我们感谢博士。 SP的Dinesh Kumar和尤尔刘TRV - VIGS载体，和Drs。查Kenerley，特里轮车，吉姆斯塔尔提供棉花种子和拜耳作物科学。这项工作是由NSF资助LS和NIH的PH

1。种植棉花幼苗<ol><li>母猪的棉花（陆地棉）各种Fibermax 832 Phytogen 425RF，Phytogen 480WR和Deltapine 90盆（直径7厘米）含地铁混合700（SunGR，Beavile，WA）的种子。 </li><li>盆保持在一个托盘用塑料圆顶覆盖23℃，120μE米-2小号-1光，在增长室12个小时的light/12小时黑暗的光照。 </li><li>删除时，有两个子叶出现圆顶。 </li><li>大约两个星期后，使用两个子叶完全展开VIGS检测的植物。在这个阶段，真叶尚未出现（图1）。 </li></ol> 2。构筑VIGS载体GrCLA1​​基因<ol><li>从G 的 cDNA文库扩增PCR与引物GrCLA1 ​​- F，5&#39; - GCCCTTTGTGCATCTTC - 3&#39;和GrCLA1 ​​- R，5&#39; - CTCTAGGGGCATTGAAG - 3“Cloroplastos alterados 1基因棉花（GrCLA1）雷蒙德叶组织。 </li><li>精华GrCLA1 ​​的PCR产物用EcoRI和KpnI和pYL156（pTRV - RNA2的）载体中插入结扎。 </li><li>屏幕含50μg/ mL的卡那霉素的LB琼脂板的克隆。经酶切和测序验证的构造。 </li><li>转换成农杆菌 GV3101中的质粒，电，恢复LB液体培养基在28 ° C。选择与LB平板转化+卡那霉素（50微克/毫升）+庆大霉素（25微克/毫升）。这种细菌可以存储在25％甘油于-80 ° C长期使用。 </li></ol> 3。执行VIGS接种<ol><li> 3天前接种，连胜携带pYL192（TRV）的农杆菌农杆菌冰冻甘油股票：RNA1，pYL156（TRV）：RNA2（矢量单独）和pYL156（TRV）：含50μg/ mL的卡那霉素的LB琼脂板RNA2 - GrCLA1和25微克/毫升庆大霉素。在28 ° C培养24小时的板块。 </li><li>每个构造前两天VIGS渗透，挑单菌落从上述板块，到5毫升50微克/ mL的卡那霉素和庆大霉素25微克/毫升为辅的LB培养基接种;细菌培养成长28 ° C在50转的速度滚鼓过夜。 </li><li>以上文化转移到烧瓶中，用50毫升LB培养基中补充50微克/毫升，卡那霉素和25微克/毫升庆大霉素，加10毫米的MES（2 - （4啉）乙烷磺酸）和20微米的乙酰丁香酮。增长在28 ° C为50转的速度在摇床过夜的文化。 </li><li>翌日，降速5分钟在4000转农细菌细胞，再暂停的文化渗透缓冲液含10毫米10毫米氯化镁 2，MES和200μM乙酰丁香酮。文化的OD 600调整为1.5。 </li><li>在板凳上3个小时保留在室温下的文化。 </li><li>在此之前Agrobacterial渗透，拳打棉花植株子叶底面没有通过子叶刺入地下25针。挥拳打向一个或两个孔（取决于组织的柔软度），每个子叶节​​（图2）。 </li><li> pYL192 Agrobacterial文化悬架（TRV）：以1:1的比例混合RNA2 - GrCLA1;手渗透从子叶下方的混合物通过使用1伤人网站：RNA1和pYL156（TRV）：RNA2或pYL156（TRV） mL的无针注射器（图3）。 </li><li>用塑料圆顶盖的植物，在昏暗的光线条件下离开一夜之间在室温下渗入植物。 </li><li>转让植物的生长房间温度为23℃，120μE米-2 S与12个小时的光/ 12小时黑暗周期-1光。 </li></ol>注：VIGS的作品，无论是23 ° C或温室（25-28℃）下的植物。当温度相对较低（23至25 ° C），它是为接种容易，表型是更均匀相比，具有较高（28-30℃）或忽高忽低的温度。植物覆盖的圆顶，也使得VIGS更有效率。 <ol start="10"><li>检查在7〜8天的渗透沉默的表型。沉默植株片真叶pYL156（TRV）：RNA2 GrCLA1 ​​开始显示白化病的表型（图4 ）。 Agrobacteria携带pYL156（TRV）：RNA2的渗入植物作为一个控制。 </li><li>验证通过检查使用控制和沉默的棉花植株中分离出的RNA逆转录聚合酶链反应（RT - PCR检测）的内源性基因表达水平的基因沉默的效率。 </li></ol>注：应根据所载的检疫条件和植物保持VIGS - ED植物应妥善蒸压和后处理的实验，作为TRV具有广泛的宿主范围，是一项须予公布的病原体。 4。代表性的成果： 手渗透Agrobacterial混合后大约两个星期，GrCLA1 ​​沉默所造成的白化的表现型上清楚地观察到真叶。沉默效率达到几乎100％在多个实验与超过50家工厂。沉默的植物显示上的新发展与镶嵌上的老叶子（图4）叶白化表型均匀分布和很强的。 <img src="/files/ftp_upload/2938/2938fig1.jpg" alt="图1" /> 图1与两个完全展开的子叶用于Agrobacterial渗透在棉花苗期约两个星期的老戏台。 <img src="/files/ftp_upload/2938/2938fig2.jpg" alt="图2" /> 图2。冲孔的棉花，用25 G针，以方便Agrobacterial渗透植物子叶下方的小孔。 <img src="/files/ftp_upload/2938/2938fig3.jpg" alt="图3" /> 图3。手伤人网站通过渗透到棉花子叶Agrobacterial混合使用无针注射器<img src="/files/ftp_upload/2938/2938fig4.jpg" alt="图4" /> 图4。白化表型上出现了VIGS沉默棉花植株。四个品种。一）Fibermax 832;二）Phytogen 480WR，C）Phytogen 425RF，D）90 Deltapine。

