作者想感谢珍妮特Schermuly编制重组的T7 RNA聚合酶和诺曼Ebelt的协助编制图1。这项工作是由德意志研究联合会（DFG FOR1680）和马普学会的资金。

1。代的长前crRNA的底物，通过PCR <ol><li>设计PCR引物定位的CRISPR群集的间隔区。添加的T7 RNA聚合酶（T7RNAP）的启动子序列（5&#39;-taatacgactcactata-3&#39;）的正向引物和用于克隆的限制性位点的两种引物（例如，BamHⅠ和HindⅢ酶切的pUC19， 图2A到载体中的PCR产物） 。 注：T7RNAP需要适当的转录起始的一个胍残留物。 </li><li>放大您的预crRNA的PCR从基因组DNA序列的。 </li><li>分离PCR产物通过琼脂糖凝胶电泳，凝胶中提取所需的频带。用限制酶消化PCR产物，创建粘性末端（例如BamHI和HindIII， 图2A）。你的PCR产物用PCR纯化试剂盒纯化，以消除裂解的副产物。 </li><li>设置了连接反应，含有T4 DNA连接酶，T4 DNA连接酶缓冲液和切割的PCR产物和相应的粘性末端的脱磷酸化的线性pUC载体的摩尔比为3:1。在16°C过夜孵育混合物。变换连接混合物通过标准的协议和主管大肠杆菌 DH5α细胞，用蓝白斑筛选，找出成功的结扎。 </li><li>隔离质粒的白色菌落，用质粒制备试剂盒。确定阳性克隆质粒测序。另外，菌落PCR可能会被用于筛查。 </li></ol> 2。代中间预crRNA基板的，通过退火的DNA寡核苷酸<ol><li>设计正向和反向所需的CRISPR重复/间隔区序列的寡核苷酸。该寡核苷酸含有序列的T7 RNA聚合酶启动子，以及终端的限制性位点（例如，BamHⅠ和HindⅢ酶切的pUC19）。终止的寡核苷酸，以确保粘性末端形式后退火的（参见图2B </s仲&gt;）。 </li><li> 5&#39;-磷酸化1 nmol的每个寡核苷酸含有5微升T4多核苷酸激酶（PNK），2微升T4 PNK 10x缓冲液，2微升ATP（10毫摩尔）在一个单独的20μl反应。孵育各样品在37℃下进行1小时</li><li>杂交的两个磷酸化的寡核苷酸。结合1μl的前进的磷酸化寡核苷酸的混合物（从2.2），1微升的磷酸化的反向寡核苷酸混合物（从2.2），1微升T4 DNA连接酶10x缓冲液在10微升反应。孵育5分钟的样品在95℃C对加热块或在沸水中，关闭热源，并让该混合物冷却至室温（约2-3小时）。 注意：在这个关键的步骤中，在缓慢冷却过程有利于相比，每个单独的寡核苷酸形成的结构内的两个寡核苷酸退火。 </li><li>结扎4微升杂交组合，1μl的消化和去磷酸化的pUC载体（0.1微克/微升）用T4 DNA连接酶，T4 DNA连接酶10x缓冲液和20微升连接混合物中的10mM ATP。在16°C过夜孵育样品。 </li><li>结扎的质粒转化感受态大肠杆菌 DH5α细胞通过标准的协议和利用蓝白色的筛选。分离质粒和消化（筛选用于插入所需的大小）和随后的质粒测序确定阳性克隆。 </li></ol> 3。通过自定义的RNA寡核苷酸合成的短CAS RNA底物的代设计短期CAS RNA底物（例如，单一的重复序列， 图2C）和利用自定义的RNA寡核苷酸合成设施。 注：列入一个脱氧核糖核苷酸的RNA寡核苷酸（ 图2C）在指定的位置，可以用来查明RNA切割的部位。 4， 在体外T7 RNA聚合酶转录<ol><li>隔离质粒与设计的结构（1.9或2.7）。，使用maxiprep质粒纯化试剂盒。 </li><li>线性化的质粒用限制性内切酶切割的片段（例如HindⅢ位）的下游。确保完全消化。 注：如果发散定义的RNA转录物的3&#39;末端的需要的话，所设计的构造应包含一个附加的特定的限制位点为“运行”转录的HindⅢ序列的上游。 </li><li>纯化的线性化的质粒用酚：氯仿（1:1）提取和乙醇沉淀。恢复通过将沉淀重新悬浮于DEPC处理过的无菌水的核酸。 </li><li>设置的T7 RNA聚合酶体外流掉的转录混合物，该混合物含有40毫摩尔的Hepes / KOH（pH 8）中，22毫摩尔MgCl 2，5mM的二硫苏糖醇，1mM亚精胺，4mM的每个核苷三磷酸（ATP，CTP，GTP，UTP ），40〜100微克/毫升的消化质粒和0.1毫克/毫升于DEPC处理过的水的T7 RNA聚合酶。孵育在37℃下进行3小时</li><li>分析得到的RNA转录变性8M尿素的12％聚丙烯酰胺凝胶（ 图3A）。