Name: a screening method for identification of heterochromatin-promoting drugs using drosophila
Uploaded: 2020-03-12T14:30:00
Description: Watch this Scientific Journal Video about A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila at JoVE.com

我们感谢J.伯克勒，E.巴赫和布卢明顿果蝇股票中心的各种果蝇菌株;国家癌症研究所（NCI）肿瘤集小分子药物库的发展治疗方案;UCSD本科生包括艾米·张、泰西克·你、杰西卡·辛格-班加、雷切尔·梅扎和亚历克斯·查韦斯。本出版物中报道的研究得到了美国胸腔学会对J.L.的研究资助和NIH的资助：R01GM131044到W.X.L.

1. 药物库准备
<ol><li>识别并准备药物库，在所需浓度（例如DMSO中为10 mM）使用适当的溶剂进行筛选。 注：药物库的一个具体示例是国家癌症研究所 （NCI） 发育治疗计划 （DTP） 的肿瘤集 III。在两个96孔PP U底板中，100%DMSO中的化合物在1000%DMSO中作为20μL提供，并储存在-20°C。NCI 板地图和每种药物的基本化学数据可在以下网站上找到： <a href="https://dtp.cancer.gov/dtpstandard/servlet/PlateMap?searchlist=4740&...

<ol>
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R., 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=Expansions+of+transgene+repeats+cause+heterochromatin+formation+and+gene+silencing+in+Drosophila.">Expansions of transgene repeats cause heterochromatin formation and gene silencing in Drosophila.</a> Cell. 77 (7), 993-1002 (1994).</li><li>Fanti, L., Dorer, D. R., Berloco, M., Henikoff, S., Pimpinelli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterochromatin+protein+1+binds+transgene+arrays.">Heterochromatin protein 1 binds transgene arrays.</a> Chromosoma. 107 (5), 286-292 (1998).</li><li>Shi, 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=JAK+signaling+globally+counteracts+heterochromatic+gene+silencing.">JAK signaling globally counteracts heterochromatic gene silencing.</a> Nature Genetics. 38 (9), 1071-1076 (2006).</li><li>Shi, 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=Drosophila+STAT+is+required+for+directly+maintaining+HP1+localization+and+heterochromatin+stability.">Drosophila STAT is required for directly maintaining HP1 localization and heterochromatin stability.</a> Nature Cell Biology. 10 (4), 489-496 (2008).</li><li>Ronsseray, S., Boivin, A., Anxolabehere, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P-Element+repression+in+Drosophila+melanogaster+by+variegating+clusters+of+P-lacZ-white+transgenes.">P-Element repression in Drosophila melanogaster by variegating clusters of P-lacZ-white transgenes.</a> Genetics. 159 (4), 1631-1642 (2001).</li><li>Loyola, A. 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异色素罗马丁是一种浓缩的DNA形式，在调节基因表达中起着核心作用。它在癌症中越来越被认识，并可能成为癌症治疗的潜在目标15，16，17，18。15,16,17,18由于人体在制造、保存和代谢方面的优势，小分子化合物在药物开发中常有。为了识别促进异种素的小分子化合物，使用DX1果蝇菌株设...

异色素罗马丁是一种浓缩的DNA形式，在基因表达、调节细胞分裂期间的染色体分离以及防止基因组不稳定1中起核心作用。异色素素被认为是一种基因抑制调节剂，在细胞线粒度22，33期间保护染色体完整性。它与系系承诺,4、5期间组蛋白H3Lysine 9（H3K9me）的二甲基化有关。4此外，使用异色素1（HP1）色域蛋白也被认为与异质素和基因表达表观遗传抑制有关6。这些蛋白质是异色素形成的基本成分和标记物。
由于基因组不稳定使细胞获得促进致癌的基因改变，异色素素在癌症开发中越来越被识别，并可能成为癌症治疗的目标77，8。8目前，没有一种药物在协助异色素形成方面得到证实。在这里，我们提出了一种简单而快速而有效的方法来筛选促进异构素形成的小分子化合物。筛查是通过用小分子药物库治疗果蝇来完成的。此方法利用受异质色素水平影响的DX1果蝇菌株中多变的眼睛颜色表型。DX1苍蝇包含七个P_lac-w]转基因的串联阵列，这些基因具有根据异质化的变异表达/抑郁，因此，眼睛颜色的变异程度反映了异色素水平。具体来说，增加异质素可以通过变色眼色（白眼）的比例上升来检测。相反，减少异构素将被P[lac-w]转基因表达（红眼）9、10、11、1210,11,12的比例上升所检测9。
因此，我们利用这种多嗜血细胞系统，产生一种变幻的眼部颜色表型，因为它的表达与目前异质素的量直接相关。一旦发现怀疑促进异构素形成的化合物，我们可以用其他方法，如西方斑点来证实这种怀疑。这些促进异种素的物质将来可以进一步开发，供患者进行临床试验。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Drosophila</td>
			<td></td>
			<td>DX1 strain</td>
			<td>DX1 flies were kindly provided by James Birchler (University of Missouri)</td>
		</tr>
		<tr>
			<td>Drosophila food media</td>
			<td>UCSD fly kitchen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methotrexate</td>
			<td>NCI drug library</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol</td>
			<td>Sigma</td>
			<td>E7023-500ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

a screening method for identification of heterochromatin-promoting drugs using drosophila

