授予没有工作是支持的。 01GU0615的“Bundesministerium献给Forschung和TECHNOLOGIE（BMBF）的。

1）细菌的shRNA的交付<ol><li>与细菌培养开始之前，人们已经准备LB培养基和LB琼脂。 </li><li> LB培养基掂量出酵母提取物（0.5％W / V），细菌用胰蛋白胨（1.0％W / V），氯化钠（0.6％W / V）和稀王水bidest这些组件。制备的溶液，在高压灭菌器消毒，然后准备使用。 </li><li>对于细菌用琼脂（1.5％W / V）的LB琼脂平板已经被添加到前杀菌的​​LB培养基。 </li><li>在微波加热直到完全溶解的LB琼脂的LB琼脂。让我们冷静下来解决方案可触及，直到瓶子很容易灼伤。选择阳性克隆进一步的步骤添加卡那霉素（100毫克/毫升）。 </li><li>吹打LB琼脂溶液20毫升到10厘米的Petri菜，准备在层流工作台（无菌条件下）的LB琼脂平板。让板冷静下来，直到液体已成为坚实的。板块现在已经准备好使用，并可以储存在4 ° C。 </li><li>编码shRNA的表达载体转化为主管大肠杆菌大肠杆菌 ceq221热休克， 使用氯化钙程序。 </li><li>含100μg/ ml卡那霉素的LB琼脂板的板转化菌，盖上盖子，用封口膜密封板和培育倒挂在37 °彗星在夜间。 </li><li>采摘生长的菌落，用牙签，接种细菌阳性克隆的迷你文化。转移到7.0毫升含100毫克/毫升卡那霉素和培养在37℃以上夜间在200转筛的新鲜LB培养基牙签。 </li><li>接种100毫升的新鲜LB培养基含37 100 mg / ml的卡那霉素1:100迷你文化在夜间，在1升的锥形瓶中，并培育℃以上夜间在200转筛。 </li><li>种子25 × 10 4（种子细胞的数量取决于根据细胞株的细胞生长速度;汇合应播种后24小时〜70-80％）人胃癌细胞每孔（EPG85 257RDB）6 -以及菜肴，并培育这些在37℃，5％的CO 2，水蒸汽在夜间饱和的气氛。 </li><li>前癌细胞感染，超过TK RNAi载体含有大肠杆菌的夜间文化大肠杆菌是衡量一个光度计来确定的OD 600。稀释细菌的解决方案，直到0.5的光密度达到 （外径600 = 0.5 = 1.6 × 10 8细菌细胞/ ml）。 </li><li>更换对FCS的培养基30分钟以前的细菌共同培养的癌细胞FCS含细胞培养液中。 </li><li> 1 × PBS洗两次细菌，并在无血清Leibovitz的L - 15培养基稀释。 </li><li>添加稀释细菌癌细胞（感染复数=每个肿瘤细胞的细菌细胞的数量），在需要的教学语言和共同培育的细菌和2个小时的癌细胞在37 ° C。 </li><li>合作培养的癌细胞后2小时内与1X x PBS洗两次，一旦与含血清的L - 15 100 U / ml青霉素，100微克/毫升链霉素，2.5微克/ 150微克/毫升霉素，莱博维茨培养基毫升庆大霉素，和100μg/ ml卡那霉素。 </li><li>继续培养癌细胞在37℃，5％的二氧化碳 ，水蒸气饱和的气氛。 </li><li>细菌治疗的效果，可以可视化的实时定量RT - PCR测定mRNA水平，荧光显微镜和免疫印迹分析蛋白水平，并与功能分析，如流式细胞仪分析，细胞增殖实验显示。 （如果需要，这些程序可以在详细描述）。 </li></ol> 2）代表性的成果如果协议是正确执行，其结果应与图所示的。 荧光显微镜图1显示了三个小时后，细菌治疗的人胃癌细胞相比，未经处理的细胞（B）（a）在。围绕处理的细胞的细胞核，可以检测出细菌。 <img src="/files/ftp_upload/2099/2099fig1.jpg" alt="图1" /> 图1：荧光显微镜细菌治疗（1:500）后人类胃癌细胞和未经处理的人胃癌肿瘤细胞作为对照，DAPI染色，DAPI带通滤波器（Λem= 640纳米），40倍的目标。 实时定量RT - PCR 一个约70％的MDR1 mRNA的下调（黑梁）图2可以看出，治疗后与治疗细菌。亲代细胞作为阳性对照，由于缺乏MDR1基因的过度表达。 257RDB P170包括细胞株的质粒表达抗多药耐药基因的shRNA小号transkingdom RNAi技术直接比较其他RNAI沉默策略。未经处理的耐药细胞株EPG85 257RDB过度表达的多药耐药，缺乏的shRNA表达质粒的细菌处理相同的细胞系，并治疗细菌携带质粒表达反MRP2的shRNA治疗这个细胞系作为阳性对照。 <img src="/files/ftp_upload/2099/2099fig2.jpg" alt="图2" /> 图2：实时定量RT - PCR MDR1基因治疗后与治疗大肠杆菌 mRNA的表达大肠杆菌 ceq221表达抗多药耐药基因的shRNA（教学语言1：500）。用看家基因醛缩酶正常化。未经处理的细胞的细胞线EPG85 257RDB MDR1/aldolase比例分别为100％。使用学生的t -检验计算P值（* = P &lt;0.05，** = P &lt;0.005，*** P &lt;0.001）。 Western blot分析 图3显示多药耐药基因也下调了细菌处理后的蛋白质水平上进行。这显示缺乏父母的药物敏感细胞株（EPG85 257P），未经处理的耐药细胞株（EPG85 257RDB）MDR1的表达和处理的样品MDR1的表达MDR1的表达。可以观察到的细菌处理的细胞MDR1的一个明显的下调。 <img src="/files/ftp_upload/2099/2099fig3.jpg" alt="图3" /> 图3：免疫印迹分析未经处理的药物敏感EPG85 - 257P MDR1的表达水平，未经处理的耐药EPG 257RDB，EPG85 - 257RDB与E.共同孵育后。 大肠杆菌 ceq221 + P43 MDR1的。小学AB C219 1:100，抗肌动蛋白二级AB抗鼠1:10 000 1:5 000。 细胞毒试验如图4所示的细胞毒性实验等功能的分析也transkingdom的RNAi的运作指标。亲本细胞株和ANIT - MDR1基因的shRNA表达质粒的细胞株，其中包含不显示任何柔红霉素的阻力。耐药细胞株EPG85 257RDB和另外两个对照组不显示在抗多药耐药基因的shRNA表达柔红霉素的阻力可以逆转约90％的细菌处理的样品相比，在电阻的显着变化。 <img src="/files/ftp_upload/2099/2099fig4.jpg" alt="图4" /> 图4：细胞毒试验。药物由一个细胞存活的细胞毒试验的具体确定的IC50值。使用学生的t -检验计算P值（* = P &lt;0.05，** = P &lt;0.005，*** P &lt;0.001）。 蒽环类药物的积累检测据降低细菌处理的样品的电阻（图4），蒽环类抗多药耐药基因的shRNA处理的细胞积累增加约90％，如图5所示。父母的细胞株和细胞系257RDB P170表现出强烈的柔红霉素积累高达100％。耐药变异显示很少任何积累。抗多药耐药基因的shRNA表达细菌处理的细胞显示anthracyline积累增加了90％。 <img src="/files/ftp_upload/2099/2099fig5.jpg" alt="图5" /> 图5：蒽环类药物的积累检测。蒽环类药物的积累通过流式细胞仪检测细菌治疗后6天的癌细胞。使用学生的t -检验计算P值（* = P &lt;0.05，** = P &lt;0.005，*** P &lt;0.001）。

