我们感谢关颖菲舍尔提供的UAS-EGFP :: MSP-300 KASH质粒。该mAB24B10抗体从NICHD的主持下制定的发展研究杂交瘤细胞银行获得由爱荷华大学生物系维持。该回购GAL4股票（BL7415）从布卢明顿库存中心在美国印第安纳大学获得的。支持美国癌症协会的机构研究资助（IRG＃58-006-53）到普渡大学癌症研究中心的感谢。敬群马是由一个农业研究普渡大学助教在粮食和农业普渡大学的支持。

1。绿色荧光蛋白的产生和表征:: KASH 果蝇 <ol><li>十字Gal4的驱动飞往UAS-EGFP :: MSP-300 KASH苍蝇。可替换地，可以使用标准的基因工程技术来产生重组苍蝇，可以同时携带Gal4驱动子和UAS-EGFP :: MSP-300 KASH转基因。这第二种方法是有利的，如果需要大量的后代。 </li><li>表征采用标准的显微技术的EGFP :: KASH标志物的表达。注意：EGFP的表达可以通过标准显微镜技术在解剖，固定果蝇组织中观察到的，或者在整个幼虫，并且不要求使用的抗体。 <ol><li>确定EGFP的表达模式:: KASH标记在整个生物体的荧光解剖显微镜下（参见材料PDF）。注：很多Gal4的驱动程序，包括图1所示的回购GAL4驱动，展览Ñ在其它幼体组织如唾液腺表达onspecific图案（见结果）。 </li><li>分析利益利用共聚焦显微镜解剖组织的EGFP :: KASH的表达模式。 <ol><li>用甲醛在4％用DAPI为0.1微克/毫升的最终浓度以可视化的EGFP的本地化:: KASH标签相对于DNA的终浓度，并染色的DNA修复所关注的组织。 </li><li>使用一个40X / 1.30油浸物镜或20X / 0.75油浸物镜，视所关注的组织的大小获取图像。下面的激发/发射条件，可用于图像采集：408 nm/425-475纳米（DAPI）; 488纳米/ 525-550毫微米（GFP）。 </li></ol></li></ol></li></ol> 2。从果蝇幼虫组织中分离出核 <ol><li>准备设备的幼虫组织解剖及随后的核隔离。需要注意的是整个dechorionated胚胎也可以用作如果需要的起始原料。它建议使用局部解剖幼虫组织，而不是整个幼虫如果感兴趣的细胞类型是存在于一个特定的组织如成虫盘。这降低了非特异性结合，并且也消除了，可以通过在非靶细胞中某些Gal4的司机的表达而引起的问题。 <ol><li>准备两对锋利的钳子（参见材料PDF），1硅化9阱玻璃板（参见材料PDF）和PBT（1×PBS，pH 7.4的0.1％的Tween-20）。 </li><li>的prebind GFP的抗体的磁珠。这一步应该在开始解剖前启动。加入10μl的磁珠（Invitrogen公司，见材料PDF）和2微克GFP抗体（罗氏公司，见材料PDF），以400微升的洗涤缓冲液（1×PBS，pH为7.4; 2.5毫摩尔MgCl 2）在1.5ml管。使用有孔玻璃珠与抗体的量可以在必要时根据目标的SAM中的量进行优化PLE和抗体与珠的结合亲和力。 </li><li>孵育珠和抗体与旋转至少30分钟，在室温下进行。 </li><li>放置1.5 ml管包含珠子和抗体的磁性机架中（参见材料PDF），并允许珠结合到磁性为约1-2分钟。