果蝇是一种极好的模型有机体，可用于筛选可能对癌症治疗有用的化合物。这里描述的方法是一种具有成本效益的体内方法，通过使用果蝇来识别异血素促进化合物。果蝇的DX1菌株，具有一种品种化的眼睛颜色表型，反映异色素形成的程度，从而提供一个工具，促进异种素促进药物筛选。在筛选方法中，根据占据眼睛部分的红色色素沉着的表面面积对眼睛的变异进行量化，以1至5的刻度进行评分。筛选方法简单明了，允许在体内测试化合物。使用这种方法进行药物筛选为识别异色素促进药物提供了一种快速而廉价的方法，这些药物可能对癌症治疗产生有益的影响。确定促进异构素形成的化合物也可能导致癌症发育的表观遗传机制的发现。

该协议成功地用于筛选促进异构素在果蝇形成的化合物，这是一个高效和低成本的体内药物开发系统（图1）。我们筛选了一个小分子药物库，肿瘤集III，这是由97FDA授权肿瘤药物组成，使用DX1菌株的果蝇黑色素气酸（图1B）。一种重要药物的结果在条形图中（图1C，D）中表示。根据筛?...

Watch this Scientific Journal Video about A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila at JoVE.com

A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila

果蝇是一种广泛使用的实验模型，适用于筛选具有癌症治疗潜在应用的药物。在这里，我们描述了使用果蝇变眼色表型作为筛选小分子化合物，促进异构素形成的方法。

使用果蝇的异色素促进药物的鉴定筛选方法

Drosophila is an excellent model organism that can be used to screen compounds that might be useful for cancer therapy. The method described here is a ...

目前，没有药物在促进异色素形成方面已建立良好。在这里，我们提出了一个简单而有效的方法来识别促进这一过程的小分子化合物。我们的筛选协议提供了一个简单，廉价，易于遵循体内的异色素促进药物鉴定系统。识别促进异色素形成的化合物可能导致发现治疗和癌症治疗的癌症机制。这种成本效益已经测量用于识别嗜血杆菌中的异色素促进化合物也可用于计算形式命中，因为它是一个预防措施，以最高的细胞池细胞筛选。我们的筛选协议也可用于快速引导屏幕，因为这个果蝇TranSheet系统产生一个变异的眼睛颜色表型，与苍蝇中存在的异色素量成反比。帮助演示这个程序的将是肯尼道，一个本科生从我们的实验室。在使用适当的溶剂和所需的浓度确定并准备药物库进行筛选后，在 25 由 95 毫米食物小瓶中与三只或三只（三只处女雌蝇）交叉三只雄蝇。与九毫米的标准果蝇食品介质。为每种药物设置三个复制小瓶。为屏幕添加一个控制小瓶。允许苍蝇在室温下产卵两天。在十字架的第二天，用短暂的二氧化碳来麻醉苍蝇。将父母用含有70%乙醇的瓶子里丢弃。接下来，稀释每个药物化合物从初始库存参加微摩尔最终浓度在33%DMSO在水中。并添加60微升的药物，或控制33%DMSO单独，在飞的食物的顶部。然后确认小瓶中不存在母蝇。并将药物溶液添加到适当的小瓶中。F1苍蝇出现两天后，一次从一个小瓶将麻醉苍蝇转移到一个多孔解剖垫上，二氧化碳从滤纸下面过滤通过。在解剖显微镜下，检查每只苍蝇，通过缺乏卷曲的 O 显性卷曲翅膀表型来识别所有异质雄性。在一到五的尺度上给每个异质雄性的眼睛颜色评分。将一个分数分配给小于 5% 的红色散射眼总表面积。向具有 6 到 25% 红斑眼总表面积的苍蝇添加两个分数。一个三飞与26至50%红斑眼总表面积。一个四飞与51至75%红斑眼总表面积。和五只苍蝇的红斑眼总表面积大于75%。然后计算每个小瓶中按三倍得分的所有雄性的所有男性的均色指数。为了确认这些化合物确实促进了异色素的形成，可以执行一种不同的验证技术，如西方印迹。在这个具有代表性的实验中，一个小分子药物库，肿瘤学集3，它由97个FDA授权的肿瘤药物组成，使用DX1菌株的果蝇黑色素进行筛选。根据筛选测定，在库中编码为E7的二氨蝶酯在Dx1菌株中引起最大的变化。暗示明氨蝶酯可能是未来癌症靶向治疗最有希望的促进异色素的药物。为了进一步确认该化合物促进异色素转化，进行了西部印迹数据。演示异铬素格式相关蛋白的上规，H3K9三甲基化，并验证筛选结果。关键步骤是根据预定的研究路线，按一到五的尺度对每只白色11-18 DX1异质雄性苍蝇的眼睛颜色进行评分。我们可以用西方的污点或免疫污迹来确认命中。发现疑似促进异色素形成的化合物，也可能导致发现癌症发育的IV遗传机制，并在人类患者中进行临床试验。

Research

JoVE Journal

Genetics

A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

科研

遗传学

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

斑马斑马模型系统中 CRISPR/Cas9-generated 基因挖空的高效生产与鉴定

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

通过重叠2方法高通量识别协同药物组合

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Screening Cotton Genotypes for Reniform Nematode Resistance

筛选抗内虫抗性棉基因型

A rapid non-destructive reniform nematode (Rotylenchulus reniformis) screening protocol is needed for the development of resistant cotton (Gossypium ...

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

使用声学纳米分配技术实现高通量DNA质粒多路复用和转染

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...

Identification of Circular RNAs using RNA Sequencing

使用RNA测序识别循环RNA

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein ...

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 ...

Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens

植物病原体分泌效应器中RNA沉默抑制器的筛选和鉴定

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins ...

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

使用基因组-规模CRISPR/Cas9基因屏幕进行细胞表面受体识别

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and ...