Najmeh Nikpour and Fiona Alum provided suggestions and edited this manuscript. Department of Biochemistry at McGill University supported HM and VM with the CIHR Training Initiative in Chemical Biology. This work was supported by Canadian Institute of Health Research (CIHR) grant 119464 (to R. S.).

下面的协议描述了用于执行基于荧光的RNA编辑分析的过程。该测定法可以在一个单一的PCR管，96孔或384孔取决于实验的范围板来进行。随后的荧光信号可以读合适的实时PCR检测系统。这里在测定中的384 - 孔板的上下文中被描述。 1，培养T。布氏细胞<ol><li>准备一个生长培养基为T。布氏 procyclic形式的细胞。 1 L培养基: <ol><li>溶解25.4克SDM-79粉末在800毫升miliQ水。 </li><li>添加2克的NaHCO 3和pH值至7.3，10 M氢氧化钠。 </li><li>加超纯水至900毫升，过滤消毒的最终体积。 </li><li>添加胎牛血清（FBS），青霉素 - 链霉素溶液和氯高铁血红素（2.5毫克/毫升）至终浓度为10％（体积/体积），分别为100单位/毫升和7.5毫克/升。 </li></ol></li><li>成长300毫升T. &lt;中EM&gt;布氏1.7A野生型（procyclic形式）细胞在28℃，在70转摇到1.5×10 7细胞/ ml的密度。注:这应该会产生3毫升活跃editosome与〜0.5毫克总蛋白，足够600编辑反应。 </li><li>离心6000×g离心10分钟收集细胞，在4℃下</li><li>用50ml冷PBSG缓冲液（10mM的磷酸氢二钠，10mM的的NaH 2 PO 4，145 mM氯化钠，以及6毫米的葡萄糖）洗涤沉淀，并再次离心收集到的细胞通过离心分离以10,000 xg在10分钟4°C。 </li></ol>原油线粒体2。隔离注意:所有的步骤应在冰上或4℃进行保存editosome活性。 <ol><li>重悬在30毫升的DTE缓冲液（1mM的Tris-HCl pH为8.0和1mM EDTA）中收获的细胞。用40毫升无菌Dounce匀浆（预冷）由抚摸着向上和向下的至少10倍于冰上，破坏细胞膜。 </LI&gt; <li>立即4.3毫升60％蔗糖（w / v的即 1.75 M）添加到匀浆至0.25米离心机的终浓度在15800×g下在4℃下10分钟，以优先减低线粒体。 </li><li>重悬线粒体沉淀4.6毫升STM缓冲液（20mM的Tris-HCl pH值8.0，250mM的蔗糖和2mM的MgCl 2）。添加13.8微升的0.1M的CaCl 2和4微升不含RNA酶的DNA酶I至终浓度分别为0.3毫米和9 U /毫升。为1小时，在冰上孵育混合物。 </li><li>添加4.6的STE缓冲液（20mM的Tris-盐酸pH值8.0，250mM的蔗糖和2mM EDTA）中在4℃以灭活DNA酶I。离心机在15800×g离心10分钟</li><li>重悬沉淀中加入400μl的裂解缓冲液（10mM的Tris-HCl（pH7.2）中的10mM的MgCl 2，100mM的氯化钾，1微克/毫升胃蛋白酶抑制素，1mM的DTT和1×完全不含EDTA的蛋白酶抑制剂），并转移到离心管。 </li><li>加10％的Triton X-100至1％的终浓度第二温育裂解物15分钟，在4℃下在管式旋转器。 </li><li>离心两次在17000×g离心15分钟，在4℃下清除线粒体裂解液;每次保留清除上清液。 </li></ol> 3。Editosome净化<ol><li>倒入10ml的10％-30％（V / V）线性梯度甘油（ 表1）中使用2个HHE梯度缓冲液（40mM的HEPES pH值为7.9，20mM的镁（醋酸）2，100mM的氯化钾超速离心管中，并2 mM EDTA）中，并通过下面的说明书的梯度壶。 </li><li>小心地从甘油梯度的顶部取出500微升的溶液，轻轻地加载500微升的清除线粒体裂解液。