我们要感谢布赫霍尔茨实验室和Sigma - Aldrich公司的RNAi队成员讨论。我们要感谢马普学会和Bundesministerium献给教化和Forschung分站赛[0315105]支持生物。 使命®是Sigma - Aldrich公司生物唱片的注册商标。

1。高品质的使命esiRNA库<ol><li>对于每一个感兴趣的基因的RNAi最敏感和最具体的目标区域是选择利用DEQOR算法（http://cluster-1.mpi-cbg.de/Deqor/deqor.html）4。DEQOR分数所有的沉默潜力可能21目标的mRNA核苷酸长的siRNA基于国家的最先进的设计约束4。此外，每一个潜在的siRNA是执行BLAST搜索对有机体的转录研究分析可能与其他基因的交叉反应。该方案，从而提供了一个分析的潜在esiRNA（300-600 bp的长度）的整体素质和跨沉默的能力。 </li><li>选择区域的目标基因的cDNA使用两旁的噬菌体RNA聚合酶启动子序列的基因特异引物进行PCR扩增。 </li><li>每任务esiRNA生产所用的PCR产物是核实身份，经测序和纯度卡尺Labchip分析。 </li><li>长双链RNA是由RNA聚合酶的转录链退火，转录PCR产品。 </li><li> esiRNAs准备长期使用RNaseIII随后通过阴离子交换色谱法，利用Q -琼脂糖凝胶离心柱纯化的双链核糖核酸酶消化有限。 </li><li> esiRNAs沉淀和TE缓冲液悬浮。收益率是衡量紫外吸收测量和浓度是由溶解在TE缓冲液中的一个适当的音量调整。最终产品的整体质量检查卡尺Labchip分析。 </li></ol> 2。选择一个单元格的行和优化筛选转染<ol><li> esiRNA（使用Eg5积极和海肾荧光素酶作为阴性对照），增加金额在384孔板转染试剂滴定金额，增加细胞孵育48小时。 Eg5是一个kinesin的电机双极纺锤体组装和枯竭所需的蛋白质，导致有丝分裂逮捕。重复此过程中，不同的转染试剂和细胞株。在这里，我们使用以下的标准程序和oligofectamine转染试剂（Invitrogen）的HeLa细胞培养。 </li><li>选择最佳的转染条件计数转染的细胞有丝分裂逮捕（圆形）与海肾荧光素酶（RLUC）esiRNA转光镜细胞总数与Eg5 esiRNAs。选择RLUC至少毒性和最明显的表型为Eg5的条件。 为优化转染条件的另一种方法涉及一个稳定的细胞系，表达EGFP（或其他合适的报告基因）的控制下构的积极推动者。转染后对EGFP（或其他报告基因）和RLUC（或其他合适的阴性对照esiRNA）的措施击倒如荧光辅助细胞分选（FACS）的疗效和毒性的esiRNA。请确保使用的EGFP esiRNA是完全互补的靶mRNA的。 </li></ol> 3。设置主屏幕5,6 <ol><li>优化自动移液工作站（如宝瓶，特餐和WellMate，矩阵）的所有组件使用的移液精度。由于吹打参数变化的解决方案，体积和板型的类型而定，优化，分别为每一步反复。如果可能的话，移液站应放置在层流罩下，以避免污染。所有的组件在使用前应进行消毒，高压灭菌，如果适用或用75％乙醇喷洒。优化洗涤协议，使交叉污染，从好到被排除了。优化自动移液的详细信息，不能给空间约束，主要取决于所使用的移液工作站。如需进一步信息，请参阅制造协议。 </li><li> 384格式的转染细胞Eg5和RLUC esiRNAs使用上述优化的条件。使用Eg5 RLUC esiRNA交替模式，覆盖了整个板块。 </li><li>孵育48小时后用冷水100％的乙醇修复细胞，补充水分的PBS 15分钟，25分钟PBS中含有1微克/毫升的DAPI和100μg/ ml的RNA酶A（QIAGEN）染色。最后用PBS和存储在4 ° C。 </li><li>如奥林巴斯ScanR系统的DAPI荧光显微镜测量强度。