这项工作是由卫生研究院（NIH）国家机构支持的补助CA127231及达蒙鲁尼恩基金会临床研究奖（CI 1502）（以SM）。我们要感谢的Zdenek德沃夏克教授帕拉茨基大学奥洛穆茨，捷克共和国的有益见解讨论这个技术移植到他们的机构和协议的标准化。

1。 PXR和SRC-1融合体在酵母载体的构建<ol><li> PCR扩增人类PXR LBD（107-434氨基酸）和人类SRC-1全长（1-1401氨基酸）。 <ol><li>使用pSG5-hPXR 8质粒作为PXR LBD模板，使用PCMX-SRC1质粒为SRC-1的模板。 </li><li>解冻PCR SuperMix，DNA模板和引物（见材料），让他们在冰上。 </li><li>加0.25微克/微升DNA模板1微升，10毫引物对2微升各自的PCR SuperMix 45微升，并添加H 2 O至使总体积为50微升。 </li><li>设置PCR在94℃下一个循环2分钟，20个循环的94℃30秒，55℃30秒，72℃2分钟，然后72℃10分钟延长一个周期。检查PCR产物用1％琼脂糖凝胶电泳。 </li></ol></li><li> PCR产物克隆到酵母双杂交载体。 <ol><li>从1％琼脂糖凝胶纯化的PCR产物。摘要PXR，LBD，PCR和Y2H载体pSH2-1BamHI和SalI位;消化SRC-1 PCR和Y2H载体pGADNOT与NotΙ和SalΙ。要做到这一点，加1微克的DNA，3微升10X反应缓冲液，1μL限制性内切酶，最后H 2 O至使总体积为30μL。 </li><li>把消解样品在37℃水浴1小时。通过在1％琼脂糖凝胶电泳检查消化。 </li><li>纯化消化的样品在1％琼脂糖凝胶上，结扎消化的PCR产物和Y2H载体和转化到DH5α感受态细胞。 </li><li>挑殖民地和隔离质粒DNA。 </li></ol></li><li>识别pSH2-1-PXR质粒DNA经BamHI / SalI位消化，找出pGADNOT-SRC-1质粒DNA用NotI位/ SalI位消化。通过测序验证一切积极质粒DNA。 </li></ol> 2。酵母双杂交试验<ol><li>接种酵母菌株到5ml YPAD孵育过夜通过不断搅拌（220转，30℃）。注意： 酿酒酵母的菌株是CTY10-5D（MAT一个ADE2 TRP 1-901 LEU2-3，112 HIS3-200 GA L4 - GAL80 - URA3 :: LEXA-lacZ的 ），其中包含一个集成的GAL1-lacZ基因与LexA操纵 10。要获得可行的酵母细胞的抗酮康唑，但那些没有进行转运改建为唑类抗18-20的原因，为CTY10-5D酵母新菌株是通过首先删除ERG3（ERG3Δ）获得，然后引入额外的缺失ERG11（ERG3Δ/ ERG11Δ）的基因通过同源重组。建筑细节在以前出版的手稿6被描述。 </li><li>第二天（早上），接种酵母（1:20）在5毫升YAPD和孵化通过不断搅拌（220转，30℃）来获取一个外径600〜0.2。 </li><li>沉淀细胞在微量离心2分钟2000转，并丢弃上清液。然后洗PELLE吨两次用无菌水，一次用0.1M的醋酸锂。 </li><li>最后一次洗涤后，弃去上清液，加入以下试剂，细胞沉淀：240微升PEG 3500 50％重量/体积，36微升1M的醋酸锂，50微升2毫克/毫升的水煮鲑鱼精子单链DNA，H 2 O的34微升。旋涡混匀，加1微克PSH-PXR和1微克pGADNOT-SRC-1的DNA混匀。 </li><li>传输组合，聚苯乙烯圆底管，搅动改造，混合（220转，30℃）30分钟。移动管子到42℃水浴中孵育另外的30分钟。 </li><li>离心管在2,000 rpm离心2分钟，除去上清液。用无菌水冲洗一次颗粒。 </li><li>吸取100微升无菌水进入管，通过手指轻轻重悬细胞攻和移液，然后镀上-His/-Leu降中（配方每升-His/-Leu差中的板材：20 G葡萄糖， 1.7克YNB，0.67克CSM-His/-Leu，5克硫酸铵，1.5％琼脂）和增量ubate在30℃下3-4天以分离转化体。 </li></ol> 3。 X-gal的筛选分析<ol><li>切硝酸纤维素膜的陪替氏培养皿的部分的大小。标签的硝酸纤维素膜上标记的方向。 </li><li>放置在酵母菌落预标记的硝酸纤维素膜，确保该膜是在与板介质的表面相接触。 </li><li>取下滤膜，将其放置菌落面朝上3毫米的滤纸，然后将膜和滤纸在-80℃下15分钟。 </li><li>让X-gal的解决方案。加50微升20毫克/毫升X-gal和15μl的β-巯基乙醇至5ml的Z-缓冲液（1升缓冲液含有16.1克的Na 2 HPO 4 7H 2 O，5.5克的NaH 2 PO 4·H 2 O，0.75克氯化钾，0.25克硫酸镁4•7H 2 O）。如果Z缓冲区保持在4℃，加入这些试剂之前，把它带到室温。 </li><li>将3毫米的滤纸两个圆在培养皿中，并用5毫升X-gal的溶液浸泡其中。排出多余的缓冲液从Petri培养皿并将其放置在硝酸纤维素膜上的菌落的一面朝上。 </li><li>盖上盖子，确保膜正与滤纸完全接触，这是完全湿透。包裹培养皿箔和孵化在37℃，30分钟至过夜。 </li></ol> 4。液体的β-半乳糖苷酶含量<ol><li>酵母生长在-His/-Leu差液体培养基中OD 600 0.7。 </li><li>转移1ml培养基至1.5 ml微量离心管中并离心以3,000 rpm持续2分钟。弃上清。 </li><li>加入1 ml的Z缓冲器中，以重悬沉淀，离心分离机在3000rpm下再持续2分钟。弃上清。 </li><li>加入150μlZ缓存与2ME重悬细胞沉淀，混匀再加入50微升氯仿和20微升0.1％SDS。大力旋涡振荡样品15秒。 </li><li>加入700μlONPG溶液（1毫克/毫升Z中的bufFER）。设置定时器，孵育在30℃下足够长的时间以使黄色开发并注意总的反应时间。 </li><li>加入500μl的1M的Na 2 CO 3使反应停止，并确定在420nm处的吸光度。空白是700微升ONPG溶液加500微升1米的Na 2 CO 3。 </li></ol>计算米勒单位如下：米勒单位=（A420 * 1,000）/（A600 *分*毫升） A420：吸光度单位在420纳米; A600：吸光度单位，在600纳米; 分钟 ：反应时间（分钟）; 毫升 ：在毫升反应体积