<ol>
	<li>Kr&#252;hn, A., Wang, A., Fruehauf, J. H., Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+short+hairpin+RNAs+by+transkingdom+RNA+interference+modulates+the+classical+ABCB1-mediated+multidrug-resistant+phenotype+of+cancer+cells.">Delivery of short hairpin RNAs by transkingdom RNA interference modulates the classical ABCB1-mediated multidrug-resistant phenotype of cancer cells.</a> Cell Cycle. 8, 3349-3354 (2009).</li><li>Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MDR1/P-glycoprotein+(ABCB1)+as+target+for+RNA+interference-mediated+reversal+of+multidrug+resistance.">MDR1/P-glycoprotein (ABCB1) as target for RNA interference-mediated reversal of multidrug resistance.</a> Curr. Drug Targets. 7, 813-821 (2006).</li><li>Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+cancer+multidrug+resistance:+a+still+unsolved+problem.">An overview of cancer multidrug resistance: a still unsolved problem.</a> Cell. Mol. Life Sci. 65, 3145-3167 (2008).</li><li>Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+RNA+interference+in+drug-resistant+cancers.">Therapeutic potential of RNA interference in drug-resistant cancers.</a> Fut. Oncol. 5, 169-185 (2009).</li><li>Nguyen, T. A., Fruehauf, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transkingdom+RNA+interference+(tkRNAi):+a+novel+method+to+induce+therapeutic+gene+silencing.">Transkingdom RNA interference (tkRNAi): a novel method to induce therapeutic gene silencing.</a> Methods Mol. Biol. 514, 27-34 (2009).</li><li>Nieth, C., Priebsch, A., Stege, A., Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+the+classical+multidrug+resistance+(MDR)+phenotype+by+RNA+interference+(RNAi.">Modulation of the classical multidrug resistance (MDR) phenotype by RNA interference (RNAi.</a> FEBS Lett. 545, 144-150 (2003).</li><li>Stege, A., Priebsch, A., Nieth, C., Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+and+complete+overcoming+of+MDR1/P-glycoprotein-mediated+multidrug+resistance+in+human+gastric+carcinoma+cells+by+RNA+interference.">Stable and complete overcoming of MDR1/P-glycoprotein-mediated multidrug resistance in human gastric carcinoma cells by RNA interference.</a> Cancer Gene Ther. 11, 699-706 (2004).</li><li>Stein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+in+vivo+reversal+of+the+multidrug+resistance+(MDR)+phenotype+by+jet-injection+of+anti-MDR1+short+hairpin+RNA-encoding+plasmid+DNA.">Complete in vivo reversal of the multidrug resistance (MDR) phenotype by jet-injection of anti-MDR1 short hairpin RNA-encoding plasmid DNA.</a> Mol. Ther. 16, 178-186 (2008).</li><li>Xiang, S., Fruehauf, J., Li, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+hairpin+RNA-expressing+bacteria+elicit+RNA+interference+in+mammals.">Short hairpin RNA-expressing bacteria elicit RNA interference in mammals.</a> Nat. Biotechnol. 24, 697-702 (2006).</li></ol>