通过绘制细胞数量的DAPI强度生成直方图和评估，由G1期细胞百分比计算与DNA含量的2N的细胞周期分布; &lt;2N&gt; 4N S期;为G2/M-phase 4N &gt; 4N非整倍体/多倍体。 </li><li>评估的结果在板的同质性。需要进一步优化，如果效果显着差异，排除位置EFFECTS。使用多孔板时常见的问题是在孵化过程中时间的边缘井增强蒸发。这导致了不同的实验条件，在不同的井，这往往转化为具体影响板位置。出于这个原因，我们建议，离开屏幕边缘井空。为了进一步减少蒸发确保孵化器在高湿度保持。我们还定期聘请障碍，如康宁透气封箱胶带，阻止水分蒸发的蒸发。其他资源位置效应也必须考虑到，如读出系统的技术变化（显微镜，酶标仪等）或液体处理的变化（渐变，不均匀性的解决方案等）。 </li><li>统计阳性​​和阴性对照之间的差异提供了就业的检测意义的一项措施。一个阴性对照（或背景噪声;模拟控制）之间较大的差异和样品才开始实际筛选确定。统计学意义的一个很好的措施是Z因素。 z因子下列公式计算： ，Z = 1 - （3 SD [样本] + 3 SD [模拟]）*（|音像[示例] - AV [模拟] |）-1;与SD：标准差; AV：平均。 z因子为1&gt; Z&gt; 0.5表示一个阴性对照组显着性的分离，从7阳性对照（和噪声）。 </li></ol> 4。主屏幕<ol><li>准备384孔组织培养板转染对利用上述优化条件下的esiRNAs。在这里，我们使用15ng esiRNA每口井在5μLTE缓冲液溶解。每盘至少有8个控制仓位应装有合适的正面和负面的生物研究过程的控制（在这里：Eg5和RLUC）为了避免位置效应边缘井装载与5μLTE缓冲液。 </li><li>添加5μLOptiMEM0.2μlOligofectamine（Invitrogen公司），其中包含每口井，混合和20分钟在室温下孵育。 </li><li>添加到转染混合物（在这里：在25个细胞/μL浓度的HeLa细胞悬液40μl;相当于1000个细胞，每孔）的细胞悬液，孵育72小时。 </li><li>测量DNA含量的自动显微镜（奥林巴斯ScanR，见上文）。评估击中选择使用的Z -得分统计：所有样品为阴性对照作为参考信号esiRNAs计算Z分数。请注意，Z - score模型是不相同的z因子。 Z分数提供了一个统计意义的样本值比较（模拟）控制措施。它是下列公式计算： Z =（值[样本] -平均[模拟]）*标准偏差[模拟] -1。 对于命中选择一个门槛已经得到应用。通常情况下，显着性标准，如：2 &lt;Z &lt;-2被使用，但根据数据集的质量，研究生物过程或简单地在屏幕的范围，可能适用不同的阈值。除了 ​​相当容易的Z - score模型的统计也更复杂的数学计算方法可用于（里面可以发现到广泛的领域筛选数据的统计评价圣马洛等8）。 </li></ol> 5。二次屏幕和命中验证<ol><li>一个次要的画面如下，以消除因实验错误或脱靶效应误报的主屏幕。对于选定命中的主屏幕使用相同的程序应反复更大的重复数（3-5），以便更好的统计评价。 </li><li>对于核实点击次要的非重叠esiRNA对在初始屏幕上确定的目标（或另一种非重叠的沉默触发）应使用。应适用相同的检测和读出主屏幕。 </li><li>最终选定的基因可以被验证的跨物种的RNAi救援9。从而，一个细菌人工染色体（BAC），如该基因的小鼠同源编码是稳定转染到细胞中。 BAC -构造保存在其基因组范围内的基因，并允许一个近乎生理的表达。对人类内源性基因的RNAi将离开鼠标转基因表达的改变抢救RNAi的表型。 RNAi的救援提供核查提供最新的RNAi表型的金标准。 </li><li>最后，经审定的候选人更详细的研究，最终推导出机械的理解。在许多情况下，RNA干扰可以使用在这些实验中，例如在次要的，更详细的分析。 </li></ol>

