Name: a facs-based protocol to isolate rna from the secondary cells of drosophila male accessory glands
Uploaded: 2019-09-05T15:00:00
Description: Watch this Scientific Journal Video about A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands at JoVE.com

我们感谢Karch实验室的成员，感谢iGE3基因组学板块，感谢日内瓦大学的流式细胞学核心设施，感谢为FACS制定协议的让-皮埃尔·奥布里-拉查纳耶博士。我们感谢卢卡·斯蒂克利帮助可视化IGV上的阅读。我们感谢自己温柔地允许重用数字，感谢编辑们促使我们在写作方面富有创造性。
这项研究由日内瓦州（CI、RKM、FK）、瑞士国家研究基金（www.snf.ch）和克拉拉兹基金会（FK）捐款资助。

1. 使用果蝇线和男性收集
<ol><li>对于此协议，使用在 SC 中表达 GFP 的雄性，而不是主细胞。在这里，使用AbdB-GAL4驱动器（在参考文献18中描述），与UAS-GFP（在染色体2上）重新组合。也可以使用其他适当的驱动程序。对于不同突变条件下的SC分离，将突变交叉成包含SC特异性GFP线的线。</li>
	<li>收集两批约25个健康，处女雄性为每个基因型和年龄他们在25°C3-4天...

<ol>
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R., Wolfner, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+post-mating+response+in+Drosophila+melanogaster+requires+multiple+seminal+fluid+proteins.">Sustained post-mating response in Drosophila melanogaster requires multiple seminal fluid proteins.</a> PLoS Genetics. 3 (12), e238 (2007).</li><li>Sitnik, J. L., Gligorov, D., Maeda, R. K., Karch, F., Wolfner, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Female+Post-Mating+Response+Requires+Genes+Expressed+in+the+Secondary+Cells+of+the+Male+Accessory+Gland+in+Drosophila+melanogaster.">The Female Post-Mating Response Requires Genes Expressed in the Secondary Cells of the Male Accessory Gland in Drosophila melanogaster.</a> Genetics. 202 (3), 1029-1041 (2016).</li><li>Avila, F. W., Wolfner, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acp36DE+is+required+for+uterine+conformational+changes+in+mated+Drosophila+females.">Acp36DE is required for uterine conformational changes in mated Drosophila females.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (37), 15796-15800 (2009).</li><li>Adams, E. M., Wolfner, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seminal+proteins+but+not+sperm+induce+morphological+changes+in+the+Drosophila+melanogaster+female+reproductive+tract+during+sperm+storage.">Seminal proteins but not sperm induce morphological changes in the Drosophila melanogaster female reproductive tract during sperm storage.</a> Journal of Insect Physiology. 53 (4), 319-331 (2007).</li><li>Singh, 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=Long-term+interaction+between+Drosophila+sperm+and+sex+peptide+is+mediated+by+other+seminal+proteins+that+bind+only+transiently+to+sperm.">Long-term interaction between Drosophila sperm and sex peptide is mediated by other seminal proteins that bind only transiently to sperm.</a> Insect Biochemistry and Molecular Biology. 102, 43-51 (2018).</li><li>Avila, F. W., Wolfner, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+the+Drosophila+seminal+protein+Acp36DE+in+mated+females+enhances+its+sperm+storage+activity.">Cleavage of the Drosophila seminal protein Acp36DE in mated females enhances its sperm storage activity.</a> Journal of Insect Physiology. 101, 66-72 (2017).</li><li>Chapman, T., Davies, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+and+analysis+of+the+seminal+fluid+proteins+of+male+Drosophila+melanogaster+fruit+flies.">Functions and analysis of the seminal fluid proteins of male Drosophila melanogaster fruit flies.</a> Peptides. 25 (9), 1477-1490 (2004).</li><li>Gligorov, D., Sitnik, J. L., Maeda, R. K., Wolfner, M. F., Karch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+function+for+the+Hox+gene+Abd-B+in+the+male+accessory+gland+regulates+the+long-term+female+post-mating+response+in+Drosophila.">A novel function for the Hox gene Abd-B in the male accessory gland regulates the long-term female post-mating response in Drosophila.</a> PLoS Genetics. 9 (3), e1003395 (2013).</li><li>Minami, R., 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+homeodomain+protein+defective+proventriculus+is+essential+for+male+accessory+gland+development+to+enhance+fecundity+in+Drosophila.">The homeodomain protein defective proventriculus is essential for male accessory gland development to enhance fecundity in Drosophila.</a> PLoS One. 7 (3), e32302 (2012).</li><li>Maeda, R. 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+lncRNA+male-specific+abdominal+plays+a+critical+role+in+Drosophila+accessory+gland+development+and+male+fertility.">The lncRNA male-specific abdominal plays a critical role in Drosophila accessory gland development and male fertility.</a> PLoS Genetics. 14 (7), e1007519 (2018).</li><li>Prince, E., 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=Rab-mediated+trafficking+in+the+secondary+cells+of+Drosophila+male+accessory+glands+and+its+role+in+fecundity.">Rab-mediated trafficking in the secondary cells of Drosophila male accessory glands and its role in fecundity.</a> Traffic. 20 (2), 137-151 (2019).</li><li>Robinson, M. D., Oshlack, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+scaling+normalization+method+for+differential+expression+analysis+of+RNA-seq+data.">A scaling normalization method for differential expression analysis of RNA-seq data.</a> Genome Biology. 11 (3), R25 (2010).</li><li>Khan, S. J., Abidi, S. N., Tian, Y., Skinner, A., Smith-Bolton, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+gentle+and+scalable+method+for+dissociation+and+fluorescent+sorting+of+imaginal+disc+cells+for+mRNA+sequencing.">A rapid, gentle and scalable method for dissociation and fluorescent sorting of imaginal disc cells for mRNA sequencing.</a> Fly (Austin). 10 (2), 73-80 (2016).</li><li>Corrigan, L., 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=BMP-regulated+exosomes+from+Drosophila+male+reproductive+glands+reprogram+female+behavior.">BMP-regulated exosomes from Drosophila male reproductive glands reprogram female behavior.</a> Journal of Cell Biology. 206 (5), 671-688 (2014).</li><li>Leiblich, 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=Bone+morphogenetic+protein-+and+mating-dependent+secretory+cell+growth+and+migration+in+the+Drosophila+accessory+gland.">Bone morphogenetic protein- and mating-dependent secretory cell growth and migration in the Drosophila accessory gland.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (47), 19292-19297 (2012).</li><li>Kubo, 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=Nutrient+conditions+sensed+by+the+reproductive+organ+during+development+optimize+male+fecundity+in+Drosophila.">Nutrient conditions sensed by the reproductive organ during development optimize male fecundity in Drosophila.</a> Genes to Cells. 23 (7), 557-567 (2018).</li><li>Immarigeon, C., Karch, F., Maeda, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FACS-based+isolation+and+RNA+extraction+of+Secondary+Cells+from+the+Drosophila+male+Accessory+Gland.">FACS-based isolation and RNA extraction of Secondary Cells from the Drosophila male Accessory Gland.</a> bioRxiv. , 630335 (2019).</li></ol>