通过Mono Q阴离子交换色谱法13可被纯化的RNA转录的RNA成分的沉淀和再悬浮于DEPC处理的无菌水，通过乙醇沉淀回收。以供将来使用，存储的RNA在-80℃下</li></ol> 5。 Cas6核酸内切酶分析<ol><li>建立一个 运行20微升T7 RNA聚合酶体外转录混合物（见4.4），它包含一个减少量为2mM ATP，并辅以2.5微升的α-[32 P]-ATP（10毫居里/毫升，5000次/毫摩尔）。通过变性8 M尿素12％聚丙烯酰胺凝胶的凝胶提取，纯化反应产物。可视化带的放射自显影。 </li><li>生产和净化目的的核证机关的蛋白质。在这个例子中，CAS6 产气荚膜梭菌嗜热通过热沉淀和Ni-NTA柱层析纯化。 </li><li>设置核酸内切酶测定反应（例如， 梭状芽孢杆菌嗜热 Cas6，反应混合物含有20毫摩尔的Hepes（KOH PH8），250 mM KCl中，2毫摩尔的MgCl 2，1mM DTT的，12000每分钟RNA底物和1μM酶液于37℃下30分钟）。 </li><li>装入5μl的反应混合物（+ 10微升RNA载样缓冲液含95％甲酰胺），8 M尿素12％聚丙烯酰胺凝胶上。可视化的裂解产物电泳后，用放射自显影。 </li></ol> 6。代表性的成果在图3A示出一个例子的RNA底物CAS核酸内切酶活性的分析。分析体外转录反应中加入100μl的5微升的等分试样进行加载。请注意，在RNA的生产效率的不同的结构之间的变化。有些因素吨帽子被观察到的影响后得到的RNA的量是（i）的初始序列+1 G的转录起始所需的，（ⅱ）的可能性转录时RNA结构的形成，及（iii）的限制位点的选择用于生成运行乳沟位置。 RNA核酸内切酶活性的调查，需要两个高度纯化的重组CAS蛋白（ 图3B）和适当的阴性对照。在理想的情况下，这种阴性对照样品相差尽可能少从调查的CAS核酸内切酶反应。这可以通过以下来实现的RNA用无CAS表达式（及之后的相同的纯化步骤）的反应缓冲液和细胞裂解液中的孵育。一个理想的阴性对照是在建议的裂解位点的脱氧核糖核苷酸的添加。在图3C中，5&#39;端标记的重复序列的裂解的梭状芽胞杆菌嗜热 Cas6所示。下<html>相同的条件下，这种重复是不基板了当一个脱氧核糖核苷酸位-9引入。此方法还提供了有关的裂解位点的信息。最后，一​​个长的内部标记的预crRNA裂解Cas6观察两个裂解片段。 <img alt="图1" src="/files/ftp_upload/4277/4277fig1.jpg" /> 图1。CRISPR / CAS活动的总体示意图。概述如下到CRISPR群集（适应），由Cas6核酸内切酶的转录和加工成小crRNAs CRISPR阵列的，摄取的crRNAs成梯级络合物和干扰的的病毒DNA的序列（protospacer）的插入重复上之间的互补crRNA和protospacer的病毒攻击。 Protospacer相邻图案（PAM）标记的的病毒protospacer序列。 IGURE“SRC =”/ files/ftp_upload/4277/4277fig2.jpg“/&gt; 图2。RNA底物的CAS蛋白的生成。该计划产生的工作流（A）长前crRNA的基板，（B）中间的前crRNA基板和（C）的简称CAS RNA底物，为前crRNA生产。例嗜热梭状芽孢杆菌的的CRISPR阵列的序列。 <a href="http://www.jove.com/files/ftp_upload/4277/4277fig2large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图3" src="/files/ftp_upload/4277/4277fig3.jpg" /> 图3。Cas6核酸内切酶的测定。 A）甲苯胺蓝染色聚丙烯酰胺凝胶的自定义设计的RNA寡核苷酸（250皮摩尔），并在体外 RNA转录体（5微升的一个典型的100μl反应）。 B.）SDS-PAGE凝胶的Cas6准备（80皮摩尔） 产气荚膜梭菌嗜热后热沉淀，在50℃1小时，Ni-NTA柱色谱法。 C.）检测的的信使Cas6活动5&#39;末端重复序列标记和预crRNA的体外转录。 -9位的dNTP的引入废除Cas6裂解短的CAS的RNA底物的（S， 图2C）。 A 5&#39;终端8个核苷酸的标记也产生为crRNA成熟从长期预crRNA基板（L， 图2A）。变性8 M尿素12％聚丙烯酰胺凝胶上分离的条带，并通过放射自显影可视化。

