr><td>Nickel Plated Pin Holder</td><td>Fine Science Tools, Heidelberg, Germany</td><td>26018-17</td><td></td></tr><tr><td>Microscope slides</td><td>VWR, Darmstadt, Germany</td><td>631-1553</td><td></td></tr><tr><td>Coverslips 22 x 22 mm (#1 Menzel-Gl&amp;auml;ser)</td><td>VWR, Darmstadt, Germany</td><td>631-1336</td><td></td></tr><tr><td>Coverslips 24 x 50 mm (0.13-0.16&amp;nbsp;mm)</td><td>Thermo Scientific, Schwerte, Germany</td><td>1076371</td><td></td></tr><tr><td>Kimtech Science Precision Wipes</td><td>Thermo Scientific, Schwerte, Germany</td><td>06-677-70</td><td></td></tr><tr><td>Dissecting stereomicroscope</td><td>Olympus, Hamburg, Germany</td><td>SZX7 (DF PLAPO 1X-4 Japan)</td><td></td></tr><tr><td>Fluorescent stereomicroscope</td><td>Olympus, Hamburg, Germany</td><td>SZX16 (SDF PLAPO 0.8X Japan) with DP72 CCD camera</td><td>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><td>Confocal microscope</td><td>Olympus, Hamburg, Germany</td><td>FV1000</td><td>Equipped with inverted IX81 microscope. Objectives: 20&amp;times; UPlan S-Apo (NA 0.85), 40&amp;times; UPlanFL (NA 1.30) and 60&amp;times; UPlanApo (NA 1.35). Lasers: UV laser Diode LD405 (50&amp;nbsp;mW), Argon laser, multi-line 457/(476)/ 488/515 (40&amp;nbsp;mW), Yellow/Green laser diode LD560 (15&amp;nbsp;mW) and Red Laser diode 635 (20&amp;nbsp;mW). Software: Fluoview 2.1c Software</td></tr><tr><td>Drosophila cooled incubator</td><td>Ewald Innovationstechnik GmbH, Bad Nenndorf, Germany</td><td>&amp;nbsp;Sanyo MIR553</td><td></td></tr><tr><td>Squirt bottle</td><td>VWR, Darmstadt, Germany</td><td>215-8105</td><td></td></tr><tr><td>BD Clay Adams Nutator Mixer</td><td>VWR, Darmstadt, Germany</td><td>15172-203</td><td></td></tr><tr><td>ELMI Digital Rocking Shaker</td><td>VWR, Darmstadt, Germany</td><td>DRS-12</td><td></td></tr><tr><td>5 PRIME Isol-RNA Lysis Reagent</td><td>VWR, Darmstadt, Germany</td><td>2302700</td><td>The protocol is compatible with other TRIzol-based reagents, &lt;em&gt;e.g.&lt;/em&gt;, TRIreagent from Sigma (Cat. Nr.T9424), TRIzol reagent from Thermofisher (Cat. Nr. 15596).</td></tr><tr><td>Diethyl pyrocarbonate/ DEPC</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>D5758</td><td>Dilute DEPC 1:1,000 in Millipore H&lt;sub&gt;2&lt;/sub&gt;O, stir overnight and autoclave.</td></tr><tr><td>UltraPure Phenol:Chloroform:Isoamyl Alcohol (25:24:1)</td><td>ThermoFischer Scientific, Invitrogen, Karlsruhe, Germany</td><td>15593-031</td><td></td></tr><tr><td>Chloroform</td><td>Merck, Darmstadt, Germany</td><td>102445</td><td></td></tr><tr><td>TURBO DNase (2 U/&amp;micro;l)</td><td>Thermo Scientific, Schwerte, Germany</td><td>AM2238</td><td></td></tr><tr><td>Invitrogen UltraPure Glycogen</td><td>ThermoFischer Scientific, Invitrogen, Karlsruhe, Germany</td><td>10-814-010</td><td></td></tr><tr><td>Sodium