<ol>
	<li>Ghildiyal, M., Zamore, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+silencing+RNAs:+an+expanding+universe.">Small silencing RNAs: an expanding universe.</a> Nat Rev Genet. 10, 94-108 (2009).</li><li>Jackson, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+profiling+reveals+off-target+gene+regulation+by+RNAi.">Expression profiling reveals off-target gene regulation by RNAi.</a> Nat Biotechnol. 21, 635-637 (2003).</li><li>Echeverri, 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=Minimizing+the+risk+of+reporting+false+positives+in+large-scale+RNAi+screens.">Minimizing the risk of reporting false positives in large-scale RNAi screens.</a> Nat Methods. 3, 777-779 (2006).</li><li>Henschel, A., Buchholz, F., Habermann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DEQOR:+a+web-based+tool+for+the+design+and+quality+control+of+siRNAs.">DEQOR: a web-based tool for the design and quality control of siRNAs.</a> Nucleic Acids Res. 32, W113-W120 (2004).</li><li>Kittler, R., Pelletier, L., Buchholz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems+biology+of+mammalian+cell+division.">Systems biology of mammalian cell division.</a> Cell Cycle. 7, 2123-2128 (2008).</li><li>Kittler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-scale+RNAi+profiling+of+cell+division+in+human+tissue+culture+cells.">Genome-scale RNAi profiling of cell division in human tissue culture cells.</a> Nat Cell Biol. 9, 1401-1412 (2007).</li><li>Zhang, J. H., Chung, T. D., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> J Biomol Screen. 4, 67-73 (1999).</li><li>Malo, N., Hanley, J. A., Cerquozzi, S., Pelletier, J., Nadon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+practice+in+high-throughput+screening+data+analysis.">Statistical practice in high-throughput screening data analysis.</a> Nat Biotechnol. 24, 167-175 (2006).</li><li>Kittler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+rescue+by+bacterial+artificial+chromosome+transgenesis+in+mammalian+tissue+culture+cells.">RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells.</a> Proc Natl Acad Sci U S A. 102, 2396-2401 (2005).</li><li>Kittler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+resources+for+endoribonuclease-prepared+short+interfering+RNAs+for+specific+loss-of-function+studies.">Genome-wide resources for endoribonuclease-prepared short interfering RNAs for specific loss-of-function studies.</a> Nat Methods. 4, 337-344 (2007).</li></ol>

作者声明有可能在提交的工作有兴趣的商业实体的财务关系。

RNA干扰已成为标准的技术研究丧失功能的表型。 RNAi的调解员的大型集合来自不同供应商提供了一个容易，成本效益和快速的基因沉默方法。这打开门系统的筛选在许多生物过程中，允许在不同的物种和细胞类型的广泛的基因组规模的角度来看关键球员。 高的假阳性和假阴性率是在RNAi屏幕的共同挑战。为了解决这个问题，已经投入了相当大的努力来提高疗效，特别是沉默的特异性触发。一个重要的发现是一个不同的siRNA针对相同的成绩单池，大大增强了目标的特殊性。因为一个非常复杂的池不同的siRNA是endoribonuclease生产的，esiRNAs高的目标特异性触发，降低假阳性率在RNAi屏幕。 esiRNAs也展示了实现高效击倒，也减少了假阴性率。 由于RNAi的屏幕技术要求高，他们可能会保持具有挑战性的执行了一段时间的例行。然而，试剂和仪器获得更好的和更多的实验室分享其专业知识，运用现代生物学的RNAi屏幕被视为在未来增加。