旋在178,000 xg离心6小时，在4℃下用超离心。 </li><li>收集500μl的级分连续地从顶部到梯度的在4℃下然后将其卡冻结使用液氮和储存在-80℃直至使用的馏分。 </li></ol><ol><li>退火含有互补序列的T7启动子序列（ 表2）与T7启动子的寡核苷酸（5&#39;-TAATACGACTCACTATAGGG-3&#39;）中，加热至90℃保持3分钟以1:1的摩尔比和冷却在RT相应DNA模板为至少10分钟。 </li><li>使用体外转录试剂盒由以下的说明书转录的RNA。 </li><li>停止转录反应通过加入7M尿素染料（7M尿素，0.05％二甲苯Cynol，和0.05％溴酚蓝）等体积。在消毒9％变性聚​​丙烯酰胺凝胶（9％丙烯酰胺，7M尿素，1×TBE）过滤器运行。 </li><li>使用紫外线（UV）遮蔽用短波紫外线灯进行定位和切除相应的RNA。将切下的凝胶块在一个离心管中，并加入400微升的凝胶洗脱缓冲液（20mM的Tris-HCl pH值7.5，250mM的醋酸钠，1mM EDTA和0.25％SDS）中。洗脱过夜在RT上一个管肩。 </li><li> PRECI通过加入1ml冰冷的100％乙醇并进行培养或在-80℃下30分钟或-20℃过夜pitate洗脱RNA。 </li><li>离心机以16,000×g离心30分钟，在4℃，以沉淀下来的核糖核酸。 </li><li>用1毫升75％乙醇洗涤沉淀。离心机以16,000×g离心20分钟，在4℃下</li><li>适当地重悬RNA沉淀在无RNA酶的水以达到所需的浓度，如表2所示。 </li></ol> 5，基于荧光的RNA编辑含量<ol><li>为一个单一的反应，结合1皮摩尔（1微升）preA6Rbz和2.5皮摩尔（1微升）gA6Rbz的（1:2.5摩尔比）在微量离心管中，孵育在70℃维持3分钟，并让其在室温下静置在至少10分钟。 </li><li>同时使用表3中制备主混合物，而不preA6Rbz和gA6Rbz，对于含有1x HHE缓冲液（25mM HEPES pH值为7.9，10mM的镁（醋酸）2，50mM的氯化钾和10mM EDTA），1 mM的编辑反应ATP，5mM的氯化钙2，圆酵母RNA，0.1％的Triton X-100和纯化的editosome 16纳克/微升。 </li><li>添加退火preA6Rbz和gA6Rbz完成主结构。 </li><li>免除18微升主混合物（ 表3）的成包含2微升不含RNA酶的水（井用无化合物）或2微升200μM的化学化合物的井，并按照图5包括对照样品中的板。 </li><li>密封板与板密封件和旋转盘下来，以消除任何气泡。孵化在28℃保温4小时。 </li><li>添加25皮摩尔gA6Rbz竞争者（2微升）到每个孔中。放置一个新密封剂，向下旋转的板，并把它实时PCR仪上。程序如下实验: 第1步:85℃5分钟;第2步:24℃，10分钟;步骤3:停止。 </li><li>加FRET底物的15皮摩尔（1微升）到每个孔中，以23微升的最终体积。用新鲜的封口机封板。快速旋转盘，并把它背在实时PCR仪上。 </li><li>设定新的实验步骤如下: 第1步:37℃1分钟;第2步:阅读;第3步:进入步骤1，40倍;步骤4:停止。 </li><li>安装板通过选择所有需要阅读和选择FAM过滤器的井。输入量为23微升并开始运行。 </li><li>计算从每个孔/样品获得通过绘制的斜坡上的条形图进行分析，以获得动力学测量值的斜率。注:动能读提高了样品和背景之间的信号噪声比;作为背景样品将有一个斜率接近于零。结束点读具有较高的背景噪音。 </li></ol>

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The authors have nothing to disclose.

一种新型的高通量筛选方法来确定对锥虫的RNA编辑复杂的抑制剂已提交，为药物发现的新工具来对抗引起的锥虫病。基于FRET的核酶法已被广泛地用于不同的用途20-22;然而，我们动用的体外监测RNA编辑活动19 FRET为基础的核酶分析的能力。该测定可能适用于其他类型的RNA编辑在真核生物，如哺乳动物23的核编码的RNA的核苷酸替换编辑。 该测定中的?...