acetate trihydrate</td><td>VWR, Darmstadt, Germany</td><td>27652.232</td><td>Prepare a 3 M Sodium acetate solution with DEPC-H&lt;sub&gt;2&lt;/sub&gt;O and adjust the pH to 5.2</td></tr><tr><td>2-Propanol</td><td>Merck, Darmstadt, Germany</td><td>109634</td><td></td></tr><tr><td>Ethanol</td><td>Merck, Darmstadt, Germany</td><td>100983</td><td>Prepare a 75% EtOH dilution with DEPC-H&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>UV/Vis-Spectrophotometre NanoDrop ND-8000</td><td>Thermo Scientific, Schwerte, Germany</td><td>ND-8000</td><td></td></tr><tr><td>Eppendorf Microcentrifuge (Refrigerated)</td><td>Thermo Scientific, Schwerte, Germany</td><td>5417R</td><td></td></tr><tr><td>Experion RNA StdSens Analysis Kit</td><td>Bio-Rad Laboratories GmbH, M&amp;uuml;nchen, Germany</td><td>7007103</td><td></td></tr><tr><td>Experion Automated Electrophoresis Station</td><td>Bio-Rad Laboratories GmbH, M&amp;uuml;nchen, Germany</td><td>7007010</td><td></td></tr><tr><td>SuperScript III Reverse Transcriptase</td><td>Thermo Scientific, Schwerte, Germany</td><td>18080044</td><td></td></tr><tr><td>Oligo d(T) Primer</td><td>Integrated DNA Technologies, Leuven, Belgium</td><td></td><td>Prepare 100 &amp;micro;M stock solutions in DEPC-H&lt;sub&gt;2&lt;/sub&gt;O and store at -20 &amp;deg;C</td></tr><tr><td>dNTP Mixture</td><td>Takara Bio Europe/Clonetech, Saint-Germain-en-Laye, France</td><td>4030</td><td>Store aliquots of 25 &amp;micro;l at -20&amp;deg;C</td></tr><tr><td>CFX96 Touch Real-Time PCR Detection System</td><td>Bio-Rad Laboratories GmbH, M&amp;uuml;nchen, Germany</td><td>1855195</td><td></td></tr><tr><td>iQ SYBR Green Supermix</td><td>Bio-Rad Laboratories GmbH, M&amp;uuml;nchen, Germany</td><td>170-8882</td><td></td></tr><tr><td>Hard-Shell PCR Plates 96-well, thin wall</td><td>Bio-Rad Laboratories GmbH, M&amp;uuml;nchen, Germany</td><td>HSP9601</td><td></td></tr><tr><td>Microseal &#039;B&#039; Film</td><td>Bio-Rad Laboratories GmbH, M&amp;uuml;nchen, Germany</td><td>MSB1001</td><td></td></tr><tr><td>&lt;em&gt;rp49&lt;/em&gt; Forward primer</td><td>Integrated DNA Technologies, Leuven, Belgium</td><td></td><td>5&#039; TCC TAC CAG CTT CAA GAT GAC 3&#039;</td></tr><tr><td>&lt;em&gt;rp49&lt;/em&gt; Reverse primer</td><td>Integrated DNA Technologies, Leuven, Belgium</td><td></td><td>5&#039; CAC GTT GTG CAC CAG GAA CT 3&#039;</td></tr><tr><td>&lt;em&gt;mmp1&lt;/em&gt; Forward primer</td><td>Integrated DNA Technologies, Leuven, Belgium</td><td></td><td>5&#039; AGG GCG ACA AGT ACT ACA AGC TGA 3&#039;</td></tr><tr><td>&lt;em&gt;mmp1&lt;/em&gt; Reverse primer</td><td>Integrated DNA Technologies, Leuven, Belgium</td><td></td><td>5&#039; ACG TCT TGC CGT TCT TGT AGG TGA 3&#039;</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...

