Name: affinity-based isolation of tagged nuclei from drosophila tissues for gene expression analysis
Uploaded: 2014-03-25T14:15:00
Description: Watch this Scientific Journal Video about Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis at JoVE.com

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...

<ol>
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Protoc. 7, 978-994 (2012).</li><li>Haenni, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+C.+elegans+intestinal+gene+expression+and+polyadenylation+by+fluorescence-activated+nuclei+sorting+and+3'-end-seq.">Analysis of C. elegans intestinal gene expression and polyadenylation by fluorescence-activated nuclei sorting and 3'-end-seq.</a> Nucleic Acids Res. 40, 6304-6318 (2012).</li><li>Barthelson, R. A., Lambert, G. M., Vanier, C., Lynch, R. M., Galbraith, D. 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B., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+INTACT+method+for+cell+type-specific+gene+expression+and+chromatin+profiling+in+Arabidopsis+thaliana.">The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana.</a> Nat. Protoc. 6, 56-68 (2011).</li><li>Deal, R. B., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+gene+expression+and+chromatin+profiling+of+individual+cell+types+within+a+tissue.">A simple method for gene expression and chromatin profiling of individual cell types within a tissue.</a> Dev Cell. 18, 1030-1040 (2010).</li><li>Steiner, F. A., Talbert, P. B., Kasinathan, S., Deal, R. B., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-type-specific+nuclei+purification+from+whole+animals+for+genome-wide+expression+and+chromatin+profiling.">Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling.</a> Genom. Res. 22, 766-777 (2012).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Henry, G. L., Davis, F. P., Picard, S., Eddy, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+genomics+of+Drosophila+neurons.">Cell type-specific genomics of Drosophila neurons.</a> Nucleic Acids res. 40, 9691-9704 (2012).</li><li>Starr, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KASH+and+SUN+proteins.">KASH and SUN proteins.</a> Curr Biol. 21, 414-415 (2011).</li><li>Fischer, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+klarsicht+has+distinct+subcellular+localization+domains+for+nuclear+envelope+and+microtubule+localization+in+the+eye.">Drosophila klarsicht has distinct subcellular localization domains for nuclear envelope and microtubule localization in the eye.</a> Genetics. 168, 1385-1393 (2004).</li><li>Patterson, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functions+of+Klarsicht+and+nuclear+lamin+in+developmentally+regulated+nuclear+migrations+of+photoreceptor+cells+in+the+Drosophila+eye.">The functions of Klarsicht and nuclear lamin in developmentally regulated nuclear migrations of photoreceptor cells in the Drosophila eye.</a> Mol. Biol. Cell. 15, 600-610 (2004).</li><li>Yu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+KASH+domain+protein+MSP-300+plays+an+essential+role+in+nuclear+anchoring+during+Drosophila+oogenesis.">The KASH domain protein MSP-300 plays an essential role in nuclear anchoring during Drosophila oogenesis.</a> Dev. Biol. 289, 336-345 (2006).</li><li>Bischof, J., Maeda, R. K., Hediger, M., Karch, F., Basler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+transgenesis+system+for+Drosophila+using+germ-line-specific+phiC31+integrases.">An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 3312-3317 (2007).</li><li>Kracklauer, M. P., Banks, S. M., Xie, X., Wu, Y., Fischer, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+klaroid+encodes+a+SUN+domain+protein+required+for+Klarsicht+localization+to+the+nuclear+envelope+and+nuclear+migration+in+the+eye.">Drosophila klaroid encodes a SUN domain protein required for Klarsicht localization to the nuclear envelope and nuclear migration in the eye.</a> Fly. 1, 75-85 (2007).</li><li>Markstein, M., Pitsouli, C., Villalta, C., Celniker, S. E., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+position+effects+and+the+gypsy+retrovirus+insulator+to+engineer+precisely+expressed+transgenes.">Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes.</a> Nat. Genet. 40, 476-483 (2008).</li><li>Sepp, K. J., Schulte, J., Auld, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+glia+direct+axon+guidance+across+the+CNS/PNS+transition+zone.">Peripheral glia direct axon guidance across the CNS/PNS transition zone.</a> Dev. Biol. 238, 47-63 (2001).</li><li>Fujita, S. C., Zipursky, S. L., Benzer, S., Ferrus, A., Shotwell, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+against+the+Drosophila+nervous+system.">Monoclonal antibodies against the Drosophila nervous system.</a> Proc. Natl. Acad. Sci. U.S.A. 79, 7929-7933 (1982).</li></ol>

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>
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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

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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