<ol>
	<li>Chen, Z. J., Scheffler, B. E., Dennis, E., Triplett, B. A., Zhang, T., Guo, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+sequencing+cotton+(Gossypium)+genomes.">Toward sequencing cotton (Gossypium) genomes.</a> Plant Physiol. 145, 1303-1310 (2007).</li><li>Dinesh-Kumar, S. P., Anandalakshmi, R., Marathe, R., Schiff, M., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-induced+gene+silencing.">Virus-induced gene silencing.</a> Methods Mol Biol. 236, 287-294 (2003).</li><li>Hamilton, A. J., Baulcombe, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+species+of+small+antisense+RNA+in+posttranscriptional+gene+silencing+in+plants.">A species of small antisense RNA in posttranscriptional gene silencing in plants.</a> Science. 286, 950-952 (1999).</li><li>Burch-Smith, T. M., Anderson, J. C., Martin, G. B., Dinesh-Kumar, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+and+advantages+of+virus-induced+gene+silencing+for+gene+function+studies+in+plants.">Applications and advantages of virus-induced gene silencing for gene function studies in plants.</a> Plant J. 39, 734-746 (2004).</li><li>Liu, Y., Schiff, M., Dinesh-Kumar, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-induced+gene+silencing+in+tomato.">Virus-induced gene silencing in tomato.</a> Plant J. 31, 777-786 (2002).</li><li>Estevez, J. M., Cantero, A., Romero, C., Kawaide, H., Jimenez, L. F., Kuzuyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+expression+of+CLA1,+a+gene+that+encodes+the+1-deoxyxylulose+5-phosphate+synthase+of+the+2-C-methyl-D-erythritol-4-phosphate+pathway+in+Arabidopsis.">Analysis of the expression of CLA1, a gene that encodes the 1-deoxyxylulose 5-phosphate synthase of the 2-C-methyl-D-erythritol-4-phosphate pathway in Arabidopsis.</a> Plant Physiol. 124, 95-104 (2000).</li><li>Mandel, M. A., Feldmann, K. A., Herrera-Estrella, L., Rocha-Sosa, M., Leon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLA1,+a+novel+gene+required+for+chloroplast+development,+is+highly+conserved+in+evolution.">CLA1, a novel gene required for chloroplast development, is highly conserved in evolution.</a> Plant J. 9, 649-658 (1996).</li><li>Gao, X., Wheeler, T., Li, Z., Kenerley, C., He, P., Shan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+GhNDR1+and+GhMKK2+compromised+cotton+resistance+to+Verticillium+wilt.">Silencing GhNDR1 and GhMKK2 compromised cotton resistance to Verticillium wilt.</a> Plant J. 66, 293-305 (2011).</li></ol>

没有利益冲突的声明。

VIGS已被证明是一个瞬时击倒内源性基因的表达，在功能基因组学分析的有力工具。在这项研究中，我们开发利用TRV -基于二进制向量农杆菌介导的VIGS。棉花CLA1（GrCLA1）基因作为一种视觉标记，监察沉默效率。我们一直得到100％的基因沉默的效率，表现出白化​​病的表型出现在所有品种的片真叶测试，大约两个星期后渗透开始。然而，CLA1沉默的棉花植株难以发展成纤维或种子阶段...