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没有利益冲突的声明。

在我们修改的酵母双杂交实验中，我们已经确定了对PXR 6酮康唑相互作用重要的残留物。因为SRC-1是共活化剂（和克隆到pGADNot矢量），我们还测试了SRC-1是否能当克隆到PSH载体系统激活lacZ的表达，以及是否会改变活动模式和/或影响的泄漏酵母双杂交实验。利用我们在ERG3Δ/ ERG11Δ酵母进行双杂交试验我们重新设计的质粒。和以前一样，我们表明lacZ基因表达（蓝色菌落）也诱导ERG3Δ...

在酵母双杂交（Y2H）法被广泛地用于发现蛋白质-蛋白质相互作用以及最近用于发现新颖的小分子干扰蛋白质-蛋白质相互作用复合体7，8，9，10，11。然而，这种分析的传统方法中，用于药物发现的或“命中”，不允许用于检测的化学品化合物的变构相互作用的残基中的蛋白-蛋白的表面，即改变仍然交互，并允许改变的残基11的询问时。事实上，这样的方法（次），如果制定可行，将使为别构相互作用的残基的蛋白质 - 蛋白质相互作用破坏关键的高通量评价一个听话的酵母系统。在药物发现的背景下，最直接的方式来建立化合物的相互作用与蛋白质会涉及结构测定（蛋白-抑制剂复合物如晶化）。这些方法暨bersome，用精心制作的资源和执行的每一个蛋白质结构研究它在技术上并不可行。 听话的基因药物筛选系统已经建立了细菌1，2和类似哺乳动物双杂交等模型系统。然而，这些系统需要最优化和类似Y2H替代系统仍然在药物发现的最测试。有迹象表明，包括灵敏度，并使用单数的方法13的相互作用的可靠性差的限制，然而，一个单一的Y2H测定法可以被修改来回答关于相互作用的残基的具体问题。在核受体的研究领域中，Y2H已被用来定义相互作用蛋白14，然而，这些蛋白质的相互作用很少被用于定义在其中的配体/受体拮抗剂与核受体-蛋白质复合物相互作用的性质。因此，我们的实验室集中精力定义一个方法，尤其是对于受体蛋白质，是不随时服从蛋白质组学为基础的方法，这将发掘新的配体/受体拮抗剂使用基于反向Y2H发现平台交互的残留物。 基于我们以前发现，酮康唑破坏PXR及其活化剂SRC-1，我们开发了一种新的反向酵母双杂交系统，使我们能够对PXR 6定义和查询酮康唑相互作用的残留物。我们的方法是基于酵母的GAL4的蛋白质，它由负责DNA结合和转录激活结构域可分离的属性。的PXR LBD蛋白被表达为融合到LexA的DNA结合结构域（DNA-BD），而全长共活化剂的SRC-1（类固醇受体共激活因子1）的蛋白质表达为融合至GAL4激活结构域（AD ）。 PXR和SRC-1融合蛋白之间的相互作用导致的含有报告基因的β-Lac的Ž已整合到酵母染色体组的GAL4结合位点的转录活化Ë。酮康唑，一个PXR受体拮抗剂，扰乱PXR和SRC-1相互作用15，16，17，我们可以检测PXR的相互作用和SRC-1的染色上筛选菌落用于X-gal的活动之后的存在或不存在酮康唑。 Y2H的原理图示于图1和实验过程总结于图2。

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Yeast Strain CTY10-5d erg3&#916;/erg11&#916;</td>
			<td>Our lab</td>
			<td></td>
			<td>CTY10-5d yeast was double knocked out ERG3 and ERG11 (erg3&#916;/erg11&#916;) genes6 .</td>
		</tr>
		<tr>
			<td>YPD Growth Medium</td>
			<td>BD Biosciences</td>
			<td>630409</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Yeast Nitrogen Base (YNB) w/o Amino Acids and Ammonium Sulfate</td>
			<td>BD Biosciences</td>
			<td>233520</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>BD Biosciences</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM-His/-Leu Complete Supplement Mixture</td>
			<td>MP Biomedicals</td>
			<td>4250-412</td>
			<td></td>
		</tr>
		<tr>
			<td>ONPG (o-Nitrophenyl &#914;-D- Galactopyranoside).</td>
			<td>Sigma-Aldrich</td>
			<td>N1127</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Broth (LB)</td>
			<td>Sigma-Aldrich</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>X-Gal</td>
			<td>Fisher</td>
			<td>BP-1615</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicated Salmon Sperm DNA boiled (10 mg/ml)</td>
			<td>Life Technology</td>
			<td>156-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Acros Organics</td>
			<td>61177</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketoconazole</td>
			<td>Sigma-Aldrich</td>
			<td>K1003</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Dimethylformamide</td>
			<td>Acros Organics</td>
			<td>326871000</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>L4158</td>
			<td></td>
		</tr>
		<tr>
			<td>50% PEG-3350 solution, filter-sterilized</td>
			<td>Sigma-Aldrich</td>
			<td>P-3640</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose Membrane</td>
			<td>Whatman</td>
			<td>10402091</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm Petri Dish</td>
			<td>Fisher</td>
			<td>875712</td>
			<td></td>
		</tr>
		<tr>
		</tr>
	</tbody>
</table>