<table border="1">
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalogue Number</td>
<td>Comments</td>
</tr>	
<tr>
<td>MISSION esiRNA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>For additional information contact: rnai@sial.com</td>
</tr>
<tr>
<td>Wellmate</td>
<td>&nbsp;</td>
<td>ThermoScientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Breathable sealing tape</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>Breathable Sealing Tape, Sterile (Product #3345)</td>
<td>&nbsp;</td>
<tr>
<td>OptiMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=E7023|SIAL&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC&cm_sp=affiliate-_-Jove-_-12346">Ethanol</a></td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>E7023</td>
<td>200 proof (absolute), for molecular biology</td>
</tr>
<tr>
<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=P5493|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC&cm_sp=affiliate-_-Jove-_-12347">Phosphate buffered saline</a></td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P5493</td>
<td>BioReagent, 10x concentrate, suitable for cell culture, for molecular biology</td>
</tr>
<tr>
<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=D8417|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC&cm_sp=affiliate-_-Jove-_-12348">4&#x2032;,6-Diamidino-2-phenylindole dihydrochloride</a></td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D8417</td>
<td>Poweder, BioReagent, suitable for cell culture, >98% (HPLC and TLC)</td>
</tr>
<tr>
<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=R6513|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC&cm_sp=affiliate-_-Jove-_-12349">Ribonuclease A from bovine pancreas</a></td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>R6513</td>
<td>For molecular biology, &ge;70 Kunitz units/mg protein, lyophilized powder</td>
</tr>
<tr>
<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=T9285|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC&cm_sp=affiliate-_-Jove-_-12350">Tris-EDTA buffer solution</a></td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T9285
</td>
<td>100 x, for molecular biology</td>
</tr>
</table>

mission esirna for rnai screening in mammalian cells

RNA干扰（RNAi）是一个基本的细胞的基因表达调控机制。 RNAi是诱发又称小干扰RNAs（siRNAs）的短双链RNA的。源自较长的双链前体蛋白的RNase III家族的内切酶Dicer酶，活性的短双链RNA。产生的碎片的RNA诱导的沉默复合体（RISC）的组成部分，它的同源靶mRNA。 RISC切割靶mRNA，从而降低了编码的蛋白质的表达1,2,3。 RNAi技术已成为一个强大而广泛用于哺乳动物细胞基因功能研究的损失，利用小分子干扰核糖核酸的实验方法。 目前两种主要方法可用于生产小分子干扰核糖核酸。一种方法涉及化学合成，而一种替代方法，采用由核糖核酸酶III 在体外 ，针对特定的长双链RNA的endonucleolytic裂解。从而，不同的寡核苷酸的siRNA池产生的，这也是已知endoribonuclease准备的siRNA或esiRNA的。化学衍生的siRNA和esiRNAs疗效比较显示，两个触发器强有力的靶基因沉默。然而，差异可以看出，当比较的特殊性。许多单一的化学合成的siRNA产生显着的脱靶效应，而esiRNAs固有的复杂混合物导致一个更具体的击倒 10 。 在这项研究中，我们目前的基因组规模的使命esiRNA库，它体现为在参与细胞周期进程的基因识别的DNA含量的屏幕RNAi筛选的利用率的设计。我们将展示如何优化转染协议和高通量筛选的检测。我们也表现出多么大的数据集，可以评估统计学和目前的方法来验证的主要命中。最后，我们给潜在的出发点，进一步验证点击功能刻画。

Watch this Scientific Journal Video about MISSION esiRNA for RNAi Screening in Mammalian Cells at JoVE.com

MISSION esiRNA for RNAi Screening in Mammalian Cells

这里我们使用一个参与细胞分裂的基因的高通量筛选人类esiRNA库。我们演示了如何设置和行为的​​一个esiRNA屏幕，以及如何分析和验证的结果。

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 ...

mission-esirna-for-rnai-screening-in-mammalian-cells

Research

JoVE Journal

Biology

16.2K 次观看.  Max Planck Institute of Molecular Cell Biology and Genetics. 这里我们使用一个参与细胞分裂的基因的高通量筛选人类esiRNA库。我们演示了如何设置和行为的​​一个esiRNA屏幕，以及如何分析和验证的结果。

视频: MISSION esiRNA for RNAi Screening in Mammalian Cells

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

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

Trypsinizing和传代的哺乳动物细胞

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

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 ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.
There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids 1. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.
The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes 2. Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 108 TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.
Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line 3, 4. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.

Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.

特派团LentiPlex汇集在哺乳动物细胞中的shRNA文库筛选

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to ...

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 ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

在哺乳动物细胞中细胞周期位置分析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

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 ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

中血红素的合成水平在哺乳动物细胞中测量

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.