除去含有未结合的抗体上清，用1毫升移液管洗涤缓冲液中。珠粒将保持绑定到1.5 ml管上相邻的磁铁的一侧的壁上。 </li><li>用1 ml洗漂珠两倍，在2.1.4节中描述的每个时间缓冲。从磁机​​架上卸下1.5 ml管和反转简单地混合珠，并在每个洗涤步骤洗涤缓冲液。 </li><li>存储在洗涤缓冲液的抗体结合珠，直到准备继续执行步骤2.7。珠粒不应该被允许干出在协议中的任何阶段。 </li></ol></li><li>解剖幼虫组织中的PBT和解剖组织转移到一井的9井菜。通常情况下，从100三龄幼虫解剖视叶和眼成虫盘提供了足够的材料为下游基因表达分析如下细胞核分离。 </li><li>冲洗分离的组织中核提取缓冲液（10mM HEPES-KOH，pH为7.5; 2.5毫摩尔MgCl 2，10 mM的KCl）中在1.5ml管中。传输孤立幼虫组织到1毫升Dounce匀浆（参见材料PDF）在冰上包含1ml新鲜核提取缓冲液。在冰上孵育5分钟。这是没有必要添加蛋白酶抑制剂如RNA是以下核隔离将被提取。 </li><li> 20笔画与松杵破坏组织，然后在冰上孵育样品10分钟以使细胞溶胀。提取细胞用15招跟紧杵细胞核。需要为不同的组织和细胞类型进行优化冲程的松和紧的杵的数目;过量均质化可导致剪切细胞核，而同质化不足，将无法从组织中提取的所有核。 </li><li>过滤匀浆液，其中含有细胞核和细胞碎片，通过一个40微米的孔隙大小的过滤器（参见材料PDF）已预清洗与核匀浆缓冲液。收集在新管的过滤匀浆。 </li><li>收集预分离样品以供后续分析，以确定核产量，细胞核完整性，并确定以原子核分离前靶基因的转录水平。 <ol><li>转移50微升过滤匀浆至新的1.5mL管中，使用移液管。这是预分离样品。加入500μlTrizol试剂（见材料PDF格式，注意事项），倒置混合，并储存在-20℃，随后的RNA分离（步骤3.1）。 </li><li>转移20微升过滤匀浆至新的1.5毫升管。 <ol><li>加DAPI为0.1μg/ ml的终浓度，并且增量在冰上10分钟ubate样品。 </li><li>转移DAPI染色细胞核到聚赖氨酸包被的盖玻片上，并放置在显微镜载玻片上。用透明指甲油密封盖玻片。 </li><li>检查细胞核完整性，及权益使用荧光显微镜GFP标记的细胞核的存在。注：这是没有必要修复细胞核，或染色的GFP，如果这些幻灯片进行了分析合理迅速下列准备工作（24小时内）。 </li></ol></li><li>取10μl匀浆过滤到一个新的1.5 ml管中，加入90微升的洗涤缓冲液（1×PBS，pH值7.4; 2.5毫米氯化镁2）。加DAPI为0.1μg/ ml的终浓度，并在冰上孵育10分钟。确定细胞核产量预分离样品中使用血球使用下一个10X的空气客观萤光计数DAPI阳性细胞核。 </li></ol></li><li>放置1.5 ml管包含（在2.1.6节的方法制备）在一个磁架的抗体结合珠，并允许磁珠绑定到磁铁至少2分钟在2.1.4节中描述。使用1毫升移液管的抗体结合珠除去洗涤缓冲液，并且从步骤2.5中添加过滤匀浆至1.5 ml管采用1毫升移液管。 </li><li>轻轻颠倒离心管数次以混匀，孵育4℃，30分钟，最终在年底搅动。避免气泡中，如果可能的，因为这些可能会导致核的聚集管。 </li><li>放置1.5 ml管含有抗体结合珠和匀浆中的磁架，并允许磁珠结合到磁体至少2分钟在第2.1.4节中描述。除去用1毫升移液管含有未结合的核部分的匀浆。 </li><li>洗涤样品3次用1 ml洗，每次缓冲。除去1.5毫升的试管含有珠子，束缚核和从机架洗涤缓冲液和颠倒混合数次，然后如上所述将管内的磁性机架中在步骤2.9，并使用1毫升移液管去除上清。 </li><li>加入150μl洗涤缓冲液至珠粒，应结合到靶细胞核。这是后分离样品。制备样品的显微分析和随后的RNA提取。 <ol><li>转移20μL 后隔离样品到新的1.5 ml管和染色用DAPI和在2.6.2节中描述分析。算的GFP + / DAPI +和GFP-/DAPI +磁珠捕获来确定富集的GFP +核的隔离后样品中的纯度的细胞核。 </li><li>加入1 ml Trizol试剂的至后隔离样品的剩余部分，颠倒混合，并储存于-20℃用于后续的RNA分离（步骤3.1）。注意：在Trizol试剂的样品可以储存数周，如果必要，在-20℃下它没有必要从使用Trizol试剂磁珠之前提取RNA洗脱核，的becaus辰珠不干扰随后的RNA分离。 </li></ol></li></ol> 3。确定转录水平的原子核从幼虫组织中分离 <ol><li>使用Trizol试剂为基于RNA提取所述的标准协议（参见材料PDF）进行了以下修改制备章节2.6.1和2.11.2 预分离和隔离后的样品分离RNA： <ol><li>以下将RNA沉淀的沉淀，加19.2微升不含RNA酶的水和0.8微升RNASecure试剂（见材料PDF），以沉淀孵育，在60℃20分钟以灭活RNA酶。 </li></ol></li><li>确定RNA浓度用荧光-RNA结合的染料，如量子比特的RNA测定试剂盒（参见材料PDF）使用QubitR 2.0荧光遵循制造商的协议。 </li><li>使用扩增逆转录酶如EpiScr合成cDNAIPT逆转录试剂盒和寡聚dT引物，按照生产商的说明进行操作。注意：通常情况下，RNA的50到100纳克产生足够的cDNA进行多重基因表达分析。 预分离和隔离后的RNA等量应该被用来生成的cDNA。 </li><li>添加180微升不含核酸酶的水至20微升cDNA样品稀释此10倍之前，实时定量PCR（定量PCR）分析。这种稀释是一个重要的步骤，因为未稀释的cDNA样品可以抑制定量PCR。 </li><li>准备一个定量PCR主混合物与聚合酶和dNTP，各前进4皮摩尔和反向引物，由用无菌水加至20微升的最终反应体积。 <ol><li>设计的qPCR引物对目标预期可被表示为感兴趣的细胞类型的基因，并在在该组织被收集的发育阶段。设计引物，以跨越一个内含子，如果可能的话，使基因组和cDNA的PCR产物可以被区分开来。注意：如果靶细胞类型的基因表达模式是未知的，针对绿色荧光蛋白 ，其应在标签的细胞核群特异性表达的引物，可以被用来优化协议为特定的细胞类型和组织（ 表1）。 </li></ol></li><li>通过比较以系列稀释的cDNA从整个组织制备确定的预分离和隔离后的样品中每个感兴趣的基因的相对转录水平。靶基因的转录水平应该归到参考基因，如RPL32（或几个较好的参考基因）。 </li></ol>