接种感染的细胞数和相应的教学语言的使用，关键取决于下观察细胞系，他们的增长速度。为了找到最佳的手机号码，播种，预实验，以确定的速度增长，强烈推荐。除此之外，不同的水分应进行测试，由于有限的延长，细胞可以站立，没有压力的死亡细菌的入侵。下观察基因的下调可能会有所不同的最佳时间点。建议执行超过一定的时间来寻找最佳条件内实验。

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Agar</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>214050</td>
<td>&nbsp;</td>
<tr>
<td>Amphotericin B</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>A2612</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Phosphate Buffered Saline-PBS (10 x)</td>
<td>&nbsp;</td>
<td>Invitrogen GmbH</td>
<td>14080048</td>
<td>&nbsp;</td>
<tr>
<td>E. coli ceq221</td>
<td>&nbsp;</td>
<td>Cequent Pharmaceuticals Inc.</td>
<td>&nbsp;</td>
<td>No catalogue available, direct order</td>
</tr>
<tr>
<td>Gentamycin</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>A2710</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen GmbH</td>
<td>&nbsp;</td>
<td>Contains 5,000 units of penicillin (base) and 5,000 &mu;g of streptomycin (base)/ml utilizing penicillin G (sodium salt) and streptomycin sulfate in 0.85% saline.</td>
</tr>
<tr>
<td>Sodium chloride</td>
<td>&nbsp;</td>
<td>Merck KgaA</td>
<td>1064060500</td>
<td>&nbsp;</td>
<tr>
<td>Tryptone</td>
<td>&nbsp;</td>
<td>Difco Laboratories</td>
<td>211705</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Extract</td>
<td>&nbsp;</td>
<td>Difco Laboratories</td>
<td>212750</td>
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bacterial delivery of rnai effectors: transkingdom rnai