细胞分离的方法从果蝇组织，如象形圆盘已经描述了23。我们试图简单地在附属腺体上使用这些程序失败，鼓励我们开发这种新协议。蛋白酶消化和机械三聚酯是我们为手术成功而烦恼的关键步骤，因此，我们在第2至第5节中放置了许多注释，以帮助实验者获得令人满意的结果。为了解脱成功，肽类细胞必须到达附属腺细胞，这些细胞受流明体中的粘性和腺体周围的肌肉层...

器官由多种细胞类型组成，每种细胞类型都有离散功能，有时表达的基因组非常不同。为了准确理解器官的功能，研究组成这个器官的每种不同的细胞类型通常是至关重要的。用于探索可能功能的主要方法之一是转录子分析。这种强大的方法提供了细胞中基因表达的快照，以揭示活跃的过程和途径。然而，对于必须从更丰富的相邻细胞中纯化的稀有细胞群来说，这种类型的分析通常是困难的。例如，果蝇男性附属腺体是主要由两种分泌细胞类型组成的器官。由于两种细胞类型的稀有细胞只占该腺体细胞的4%，因此没有使用细胞类型特定的转录组分析来帮助确定这些细胞的功能。
附属腺体（AGs）是昆虫中男性生殖道的器官。它们负责生产的大部分蛋白质（蛋白（ SFPs ）和附属腺蛋白（ ACPs ）。其中一些SFP已知诱导交配雌性生理和行为反应，通常称为交配后反应（PMR）。一些PmR包括：排卵率和卵子产卵率增加，精子的储存和释放，女性饮食的变化，以及女性对二级求亲男性1，2的接受率下降。由于昆虫影响到从人类健康（作为致命疾病的载体）到农业的许多重大社会问题（昆虫可以是害虫，但对授粉和土壤质量至关重要），了解昆虫繁殖是一个重要的研究领域。与模型有机体果蝇黑色素酶一起，对GS和ACPs的研究有了显著进展。这些研究强调了AGs和它们产生的一些单个蛋白质在创建PMR中的作用，影响了其他物种的工作，如病媒伊蚊3、4和其他昆虫1 ， 5.此外，当AG分泌精液1，6的成分时，它们通常被认为是哺乳动物前列腺和精质囊泡的功能模拟。这种功能相似性结合两个组织类型的分子相似性，使AGs成为苍蝇7前列腺的模型。
在果蝇雄性，有两个附属腺叶。每个叶可以被看作是一个囊状结构，由围绕中央流明的单层分泌细胞组成，由光滑的肌肉包裹。如上所述，有两种形态、发育和功能上截然不同的分泌细胞类型组成这个腺体：多边形主细胞（占细胞的96%），以及较大的圆形二次细胞（SC）（组成剩余的4%的细胞，或大约40个细胞每叶）。已经表明，两种细胞类型产生不同的ASP集，以诱导和维持PMR。迄今为止获得的大部分数据都强调了单一蛋白质在触发PMR大多数特征行为中的作用。这种蛋白质，性肽，是一种小的，36个氨基酸肽，由主细胞8，9，10分泌。虽然性肽似乎在PMR中起主要作用，但主细胞和继发细胞产生的其他AcPs也已被证明影响PMR11、12、13、14的各个方面。 ，15，16，17.例如，根据我们目前的知识，SC，通过它们产生的蛋白质，似乎需要在第一天18日之后使SP信号永久化。
鉴于SCs的稀有性（每只男性只有80个细胞），我们关于这些细胞及其产生的蛋白质的所有知识都来自遗传学和候选方法。到目前为止，只有相对较少的基因列表被证明是SC特异性的。此列表包括地域蛋白缺陷已验证的三叶草 （Dve）19，lncRNA MSA20， Rab6， 7， 11和1921， CG1656和CG1757511， 15，21和主箱转录因子腹 - B （Abd-B）18.以前，我们已经表明，在二次细胞（iab-6cocuD1突变体）中，Abd-B和lncRNA MSA的表达存在突变缺陷，将PMR的长度从+10天缩短到只有一天12,18,20.这种表型似乎是由于SP在女性生殖道12、18、20中储存不当所致。在细胞水平上，这种突变体的次生细胞表现出异常形态，失去其特有的真空状结构18，20，21。使用这种突变线，我们以前试图通过比较来自野生类型或突变附件腺体12的整个GS的转录配置文件来识别涉及SC功能的基因。此外，其他实验室显示，SC数，形态和真空含量取决于男性的饮食，交配状态和年龄21，25，26。
虽然这些方法取得了积极进展，但SC全文远未实现。这些细胞在这个器官中的稀有性使得即使从野生型细胞也很难进一步进步。对于测试基因表达，这些细胞在特定环境刺激后的变化将更加困难。因此，需要一种分离和纯化SC RNA的方法，该方法足够快速和简单，足以在不同的遗传背景和环境条件下发挥作用。
Abd-B和MSA基因都需要从果蝇Bithorax复合物（称为D1增强剂）的特异性1.1kb增强剂，以在SCs18，20中表达。此增强剂以前曾用于创建GAL4驱动程序，当该驱动程序与UAS-GFP关联时，能够专门在 SC 中驱动强大的 GFP 表达式。因此，我们使用此行作为 FACS 协议的基础，将这些细胞从野生类型和iab-6cocuD1 AGs 中分离。由于iab-6cocuD1突变S显示不同的细胞形态，我们表明，该协议可用于分离细胞，以确定其转录组从这种罕见的细胞类型在完全不同的条件下。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-wells Tissue Culture plate</td>
			<td>VWR</td>
			<td>734-2325</td>
			<td></td>
		</tr>
		<tr>
			<td>Binocular microscope for dissection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Binocular with light source for GFP</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Draq7 0.3 mM</td>
			<td>BioStatus</td>
			<td>DR71000</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic microtubes 1.5 mL</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FACS</td>
			<td>Beckman Coulter</td>