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作者什么都没有透露。

这个协议可以用于分离或富集高度从在果蝇胚胎或幼虫组织的混合细胞群特异性标记细胞核。使用此协议，原子核可以从解剖组织中约1小时隔离。分离的核的纯度和产率，必须为每种细胞类型由实验确定。它可以是难以量化，因为在样品中存在的珠的珠结合的原子核在使用血细胞计数协议的隔离后阶段。因此，如果所需的下游应用核的确切数目，这将是必要估计的基础上的另一种方法，如...

果蝇组织例如中枢神经系统包含的细胞类型组成的复杂混合物。因此，分析从果蝇组织细胞特异性的基因表达图谱，它首先要分离特定细胞的同源性的群体以足够的数量，以使下游应用。方法来分离从完整组织细胞包括激光显微切割和荧光激活细胞的全细胞的分选（FACS）。而FACS已用于分离细胞和细胞核从果蝇胚胎和秀丽隐杆线虫基因表达和染色质分析1-3，流式细胞仪和激光显微切割可能难以在包含高度混杂的细胞类型或包含细胞的组织中成功地执行不规​​则形态，如神经元。为了克服这个困难，核，而不是细胞可以从特定的细胞类型中分离出来，并用于随后根E蛋白表达谱。重要的是，利用核RNA样品微阵列基因表达分析显示，总体比较的结果与使用总RNA 4，5的执行。此外，当使用核RNA的基因表达分析已经成功地用于研究基因表达的多种生物，包括C。线虫 ， 拟南芥 ， 果蝇和人4，5 2，3。 几种方法最近已被描述为从果蝇组织是适合于基因表达分析和/或染色质免疫标记的细胞核的特定人群的隔离。组织特异性染色质免疫沉淀（BITS-CHIP）方法的批次隔离采用流式细胞仪分离固定核细胞类型特异性核本地化的绿色荧光蛋白2表达的基础上。这种做法一直苏ccessfully用于分析的组蛋白修饰和转录因子的使用分离的细胞核的染色质免疫沉淀从果蝇胚胎2的中胚层的分布。然而，基于FACS的方法可能不太适合于分离构成，由于需要获得合适的数量为下游应用的增加排序时间只有一小部分的混合群体的标记的细胞核。为了克服这些限制，几个小组已经利用基于亲和力的分离技术来净化该被标记为在特定细胞类型的特定表位的核。细胞核标记在特定细胞类型（完整）的方法在拟南芥6,7中使用而开发的隔离最近已适于在果蝇 8使用。在该方法中，核膜融合蛋白的底物在体内生物素化是coexpresSED与大肠杆菌生物素连接酶BirA的在特定细胞类型。生物素标记的细胞核可以通过使用链霉亲和亲和隔离混合人群随后纯化。使用这种方法，细胞核被成功标记和分离，其中，核膜融合蛋白，下一个胚层特异性增强子8的控制下表达果蝇胚胎的中胚层。作者还生成可以在任何细胞类型下的Gal4的调控序列，9无人机系统的控制下表达核膜融合蛋白。这种方法能够快速分离由混合种群标记的细胞核的子集，但需要三个独立的转基因构建体，因此可能不适合于特定基因的应用。最近，一个方法已经被描述，其中孙（S AD1和UN C-84），该定位于核膜的内膜结构域的蛋白，具有标记荧光蛋白，并根据GAL4/UAS系统10的控制下表达。细胞核中分离出的非离子型洗涤剂的存在下除去核膜的外膜，并使用连接到抗GFP抗体的磁珠亲和纯化。这种方法被成功用于隔离果蝇 10的成年大脑内特定神经元亚型标记的细胞核的小种群。 这里，一个协议，用于从果蝇幼虫的组织细胞的混合群标记的细胞核的分离进行说明。这种方法是独立开发的，但是类似于由Henry 等人 10首先描述的方法中，细胞核被标记为与表示仅在果蝇特定的细胞类型，以方便标签的原子核的随后分离从混合群体的荧光标记用亲和力为基础的方法。要标记细胞核，该KASH（K larsicht / A NC-1 / S炔ħomology）域名已动用。该KASH域是定位于核膜通过与核周腔11内SUN结构域的蛋白相互作用的外膜，部分的跨膜结构域。蛋白质，例如果蝇 MSP-300和Klarsicht的C-末端KASH结构域锚定这些蛋白质与外核膜，而它们的N-末端结构域与细胞骨架蛋白，如肌动蛋白或微管在细胞质12-14交互。生成构建体，其中果蝇 MSP-300的KASH结构域融合到EGFP的C-末端，在Gal4的调控序列，UAS在pUAST-attB位向量15，16的控制。使用phiC31位点特异性整合，产生的转基因果蝇其中UAS-EGFP :: MSP-300 KASH的基因被插入到染色体上的基因座attP2位3L 17。在UAS-EGFP :: MSP-300 KASH苍蝇可越过与果蝇表达Gal4驱动子在特定的细胞类型，产生的绿色荧光蛋白在感兴趣的细胞类型的外核膜的靶向表达。标记的细胞核然后可以从使用​​抗GFP耦合到磁珠混合细胞群纯化。在这种方法中，不需要使用非离子洗涤剂是因为EGFP标记被定位于外核膜的胞质侧，因此接触到的抗体。 下面描述的协议可以被用于分离/从果蝇幼虫组织的特定细胞类型丰富EGFP-标记的细胞核，以量化的纯度和分离的细胞核的产量，并提取核RNA适于定量逆转录聚合酶链反应（实时定量-PCR）的基因表达分析。细胞核的分离和亲和纯化（不含布甲Ë夹层）可以在​​不到一小时内完成。结果列显示，胶质细胞核可以成功地从幼虫视叶和眼成虫盘分离，并用于随后的基因表达分析。可以预料，这种方法将是标记的细胞核的从其中靶细胞构成总人口的5％以内的胚胎和幼虫组织的分离/富集有用。 果蝇的所有股票和在这项研究中产生的质粒可从应要求作者。