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

Universidad de Cartagena UDEC

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

Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers. To robustly and efficiently identify oncogenic &#8220;drivers&#8221; among these tumors and define their complex relationships with concurrent genetic alterations during tumor pathogenesis remains a daunting task. Recently, zebrafish have emerged as an important animal model for studying human diseases, largely because of their ease of maintenance, high fecundity, obvious advantages for in vivo imaging, high conservation of oncogenes and their molecular pathways, susceptibility to tumorigenesis and, most importantly, the availability of transgenic techniques suitable for use in the fish. Transgenic zebrafish models of cancer have been widely used to dissect oncogenic pathways in diverse tumor types. However, developing a stable transgenic fish model is both tedious and time-consuming, and it is even more difficult and more time-consuming to dissect the cooperation of multiple genes in disease pathogenesis using this approach, which requires the generation of multiple transgenic lines with overexpression of the individual genes of interest followed by complicated breeding of these stable transgenic lines. Hence, use of a mosaic transient transgenic approach in zebrafish offers unique advantages for functional genomic analysis in vivo. Briefly, candidate transgenes can be coinjected into one-cell-stage wild-type or transgenic zebrafish embryos and allowed to integrate together into each somatic cell in a mosaic pattern that leads to mixed genotypes in the same primarily injected animal. This permits one to investigate in a faster and less expensive manner whether and how the candidate genes can collaborate with each other to drive tumorigenesis. By transient overexpression of activated ALK in the transgenic fish overexpressing MYCN, we demonstrate here the cooperation of these two oncogenes in the pathogenesis of a pediatric cancer, neuroblastoma that has resisted most forms of contemporary treatment.

The goal of this study is to demonstrate how the mosaic transgenesis strategy can be used in zebrafish to rapidly and efficiently assess the relative contributions of multiple oncogenes in tumor initiation and progression in vivo.

马赛克斑马鱼转基因的候选基因合作在肿瘤发病机制的功能基因组分析

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers.  To robustly and efficiently ...

科研

发育生物学

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 ...

癌症研究

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

形态分析果蝇幼虫外周感觉神经元树突和轴突使用遗传马赛克

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

神经科学

Tumor Allotransplantation in Drosophila melanogaster with a Programmable Auto-Nanoliter Injector

This protocol describes the allotransplantation of tumors in Drosophila melanogaster using an auto-nanoliter injection apparatus. With the use of an autoinjector apparatus, trained operators can achieve more efficient and consistent transplantation results compared to those obtained using a manual injector. Here, we cover topics in a chronological fashion: from the crossing of Drosophila lines, to the induction and dissection of the primary tumor, transplantation of the primary tumor into a new adult host and continued generational transplantation of the tumor for extended studies. As a demonstration, here we use Notch intracellular domain (NICD) overexpression induced salivary gland imaginal ring tumors for generational transplantation. These tumors can first be reliably induced in a transition-zone microenvironment within larval salivary gland imaginal rings, then allografted and cultured in vivo to study continued tumor growth, evolution, and metastasis. This allotransplantation method can be useful in potential drug screening programs, as well as for studying tumor-host interactions.

Tumor Allotransplantation in Drosophila melanogaster with a Programmable Auto-Nanoliter Injector

This protocol provides detailed guidance for the initial and continued generational allotransplantation of Drosophila tumors into the abdomen of adult hosts for studying various aspects of neoplasia. Using an autoinjector apparatus, researchers can achieve improved efficiency and tumor yields compared to those achieved by traditional, manual methods.

使用可编程自动纳升注射器在 黑色果蝇 中植入肿瘤异体

This protocol describes the allotransplantation of tumors in Drosophila melanogaster using an auto-nanoliter injection apparatus. With the use of an ...

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Drosophila has illuminated our understanding of the genetic basis of normal development and disease for the past several decades and today it continues to contribute immensely to our understanding of complex diseases 1-7. Progression of tumors from a benign to a metastatic state is a complex event 8 and has been modeled in Drosophila to help us better understand the genetic basis of this disease 9. Here I present a simple protocol to genetically induce, observe and then analyze the progression of tumors in Drosophila larvae. The tumor induction technique is based on the MARCM system 10 and exploits the cooperation between an activated oncogene, RasV12 and loss of cell polarity genes (scribbled, discs large and lethal giant larvae) to generate invasive tumors 9. I demonstrate how these tumors can be visualized in the intact larvae and then how these can be dissected out for further analysis. The simplified protocol presented here should make it possible for this technique to be utilized by investigators interested in understanding the role of a gene in tumor invasion.