<table border="1"><tbody><tr><th>试剂和设备的名称</th><th>公司</th><th>目录/序列号</th><th>评论</th></tr><tr><td>滚子鼓</td><td> GLAS - COL，LLC。 </td><td> 099A TC108 </td><td>农业细菌培养</td></tr><tr><td>孵化器</td><td>谢尔顿制造公司</td><td> 01046209 </td><td>农业细菌培养</td></tr><tr><td>紫外/可见分光光度计</td><td> Beckman Coulter公司</td><td>型号：DU530 </td><td>测量外径</td></tr><tr><td>基因脉冲</td><td> BioRad公司</td><td>型号：编号：1652076; 154BR3880 </td><td>电穿孔</td></tr><tr><td>脉冲控制器</td><td> BioRad公司</td><td>型号：编号：1652098; 232BR4833 </td><td>电穿孔</td></tr><tr><td> Micropulser电穿孔的比色皿</td><td> BioRad公司</td><td> 165-2081 </td><td>电穿孔</td></tr><tr><td> 1 ml注射器</td><td> BD公司</td><td> 30962 </td><td>农业细菌接种</td></tr><tr><td>地铁混合700 </td><td> SUNGR </td><td> SKU＃553001 </td><td>育苗</td></tr><tr><td>兵马俑锅</td><td> TOPlastics </td><td>全球定位系统3001B2 </td><td>育苗</td></tr><tr><td> MES的水合物</td><td> USB </td><td> 18886 </td><td>渗透缓冲区</td></tr><tr><td>乙酰丁香酮</td><td>西格玛</td><td> D134406 </td><td>渗透缓冲区</td></tr></tbody></table>

agrobacterium-mediated virus-induced gene silencing assay in cotton

棉花（ 陆地棉 ）是全球最重要的农作物之一。分子育种新品种已相当大的努力。大规模基因功能分析棉花已经落后于最现代的植物物种，可能是由于其基因组，基因复制和多倍体，生长周期长和遗传转化 1顽抗的大尺寸。为了方便的高通量功能基因/基因棉花的研究，我们尝试发展快速，高效的瞬态分析评估棉花基因的功能。 病毒诱导的基因沉默（VIGS）是一个强大的技术开发基于主机的转录后基因沉默（PTGS）抑制病毒增殖2,3。农杆菌介导的VIGS已被成功地应用在双子叶植物， 如茄科，拟南芥和豆科植物物种的广泛，单子叶植物物种，其中包括大麦，小麦和玉米， 各种功能基因组研究3,4。由于这种快速和有效的方法避免了厂房改造和克服的功能冗余，这是特别有吸引力，适合在作物品种，如棉花不适合转型功能基因组学研究。 在这项研究中，我们报告VIGS在棉花农杆菌介导的系统的详细协议。其中几种病毒的VIGS载体，烟草脆裂病毒（TRV）侵入的主机范围广，能够传遍整个工厂的大力尚未产生症状轻微的hosts5。要监视的沉默效率，GrCLA1 ​​alterados 1基因棉花（AtCLA1），已被克隆，并插入到VIGS二进制向量pYL156。CLA1基因参与叶绿体发展 6，和以往的研究表明，拟南芥 Cloroplastos的同源基因， AtCLA1损失的功能，导致7片真叶白化病的表型，沉默效率提供了一个良好的视觉标记。 农杆菌浸润在大约两个星期后，白化病的表型开始出现片真叶，与100％，在所有实验复制的沉默效率。内源性基因表达的沉默，也证实了RT - PCR分析。值得注意的是，沉默potently可能发生在我们测试的所有品种，包括各种商业化种植的品种在得克萨斯州。这种快速，高效的的农杆菌介导的VIGS检测，提供了一个快速大规模的棉花全基因组水平的基因功能分析非常强大的工具。

Watch this Scientific Journal Video about Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton at JoVE.com

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

我们目前为农杆菌介导的病毒诱导的基因沉默（VIGS）棉花检测的详细协议。烟草脆裂病毒（TRV）派生的VIGS载体部署GrCLA1​​棉花，Cloroplastos alterados 1基因诱导RNA沉默。白化沉默GrCLA1​​引起的表型观察接种后2个星期内，在苗期。

农杆菌介导的病毒诱导的基因沉默含量在棉花

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

agrobacterium-mediated-virus-induced-gene-silencing-assay-in-cotton

Research

JoVE Journal

Biology

24.2K 次观看.  Texas A&M University. 我们目前为农杆菌介导的病毒诱导的基因沉默（VIGS）棉花检测的详细协议。烟草脆裂病毒（TRV）派生的VIGS载体部署GrCLA1​​棉花，Cloroplastos alterados 1基因诱导RNA沉默。白化沉默GrCLA1​​引起的表型观察接种后2个星期内，在苗期。

视频: Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

病毒诱导的基因沉默（VIGS）烟草benthamiana和番茄

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

科研

生物学

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

活细胞钙成像相结合的siRNA介导的基因沉默标识CA 2 +泄漏通道内质网膜和监管机制

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

在细胞培养中缺氧诱导和测试

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

优化和利用<em&gt;农杆菌</em&gt;介导的瞬态蛋白生产中<em&gt;烟草</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

壳聚糖/干扰RNA纳米介导的基因沉默的疾病病媒蚊幼虫

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

减数分裂纺锤体评估在小鼠卵母细胞的siRNA介导沉默

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

沉默的火花：CRISPR/Cas9基因组编辑在弱电鱼

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axig1) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/loxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Plant morphogenic genes can be used to improve genetic transformation of recalcitrant genotypes. Described here is an Agrobacterium-mediated genetic transformation (QuickCorn) protocol for three important public maize inbred lines.

使用形态基因的农业细菌 -顽固玉米近亲繁殖系的介导未成熟胚胎转化

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) ...