<table border="0" cellpadding="0" cellspacing="0" width="1884">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:599px;">Anti-chaoptin antibody (mouse, monoclonal)</td>
			<td style="width:263px;">Developmental Studies Hybridoma Bank</td>
			<td style="width:195px;">mAB24B10</td>
			<td style="width:828px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-GFP antibody (mouse, monoclonal)</td>
			<td>Roche</td>
			<td>11814460001</td>
			<td>This specific antibody is recommended for this protocol. However, alternative antibodies could also be used.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:599px;">2X Bullseye EvaGreen qPCR Mastermix with ROX</td>
			<td>MidSci</td>
			<td>BEQPCR-R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI (4&#39;,6-diamidino-2-phenylindole, dihydrochloride salt)</td>
			<td>Biotium</td>
			<td>40011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dounce Homogenizer (1 ml)</td>
			<td>VWR</td>
			<td>62400-595</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dumont #5 Tweezers, 11 cm</td>
			<td>World Precision Instruments</td>
			<td>14095</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dynabeads Protein G for Immunoprecipitation</td>
			<td>Invitrogen</td>
			<td>10003D</td>
			<td>This specific brand of magnetic beads is recommended for this protocol.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EpiScript Reverse Transcriptase</td>
			<td>Epicentre</td>
			<td>ERT12910K</td>
			<td><a href="http://www.epibio.com/docs/default-source/protocols/episcript-reverse-transcriptase.pdf">http://www.epibio.com/docs/default-source/protocols/episcript-reverse-transcriptase.pdf</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Falcon cell strainers, 40 &#956;m pore size</td>
			<td>VWR</td>
			<td>21008-949</td>
			<td>Alternative pore sizes could be used, depending on the size of the target cell nuclei.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnetic stand (8 x 1.5 ml tubes)</td>
			<td>Millipore</td>
			<td>LSKMAGS08&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:599px;">Mini tube rotator</td>
			<td style="width:263px;">Genemate</td>
			<td style="width:195px;">H-6700</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:599px;">Qubit starter kit</td>
			<td style="width:263px;">Life Technologies</td>
			<td style="width:195px;">Q32871</td>
			<td style="width:828px;">Similar fluorescence-based methods for quantifying RNA yield could be used. <a href="http://tools.invitrogen.com/content/sfs/manuals/mp32852.pdf">http://tools.invitrogen.com/content/sfs/manuals/mp32852.pdfZ</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:599px;">RNAsecure (Ambion)</td>
			<td style="width:263px;">Life Technologies</td>
			<td style="width:195px;">AM7010</td>
			<td style="width:828px;">The use of this reagent to inactivate RNAses is optional.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:599px;">RT-PCR machine Thermal Cycler</td>
			<td style="width:263px;">BioRad</td>
			<td style="width:195px;">CFX Connect Thermal Cycler</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Siliconized 9 well glass plate</td>
			<td>Hampton Research</td>
			<td>HR3-134</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:599px;">Stereomicroscope Fluorescence Adapter (Royal Blue (440 - 460nm) excitation + yellow barrier</td>
			<td style="width:263px;">Nightsea</td>
			<td style="width:195px;"></td>
			<td>This adaptor enables GFP fluorescence to be examined using a typical dissecting microscope (eg Nikon)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:599px;">StereoZoom Microscope 83604 Set with 10X Wide Field Eyepieces and Universal Boom Stand</td>
			<td style="width:263px;">Nikon</td>
			<td style="width:195px;">SMZ 745</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:599px;">Thermal Cycler</td>
			<td style="width:263px;">BioRad</td>
			<td style="width:195px;">T100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trizol reagent</td>
			<td style="width:263px;">Life Technologies</td>
			<td>15596018</td>
			<td>CAUTION; Alternative RNA extraction methods can be used. <a href="http://tools.invitrogen.com/content/sfs/manuals/trizol_reagent.pdf" >http://tools.invitrogen.com/content/sfs/manuals/trizol_reagent.pdf</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tween 20</td>
			<td>Amresco</td>
			<td>0777-1L</td>
			<td></td>
		</tr>
	</tbody>
</table>