RNA干扰（RNAi）代表一个特定的mRNA表达抑制的高效机制。除了其作为一个功能强大的实验室工具的潜力，RNAi途径似乎是有希望的治疗利用。对于RNA干扰（RNAi）技术为基础的治疗，交付的发展RNA干扰调解剂对靶细胞的主要障碍之一。一种新的策略，以克服这一障碍是transkingdom RNA干扰（RNAi的TK）。这种技术采用非致病性细菌，如大肠杆菌 ，产生并传递到靶细胞治疗短发夹RNA（shRNA）诱导的RNAi。第一代TK的RNAi介导载体，跳闸，含有噬菌体T7启动子表达调控治疗的shRNA利益。此外，行程由耶尔森氏菌pseudotuberculosis发票编码invasin的轨迹，允许自然的非侵入性细菌进入β1整合素阳性哺乳动物细胞和单核细胞增生李斯特菌HlyA基因，产生listeriolysin O.这种酶可以使治疗的shRNA摆脱项囊泡内的靶细胞的细胞质中。 TRIP结构是一个称职的非致病性大肠埃希氏菌菌株，编码T7 RNA聚合酶对T7启动子驱动的shRNA的合成所必需的引入。一个良好的特点与癌症相关的，针对不同的RNAi战略目标分子是ABCB1（MDR1/P-glycoprotein，MDR1/P-gp）。这ABC转运行为，作为药物挤压泵和调解“经典”ABCB1 -介导的多药耐药（MDR）的特点是由一个特定的交叉耐药模式的人类癌症细胞的表型。不同的ABCB1表达MDR肿瘤细胞治疗与反ABCB1 shRNA表达载体轴承 E. 大肠杆菌 。此过程导致癌细胞内的RNAi途径激活一个相当大的下调ABCB1编码的mRNA以及相应的药物挤出泵的。因此，增强药物蓄积在质朴的耐药癌细胞MDR表型逆转。通过这种模式，数据提供的证据的概念，TK RNAi是适用于癌症相关的因素，如ABCB1在人类癌细胞中，调制。

Watch this Scientific Journal Video about Bacterial Delivery of RNAi Effectors: Transkingdom RNAi at JoVE.com

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA干扰（RNAi）技术为基础的疗法的发展，一种新型的战略发展，transkingdom RNA干扰（tkRNAi）。该技术使用非致病性细菌产生并传递到靶细胞治疗短发夹RNA（shRNA）。在这里，tkRNAi成功地应用于古典ABCB1介导的多药耐药（MDR）的癌细胞逆转。

细菌提供的RNAi效应：Transkingdom RNA干扰

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

bacterial-delivery-of-rnai-effectors-transkingdom-rnai

Research

JoVE Journal

Biology

13.4K 次观看.  Charité Campus Mitte. RNA干扰（RNAi）技术为基础的疗法的发展，一种新型的战略发展，transkingdom RNA干扰（tkRNAi）。该技术使用非致病性细菌产生并传递到靶细胞治疗短发夹RNA（shRNA）。在这里，tkRNAi成功地应用于古典ABCB1介导的多药耐药（MDR）的癌细胞逆转。

视频: Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

成蚊（冈比亚按蚊的RNAi实验议定书）

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

科研

生物学

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls.
The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of ~50-150 &#181;m and extends over ~15 &#181;m around the mean penetration depth, which is adjustable between 0 and 50 &#181;m. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches.
For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery.
When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.

In this video, we show a procedure for an accurate biolistic delivery of reagents into live tissue with a novel miniature gene gun. We are knocking down the expression of the axon guidance molecule Netrin in leech embryos by delivering molecules of dsRNA into the ventral body wall and ganglia of single segments.

本地化的RNAi和药用水蛭异位基因表达

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

细菌III型效应，在酵母中的表达诱导的生长抑制的表型鉴定

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

在哺乳动物细胞的RNAi筛选使命esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

RNAi的干扰双链RNA注射到果蝇胚胎

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

通过显微注射介导的RNAi Necromenic线虫基因敲除和转基因 Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

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 ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

定量和自动化高通量的全基因组RNAi屏幕 C。线虫</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

RNAi介导的基因敲除和<em&gt;在体内</em在成年女性利尿含量<em&gt;埃及伊蚊</em&gt;蚊子

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

啮齿动物附睾精子磷酸分析

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...