<table border="1">
	<tbody>
		<tr>
			<td>5&#39;-ACCGGATCCCGATGAAGA AGGAGATGATCATGTCC-3&#39;</td>
			<td>our lab</td>
			<td></td>
			<td>PXR LBD forward primer for pSH2-1</td>
		</tr>
		<tr>
			<td>5&#39;-AGAGTCGACTCAGCTA CCTGTGATGCC -3&#39;</td>
			<td>our lab</td>
			<td></td>
			<td>PXR LBD reverse primer for pSH2-1</td>
		</tr>
		<tr>
			<td>5&#39;-TATAGC GGCCGCATGAGTG GCCTCGGGGACAGTTCATCC -3&#39;</td>
			<td>our lab</td>
			<td></td>
			<td>SRC-1 forward primer for pGADNOT</td>
		</tr>
		<tr>
			<td>5&#39;-GCGGTCGACTTATTCAGTCA GTAGCTG -3&#39;</td>
			<td>our lab</td>
			<td></td>
			<td>SRC-1 reverse primer for pGADNOT</td>
		</tr>
		<tr>
			<td>Platinum PCR Supermix</td>
			<td>Invitrogen</td>
			<td>11306-016</td>
			<td></td>
		</tr>
		<tr>
			<td>BamHI</td>
			<td>our lab</td>
			<td>R0136</td>
			<td></td>
		</tr>
		<tr>
			<td>SalI</td>
			<td>our lab</td>
			<td>R0138</td>
			<td></td>
		</tr>
		<tr>
			<td>NotI</td>
			<td>our lab</td>
			<td>R0189</td>
			<td></td>
		</tr>
	</tbody>
</table>

reverse yeast two-hybrid system to identify mammalian nuclear receptor residues that interact with ligands and/or antagonists

由于药物代谢和炎症反应的重要调节器，孕烷X受体（PXR），它在疾病的病理生理过程中起重要作用联系起来代谢和炎症（ 如脂肪肝）1,2。出现了在激动剂配体为PXR的识别很大的进展，但是，也有类药拮抗剂和PXR 3,4,5其结合位点的有限的描述。一个关键的障碍已经无法有效地净化全长蛋白与拮抗剂的结构研究尽管PXR克隆和特征1998。我们实验室开发了一种新的高通量酵母基于双杂交法来定义的拮抗剂，酮康唑的，上PXR 6结合的残基。我们的方法包括创建突变库，将营救的单一突变对PXR的AF-2面有望与酮康唑交互的效果。事态抢救或“功能获得的”塞科第二突变可制成使得关于酮康唑对PXR的遗传相互作用和表面残基的结论是可行的。因此，我们开发PXR突变体及其共激活因子，SRC-1相互作用的高通量双杂交酵母屏幕。使用这种方法，其中的酵母进行了修改，以适应抗真菌药，酮康唑的研究中，我们能够证明对PXR的特定突变富集的克隆不能结合到酮康唑。通过反向逻辑，我们的结论是原来的残留物是直接的互动残酮康唑。此法是一种新型的，听话的基因检测，以屏幕上的核受体拮抗剂的表面结合位点。该测定法可以适用于任何药物，无论其细胞毒性潜力的酵母以及细胞蛋白（s）表示，不能使用标准结构生物学或基于蛋白质组学方法，研究。潜在的缺陷包括解释数据的（互补的方法很有用），可靠ANCE单Y2H的方法，在处理酵母或进行酵母双杂交试验的专业知识，并检测优化。

我们进行了一个实验，看看我们是否可以检测PXR和类固醇受体共激活因子-1（SRC-1）的协会的比色读数。由于酵母具有显著甾醇的生产，它先前已经表明，在酵母中的lacZ的表达，而不需要额外的外源性配体所诱导。我们发现，lacZ的表达（蓝色菌落）也感应出的酵母菌株转化的PXR和SRC-1，但是，没有诱导LacZ的表达（白色菌落）在酵母转化的空载体，PXR的或单独的SRC-1 （ 图3）。以确定酮...