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Cooperation between an activated oncogene like RASV12 and mutations in cell polarity genes like scribbled, result in tumor growth in Drosophila where tumor cells also display invasive behaviors. Here a simple protocol for the induction and observation of the benign and invasive tumors is presented.

一个协议，用于基因诱导和良性和浸润性肿瘤可视化的头侧配合<em&gt;果蝇</em

Drosophila has illuminated our  understanding of the genetic basis of normal development and disease for the  past several decades and today it continues ...

医学

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described here is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

普帕尔阶段发育性皮瘤中组织定向和生长动力学的成像与分析

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

激光显微切割的从细胞的一个子集基因表达分析中的应用果蝇永光盘

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

生物学

A Drosophila Model to Study Wound-induced Polyploidization

Polyploidy is a frequent phenomenon whose impact on organismal health and disease is still poorly understood. A cell is defined as polyploid if it contains more than the diploid copy of its chromosomes, which is a result of endoreplication or cell fusion. In tissue repair, wound-induced polyploidization (WIP) has been found to be a conserved healing strategy from fruit flies to vertebrates. WIP has several advantages over cell proliferation, including resistance to oncogenic growth and genotoxic stress. The challenge has been to identify why polyploid cells arise and how these unique cells function. Provided is a detailed protocol to study WIP in the adult fruit fly epithelium where polyploid cells are generated within 2 days after a puncture wound. Taking advantage of D. melanogaster&#39;s extensive genetic tool kit, the genes required to initiate and regulate WIP, including Myc, have begun to be identified. Continued studies using this method can reveal how other genetic and physiological variables including sex, diet, and age regulate and influence WIP&#39;s function.

A Drosophila Model to Study Wound-induced Polyploidization

Wound-induced polyploidization is a conserved tissue repair strategy where cells grow in size instead of dividing to compensate for cell loss. Here is a detailed protocol on how to use the fruit fly as a model to measure ploidy and its genetic regulation in epithelial wound repair.

研究伤口引起的多倍体化的果蝇模型

Polyploidy is a frequent phenomenon whose impact on organismal health and disease is still poorly understood. A cell is defined as polyploid if it ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

番茄/ GFP-FLP / FRT方法马赛克成人的实时成像<em&gt;果蝇</em&gt;感光细胞

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

解剖和免疫染色成虫盘的<em&gt;果蝇</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

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马赛克斑马鱼转基因的候选基因合作在肿瘤发病机制的功能基因组分析

诱导和诊断肿瘤

形态分析果蝇幼虫外周感觉神经元树突和轴突使用遗传马赛克

使用可编程自动纳升注射器在黑色果蝇中植入肿瘤异体

一个协议，用于基因诱导和良性和浸润性肿瘤可视化的头侧配合

普帕尔阶段发育性皮瘤中组织定向和生长动力学的成像与分析

激光显微切割的从细胞的一个子集基因表达分析中的应用果蝇永光盘

研究伤口引起的多倍体化的果蝇模型

番茄/ GFP-FLP / FRT方法马赛克成人的实时成像

解剖和免疫染色成虫盘的

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摘要

探索更多视频

此视频中的章节

马赛克斑马鱼转基因的候选基因合作在肿瘤发病机制的功能基因组分析

诱导和诊断肿瘤

形态分析果蝇幼虫外周感觉神经元树突和轴突使用遗传马赛克

使用可编程自动纳升注射器在 黑色果蝇 中植入肿瘤异体

一个协议，用于基因诱导和良性和浸润性肿瘤可视化的头侧配合

普帕尔阶段发育性皮瘤中组织定向和生长动力学的成像与分析

激光显微切割的从细胞的一个子集基因表达分析中的应用果蝇永光盘

研究伤口引起的多倍体化的果蝇模型

番茄/ GFP-FLP / FRT方法马赛克成人的实时成像

解剖和免疫染色成虫盘的

使用可编程自动纳升注射器在黑色果蝇中植入肿瘤异体