			<td>MoFlo Astrios</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine dissection forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal bovine serum</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td>heat inactivated prior to use</td>
		</tr>
		<tr>
			<td>Glass dishes for dissection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ice bucket</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImProm-II Reverse Transcription System</td>
			<td>Promega</td>
			<td>A3800</td>
			<td></td>
		</tr>
		<tr>
			<td>MasterPure RNA Purification Kit</td>
			<td>Epicentre</td>
			<td>MCR85102</td>
			<td></td>
		</tr>
		<tr>
			<td>Nextera XT kit</td>
			<td>illumina</td>
			<td></td>
			<td><a href="https://emea.illumina.com/products/by-type/sequencing-kits/library-prep-kits/nextera-xt-dna.html">https://emea.illumina.com/products/by-type/sequencing-kits/library-prep-kits/nextera-xt-dna.html</a></td>
		</tr>
		<tr>
			<td>P1000</td>
			<td>Gilson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P20</td>
			<td>Gilson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P200</td>
			<td>Gilson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td></td>
			<td></td>
			<td>50U/mL stock</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td>home made</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>PolydT primers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Random hexamer primers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RNA 6000 Pico kit</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Schneider&#8217;s Drosophila medium</td>
			<td>Gibco</td>
			<td>21720-001</td>
			<td></td>
		</tr>
		<tr>
			<td>SMARTer cDNA synthesis kit</td>
			<td>Takara</td>
			<td></td>
			<td><a href="https://www.takarabio.com/products/cdna-synthesis/cdna-synthesis-kits/smarter-cdna-synthesis-kits">https://www.takarabio.com/products/cdna-synthesis/cdna-synthesis-kits/smarter-cdna-synthesis-kits</a></td>
		</tr>
		<tr>
			<td>SYBR select Master mix for CFX</td>
			<td>applied biosystems</td>
			<td>4472942</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Shaker</td>
			<td>Hangzhou Allsheng intruments</td>
			<td>MS-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tipone 1250 &#956;l graduated tip</td>
			<td>Starlab</td>
			<td>S1161-1820</td>
			<td></td>
		</tr>
		<tr>
			<td>Tipone 200 &#956;l bevelled tip</td>
			<td>Starlab</td>
			<td>S1161-1800</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme</td>
			<td>Gibco</td>
			<td>12604013</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a facs-based protocol to isolate rna from the secondary cells of drosophila male accessory glands