affinity-based isolation of tagged nuclei from drosophila tissues for gene expression analysis

果蝇胚胎和幼虫组织往往含有细胞类型的一种高度非均质混合物，其可以在这些组织中复杂的基因表达的分析。因此，分析从果蝇组织细胞特异性基因的表达谱，这可能是必要的隔离具有高纯度，并在足以产量为下游应用，如转录谱和染色质免疫沉淀特定的细胞类型。然而，在组织中，如对中枢神经系统的不规则细胞形态，再加上在这些组织中的特定细胞类型的罕见群体，可以构成挑战的细胞分离分拣的传统方法，如激光显微切割和荧光激活细胞分选术（FACS） 。这里，另一种方法使用标记的核的基于亲和力的隔离，而不是整个细胞特征的细胞特异性基因表达图谱进行说明。在特定的C核感兴趣的ELL类型的基因标记使用GAL4/UAS双元表达系统中的核膜本地化EGFP标记。这些EGFP-标记的细胞核可以使用抗GFP被耦合到磁性小珠分离。在这个协议中描述的方法使晶核一致隔离从果蝇幼虫中枢神经系统在高纯度和足够的水平进行表达分析特定类型的细胞，即使这些细胞类型中包括的总的细胞群体的少于2％组织。这种方法可以被用来从各种各样的果蝇胚胎，并采用特定的Gal4驱动幼虫细胞类型的分离的细胞核，并且可以是从那些不适合用于FACS或激光显微切割的细胞类型中分离细胞核有用。

回购-GAL4驱动程序（布卢明顿库存号7415）在神经胶质细胞在果蝇神经系统在发育18的多个阶段特异性表达。被苍蝇产生稳定表达的UAS-EGFP :: MSP-300 KASH下使用标准的基因工程技术生产的回购GAL4驱动程序的控制权。表征EGFP的这些苍蝇的表达模式:: KASH转基因，绿色荧光蛋白的表达从三龄幼虫的解剖视叶和眼成虫盘的格局是利用共聚焦显微镜观察。解剖组织都...

Watch this Scientific Journal Video about Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis at JoVE.com

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis | Biology | Video

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

果蝇组织中往往含有细胞类型的非均相混合物。为了检验从一个特定的组织特异性细胞类型的基因表达，细胞核可遗传标记，随后使用具有亲和力为基础的方法分离出来。分离的细胞核，可用于下游应用，如基因表达分析和染色质免疫沉淀。

标记的细胞核从亲和力为基础的隔离<em&gt;果蝇</em&gt;组织的基因表达分析

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

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Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

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14.6K 次观看.  Purdue University.  果蝇组织中往往含有细胞类型的非均相混合物。为了检验从一个特定的组织特异性细胞类型的基因表达，细胞核可遗传标记，随后使用具有亲和力为基础的方法分离出来。分离的细胞核，可用于下游应用，如基因表达分析和染色质免疫沉淀。 

视频: Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Bacterial Gene Expression Analysis Using Microarrays

使用微阵列的细菌基因表达分析

科研

生物学

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

从胚胎斑马鱼和cDNA合成的基因表达分析的RNA分离

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

机器人技术和植物组织的基因表达研究的动态图像分析

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.