RNA编辑，转录后修饰的mRNA的过程中，首先被锥虫1发现。从那时起，大量的工作已经在后面学习中的RNA编辑布氏锥虫 2,3机制进行的。在一系列的酶反应中，editosome，约20蛋白核心复合物，产生成熟的mRNA线粒体的能量产生的氧化磷酸化系统的多个组件。催化事件的顺序是信使核糖核酸，尿苷酸（U）增加或删除，并结扎，所决定的指导的RNA（gRNAs）4。 除了 ​​核心editosome复杂的蛋白质，一些辅助因子也已确定5-7。这些蛋白质大多见于分组独立的复合物。然而，蛋白质装配在芯editosome复杂的顺序和核心复合物与该配件的交互模式配合物尚未确定。针对RNA编辑的过程中锥虫可提供化学解剖，援助在研究editosome复杂的装配和功能。此外，在几个editosome蛋白质功能的研究表明在不同人生阶段的必要性，指出其潜在的药物靶点8-12。因此，editosome的发现抑制剂也可作为对锥虫的先导化合物。这是及时的，因为目前市面上所引致的锥虫病的药物是有毒的，低效和昂贵13,14。 一个高效，便捷的体外试验要探索的化学宇宙，阻止RNA编辑特异性抑制剂。三个实验已经开发并用于监视editosome活动:（a）全轮体外 RNA编辑分析15（二）预切割体外 RNA编辑实验16,17，一ND（三）锤头状核酶（HHR）为基础的检测18。前两个实验依靠编辑的产品（ATP酶6基因）与放射性的帮助下直接观察。该HHR基测定法使用了被建模的行为作为经编辑的核酶的ATP酶6 mRNA的修改版本。功能核酶则特异性切割放射性标记的RNA底物，作为一名记者。最近，MOSHIRI 等人开发了一种"混合，并测量"HHR-基于体外报告基因分析来监测RNA编辑其中的放射性标记的RNA底物被替换为荧光共振能量转移（FRET）底物19。该测定法的原理的优点是:（a）它是一种快速和方便的混合和测定法的测量类型，为生产活性核酶和底物裂解的同时出现在同一试管中低体积（ 即 20微升），（二）它避免了使用放射性标记的物质，（C）的灵敏度是一个通过荧光仪在微滴定板格式，以及（d）一个高信噪比fforded。使用这种测定法，对纯化的editosome已知的RNA编辑连接酶抑制剂的作用被证实19。这个实验验证了分析的快速鉴定RNA编辑酶抑制剂，主要针对来自T.整个editosomes 布氏 。 图1是一个详细的一步一步的示意性的基于荧光的体外 RNA编辑测定。这个协议可以被用于监测在体外 RNA编辑或很容易地适应用于筛选各种比例的化合物库。