为了理解器官的功能，理解其组成细胞群的作用通常很有用。不幸的是，单个细胞群的稀有性往往使得很难获得足够的材料用于分子研究。例如，果蝇雄性生殖系统的附属腺体包含两种截然不同的分泌细胞类型。主细胞占腺体分泌细胞的96%，而次级细胞（SC）占细胞的其余4%（每个男性约80个细胞）。虽然这两种细胞类型都产生的重要成分，但已知只有少数基因是 SC 特有的。到目前为止，SC的稀有性阻碍了这一重要细胞类型的转录组分析研究。这里提出了一种方法，允许纯化用于RNA提取和测序的SC。该协议包括首先从苍蝇中解剖腺体，表达SC特异性GFP报告器，然后将这些腺体置于蛋白酶消化和机械分离中，以获得单个细胞。按照这些步骤，使用荧光活细胞分拣机（FACS）对单个、活体、GFP标记的细胞进行分类，用于RNA纯化。此程序产生 SC 特异性 RNA 从 +40 男性每个条件在一天内的下游RT-qPCR和/或RNA测序。程序的快速性和简单性允许从不同的基因型或环境条件，在短时间内确定许多不同的苍蝇的转录。

这里提出的协议使一个实验者能够从果蝇雄性附属腺中提取二级细胞，并在一天内提取其RNA（图1）。
我们使用Abd-B-GAL4构造18来标记辅助细胞 （SC），而不是用 GFP 标记主细胞 （MC） （图 2A）。此过程的一个目标是获得 SC 的野生类型转录?...

Watch this Scientific Journal Video about A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands at JoVE.com

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

在这里，我们提出一个协议，从果蝇雄性附属腺体（辅助细胞）分离和排序特定细胞群，用于RNA测序和RT-qPCR。细胞分离是通过FACS纯化GFP表达的二次细胞后，需要解剖，蛋白酶消化和机械分散多步骤解散过程。

从果蝇雄性附件腺的二级细胞中分离RNA的FACS协议

To understand the function of an organ, it is often useful to understand the role of its constituent cell populations. Unfortunately, the rarity of ...