Watch this Scientific Journal Video about Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists at JoVE.com

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

酮康唑结合并拮抗孕烷X受体（PXR）激活。 PXR突变体的酵母高通量筛选定义一个独特的区域为酮康唑约束力。这种酵母为基础的遗传学方法发现与具有表面结合位点相关联的配体小说核受体相互作用。

反向酵母双杂交系统，以确定哺乳动物核受体残留的互动与配体和/或拮抗剂

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

reverse-yeast-two-hybrid-system-to-identify-mammalian-nuclear

Research

JoVE Journal

Biology

12.7K 次观看.  Albert Einstein College of Medicine. 酮康唑结合并拮抗孕烷X受体（PXR）激活。 PXR突变体的酵母高通量筛选定义一个独特的区域为酮康唑约束力。这种酵母为基础的遗传学方法发现与具有表面结合位点相关联的配体小说核受体相互作用。 

视频: Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

蛋白质组学鉴定蛋白质，P2X2配体门控的阳离子通道交互

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

科研

生物学

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

基于分体式泛素膜酵母双杂交（神话）系统：一个强大的工具用于识别蛋白质 - 蛋白质相互作用

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

在哺乳动物细胞通过显微注射mRNA的核出口动力学分析

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

改良酵母双杂交系统，以确定蛋白与生长因子Progranulin的相互作用

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine disruption. Estrogenic ligands include both natural and manmade &#34;Environmental Estrogens&#34; (EEs) found in many consumer goods including Personal Care Products (PCPs), plastics, pesticides, and foods. EEs disrupt hormone signaling in humans and other animals, potentially reducing fertility and increasing disease risk. Despite the importance of EEs and other Endocrine Disrupting Chemicals (EDCs) to public health, endocrine disruption is not typically included in undergraduate curricula. This shortcoming is partly due to a lack of relevant laboratory activities that illustrate the principles involved while also being accessible to undergraduate students. This article presents an optimized YES for quantifying ligands in personal care products that bind estrogen receptors alpha (ER&#945;) and/or beta (ER&#946;). The method incorporates one of the two colorimetric substrates (ortho-nitrophenyl-&#946;-D-galactopyranoside (ONPG) or chlorophenol red-&#946;-D-galactopyranoside (CPRG)) that are cleaved by &#946;-galactosidase, a 6-day refrigerated incubation step to facilitate use in undergraduate laboratory courses, an automated application for LacZ calculations, and R code for the associated 4-parameter logistic regression analysis. The protocol has been designed to allow undergraduate students to develop and conduct experiments in which they screen products of their choosing for estrogen mimics. In the process, they learn about endocrine disruption, cell culture, receptor binding, enzyme activity, genetic engineering, statistics, and experimental design. Simultaneously, they also practice fundamental and broadly applicable laboratory skills, such as: calculating concentrations; making solutions; demonstrating sterile technique; serially diluting standards; constructing and interpolating standard curves; identifying variables and controls; collecting, organizing, and analyzing data; constructing and interpreting graphs; and using common laboratory equipment such as micropipettors and spectrophotometers. Thus, implementing this assay encourages students to engage in inquiry-based learning while exploring emerging issues in environmental science and health.

This article presents an optimized yeast estrogen screen for quantifying ligands in Personal Care Products (PCPs) that bind estrogen receptors alpha (ER&#945;) and/or beta (ER&#946;). The method incorporates two colorimetric substrate options, a six-day refrigerated incubation for use in undergraduate courses, and statistical tools for data analysis.

应用酵母雌激素筛检优选本科教学实验室的个人护理产品中的雌激素配体

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine ...

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor&#39;s function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).

一种基于流式细胞仪的检测方法, 用于识别破坏荧光标记的 CXC 趋化因子配体12到 CXC 趋性因子受体4的化合物

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

荧光活细胞显微镜对微孔裂变酵母核动力学的检测

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

结合表观遗传修饰和生物力学线索的两步策略，以产生哺乳动物多能细胞

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...