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			<td height="20" style="height:20px;">SDM-79 Medium</td>
			<td>Gibco by life technologies</td>
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			<td>life technologies</td>
			<td style="width:139px;">12483-020</td>
			<td>heat inactivation at 550C for 1 h</td>
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			<td height="21" style="height:21px;width:391px;">Hemin, minimum 80%</td>
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			<td style="width:139px;">H5533-10G</td>
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			<td height="21" style="height:21px;width:391px;">Penicillin-Streptomycin Sollution</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:139px;">MT-30-002-CI</td>
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			<td height="21" style="height:21px;width:391px;">Dnase 1 recombinant, Rnase Free&#160;</td>
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			<td style="width:139px;">4716728001</td>
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			<td height="21" style="height:21px;width:391px;">T7 RiboMax Express Large&#160; scale RNA&#160; production system</td>
			<td style="width:203px;">Promega</td>
			<td style="width:139px;">P1320</td>
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			<td height="21" style="height:21px;width:391px;">Kimble Kontes Dounce Tissue Grinders&#160;&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:139px;">K885300-0040&#160;&#160;</td>
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			<td height="21" style="height:21px;width:391px;">Gradient Master, ver 5.25&#160;</td>
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			<td style="width:139px;">107-201M</td>
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			<td height="21" style="height:21px;width:391px;">Ultra Clear Tube, 13.2ML</td>
			<td style="width:203px;">Beckman Coulter</td>
			<td style="width:139px;">344059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Optima L-100XP&#160; Ultracentrifuge&#160;</td>
			<td style="width:203px;">Beckman Coulter</td>
			<td style="width:139px;">392052</td>
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			<td height="21" style="height:21px;width:391px;">SW 41 Ti ROTOR</td>
			<td style="width:203px;">Beckman Coulter</td>
			<td style="width:139px;">331336</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">MicroSeal &#39;B&#39; Seal, Seals</td>
			<td style="width:203px;">Biorad</td>
			<td style="width:139px;">MSB1001</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:391px;">CFX 384 Touch Real-Time PCR Detection System</td>
			<td style="width:203px;">Biorad</td>
			<td style="width:139px;">185-5484</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Acryl/Bis solution (19: 1), 40% (w/v)</td>
			<td style="width:203px;">Bio Basic</td>
			<td style="width:139px;">A0006-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Urea, Molecular biology grade, 1Kg</td>
			<td style="width:203px;">life technologies</td>
			<td style="width:139px;">AM9902</td>
			<td style="width:248px;"></td>
		</tr>
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rna catalyst as a reporter for screening drugs against rna editing in trypanosomes

实质性的进展已经取得了确定的线粒体RNA编辑在锥虫的机制。同样，相当大的进展已经取得了识别editosome络合物的催化RNA编辑的组件。但是，目前还不清楚这些蛋白质是如何协同工作。从对editosome高通量筛选获得的化学化合物可以阻止或影响在编辑周期的一个或多个步骤。因此，新化合物的鉴定将产生有价值的分子探针解剖editosome功能和组装。在以前的研究中， 在体外测定法的编辑进行了使用放射性标记的RNA。这些测定法是耗时，低效和不适合于高通量的目的。在这里，均匀的荧光为基础的"混合和计量"锤头状核酶体外记者分析监测RNA编辑，呈现。仅作为RNA编辑的结果锤头状核酶荧光共振能量转移（FRET）寡基板发生裂解。这反过来导致荧光团与猝灭剂，从而产生一个信号分离。与此相反，当editosome功能受到抑制，荧光信号会被淬灭。这是一个高度敏感和简单的分析，应该是普遍适用于体外 RNA编辑或化学物质，可以抑制editosome功能的高通量筛选监控。

以证明需要设立一个大型屏幕必要的步骤， 图2-5是与测定的质量代表对照实验。这些都是必不可少的对照实验为一致的测定过筛选的几天或不同的屏幕之间的对比。 评估荧光信号噪声比 以确保在一个大型安装的荧光素标记的寡核糖核苷酸底物的稳定性和质量，Z&#39;因子，其定义为在测定背景和最大信号之间的差用的是活性核酶...

Watch this Scientific Journal Video about RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes at JoVE.com

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

RNA催化剂作为记者为筛选药物对RNA编辑在锥虫

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been ...

rna-catalyst-as-reporter-for-screening-drugs-against-rna-editing

Research

JoVE Journal

Biology

8.4K 次观看.  McGill University. A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

视频: RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

可视化的RNA中的本地化爪蟾卵母细胞

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

科研

生物学

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

在体外转录和Gaussia荧光素酶基因的上限由HeLa细胞转染

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA纯化的染色质隔离（CHIRP）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

作为寄主植物挥发物的筛选工具electroantennographic生物活性

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

CRISPR / CAS系统内切酶的底物生成

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

核糖体RNA的蚊子肠道宏基因组RNA-seq的枯竭

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em在原位</em&gt;改编为海洋软骨鱼类杂交整装胚胎发育和细胞组织学

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

对糖皮质激素信号A化学筛选程序与斑马鱼幼虫的荧光素酶报告系统

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

对于耐药性突变对激酶抑制剂的筛选和验证方法

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

RNA结合蛋白的检测通过<em&gt;体外</em在脂肪细胞培养&gt; RNA下拉

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...