这种方法允许对来自果蝇雄性附属腺体的罕见细胞种群进行转录分析，称为继发细胞，并发现基因，使它们成为生殖成功的关键。在一天中，该技术允许从不同基因型中分拣二级细胞，并提取总RNA，以便随后进行qPCR或RNA测序。有时，项目受到起始材料的罕见性限制。此方法可以为面临处理罕见细胞群的挑战的其他组提供基础。因为这个程序需要积累40个腺体，所以进行解剖很重要。准备自制的移液器提示也很重要，所以你应该把它可视化到视频上，如何制作它，并提前准备。在开始手术之前，切割和火焰圆，宽，200微升提示处理腺体，并通过多个1000微升尖端打开附近的火焰不到一秒钟，以缩小开口。然后，根据修改的提示，根据它们的渴望速度，从更窄到更宽。对于进入性腺解剖，将20至25名男性果蝇放在冰的玻璃盘中，并使用钳子和解剖显微镜去除一只雄性苍蝇的生殖道。从所有其他组织（包括睾丸和脱毛球球）中清除附件腺对，并在室温下将附件腺体转移到含有血清补充介质的玻璃板上。收集20对附属腺体后，在室温下将腺体在PBS盘中清洗一至两分钟。在洗涤结束时，将腺体转移到新鲜准备好的分离溶液中，并在解剖显微镜下使用锋利的钳子牢牢捏住腺叶的中间。使用第二对钳子，用锋利的尖端切割组织，以分离附属腺体的近端区域和伊开精管从含有二次细胞的腺体部分。切割腺体，使乳腺可以到达由外肌层和腺体流明中的粘性保护的细胞。然后，使用预湿、火焰圆、宽、200 微升移液器尖端将腺体转移到 1.5 毫升管中。对于组织消化，将管子放在37摄氏度的摇床中，每分钟旋转1000次，60分钟。在孵化结束时，停止消化用一毫升血清补充介质，并使用预湿，火焰圆，宽，1000微升移液器尖端转移到24井板样品。在荧光显微镜下检查细胞的GP表达。大多数腺体尖端应看起来完好无损，一些二级细胞应分离。使用圆圆的、窄的、1000微升的移液器尖端轻轻三到五次三次，以破坏附属腺体组织。然后，使用非常窄的，圆润的，1000微升移液器尖一到两次，以个性化细胞。让细胞沉淀15分钟。确认细胞在显微镜下看起来健康，并去除多余的介质。然后，将细胞转移到一个新的1.5毫升管中，然后用新鲜的介质冲洗孔，以恢复任何剩余的细胞。Abd-B-GAL4 构造可用于这些实验，用 GFP 标记辅助细胞，而不是主细胞。在这项研究中，这两种细胞类型都从野生类型和 iab-6cocuD1 突变附属腺体中分类。此 D1 突变体缺乏 Abd-B 和 MSA 转录的表达，影响继发细胞发育、形态和功能。细胞分离后，FACS用于选择基于 DRAQ7 的活细胞，GFP 荧光水平用于分离二级细胞和主细胞。从每个细胞群体中提取RNA，并调整为每个样本两纳米，用于后续的RT-qPCR和测序。通过野生型主细胞和继发细胞提取物的实时定量PCR对特定基因的表达进行定量，表明仅从二级细胞提取物中检测出二次细胞基因Rab19和MSA，而主细胞基因性肽仅从主细胞样本中检测。对三种野生类型和三种突变体复制的RNA测序数据的主要成分分析表明，野生类型复制的两个聚类彼此紧密地结合，远离突变样本。与特定基因对齐的可视化读取显示，内务管理基因，如行为素5C，和继发细胞特异性基因Rab19和Dve在两种基因型中均表达。然而，对于MSA，在野生型苍蝇中观察到的基因的强烈表达在突变菌株中丢失。获得足够的活体分离的继发细胞对于成功的手术很重要。因此，验证了小规模实验中的解剖和分离方案。这种方法允许从少量苍蝇中分拣两种细胞类型的附属腺体，从而研究这些细胞如何对多种遗传或环境条件进行转录反应。男性生殖成功已知受到许多事物的影响，包括基因型、年龄和交配状态。这种方法的易用性应该使我们能够研究这些因素如何影响附属腺体转录。

Research

JoVE Journal

Genetics

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

使用生物素化补骨脂素全球定位RNA-RNA相互作用

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

科研

遗传学

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

一种从任意 DNA 源进行大规模 gRNA 库生产的通用协议

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

整合酶缺陷慢病毒载体载体的制备及其在细胞 CRISPR/Cas9-mediated 基因敲除中的表达

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

rna 下拉法识别长非编码 rna 的 rna 靶点

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding ...

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

回顾性 MicroRNA 排序: 用福尔马林固定石蜡嵌入 RNA 标本补充 DNA 文库制备协议

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

TreadWheel:果蝇轻度诱导运动的间歇训练协议

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and ...

Purification of Low-abundant Cells in the Drosophila Visual System

果蝇视觉系统中低丰细胞的纯化

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small ...

Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

从活细胞中的单端粒表达的癌症细胞克隆的生成, 以可视化含有端粒重复的 rna terra

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in ...

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

RNA足迹的识别:通过RNA免疫沉淀在串联中的蛋白质复合物,然后测序(RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Use of Drosophila S2 Cells for Live Imaging of Cell Division

使用果蝇S2细胞进行细胞分裂的活成像

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a ...