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没有利益冲突的声明。

所提出的方法使产生的CAS核酸内切酶底物不同尺寸范围不同序列设计的自由。用于产生合成的RNA寡核苷酸基板的最直接的方法是有限的短RNA的设计，由于增加了成本和技术的局限性，在创建更长的RNA的低聚物。自定义的RNA合成的实际和经济的最大在于虽然成功RNA合成已被报道用于超过100个核苷酸长度的未修饰RNA的低聚物，在40个核苷酸以下。然而，任何给定的序列可以合成和有针对性地引入经修饰的核苷酸（如脱氧核糖核苷酸），可以被用来分析详细的裂解位点。停止前的cRNA应通过体外转录产生。 含有T7 RNA聚合酶启动子的寡核苷酸退火允许生产的中间长度预crRN为。例行和经济上的合成的DNA寡核苷酸的最大长度仅仅是150个碱基以上，代表最大的合成预crRNAs通过此方法。几个退火，形成粘性末端的DNA寡核苷酸对相互的组装可以延长这个最大的长度，但必须在克隆的构造越来越多的挑战。这种方法的主要优点是生成crRNA预构造（及因此CAS基板）核酸内切酶的能力与任何所需的顺序。这允许测试合成crRNA设计。 最后，可以得到更大的预crRNAs从PCR amplificates整个基因组的CRISPR元素或其馏分。改建的体外转录的模板质粒可以通过位点定向诱变引入用于生成预crRNA变种。这些结构可以被用来分析全球信使核糖核酸图案内的整个前crRNA。 通过T7 RNA聚合酶体外转录的未修饰RNA，用于生产的先驱工作的基础上转移RNA 14，雀麦花叶病毒RNA 15。 T7 RNA聚合酶启动子的共识由识别域（-17至-5）和一个起始域（-4至6）在一个基本的鸟苷1 16的转录起始。设备的持仓+1的下游可以改变，它允许为几乎任何所需的RNA序列的转录。所提出的方法在体外决胜转录模板用于产生允许的完整的预crRNAs的匹配的基因组的的CRISPR区域的或合成的的预crRNA变种在体外合成。转录产生的具有5&#39;-末端三磷酸本除非浸过水的GMP的转录反应，得到5&#39;单磷酸末端14。这种总站的需要，如果是被标记的成绩单用T4多核苷酸激酶和γ-[32 P]-ATP。的Cas6和Cas6样酶的裂解活性生成的crRNA包含5&#39;-羟基和2&#39;，3&#39;-环磷酸末端。然后，这些RNA进行分析，以识别的级联复杂的。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>南极磷酸酶</td><td> NEB </td><td> M0289L </td><td></td></tr><tr><td> 在的体外转录级超纯水三磷酸核苷</td><td>耶拿生物科学</td><td> NU-1010 - NU-1013 </td><td></td></tr><tr><td>液相色谱，ktapurifier 100; </td><td> GE医疗集团</td><td></td><td></td></tr><tr><td> DNA寡核苷酸</td><td> MWG操纵子</td><td></td><td></td></tr><tr><td> RNA寡核苷酸</td><td> MWG操纵子</td><td></td><td></td></tr><tr><td>凝胶提取试剂盒</td><td> QIAGEN </td><td> 28704 </td><td></td></tr><tr><td> PCR纯化试剂盒</td><td> QIAGEN </td><td> 28104 </td><td></td></tr><tr><td> Spin Miniprep试剂盒</td><td> QIAGEN </td><td> 27106 </td><td></td></tr><tr><td>质粒试剂盒</td><td> QIAGEN </td><td> 12163 </td><td></td></tr><tr><td> BamHI位</td><td> NEB </td><td> R0136L </td><td></td></tr><tr><td> HindIII位</td><td> NEB </td><td> R0104L </td><td></td></tr><tr><td> T4 DNA连接酶</td><td> NEB </td><td> M0202S </td><td></td></tr><tr><td> T4多聚核苷酸激酶</td><td> Ambion公司</td><td> AM2310 </td><td></td></tr><tr><td> T7 RNA聚合酶</td><td>自己编写</td><td></td><td></td></tr><tr><td>脱氧解决方案组合</td><td> NEB </td><td> N0447L </td><td></td></tr><tr><td>迷你的PROTEAN利电泳系统</td><td> Bio-Rad公司</td><td> 165-8001 </td><td></td></tr><tr><td>风暴840扫描仪</td><td&gt; GE医疗</td><td> 163723 </td><td></td></tr><tr><td>存储Phosphorscreen </td><td>分子动力学</td><td> 63-0034-81 </td><td></td></tr><tr><td>单Q 5/50 GL </td><td> GE医疗集团</td><td> 17-5166-01 </td><td></td></tr><tr><td> phusion高-Fidelity DNA聚合酶</td><td> NEB </td><td> M0530L </td><td>使用GC缓冲液</td></tr><tr><td>甲苯胺蓝</td><td>西格玛</td><td> T3260 </td><td></td></tr><tr><td> pUC19中</td><td> NEB </td><td> N3041S </td><td></td></tr><tr><td> γ-[32 P]-ATP，α-[32 P]-ATP </td><td>哈特曼分析</td><td> SRP，SRP-401-307 </td><td></td></tr></tbody></table>

substrate generation for endonucleases of crispr/cas systems

病毒和它们的原核宿主的相互作用塑造细菌和古细菌生活的演变。原核生物开发了多种策略逃避病毒攻击，包括限制修饰，流产感染和CRISPR / CAS系统。这些发现在许多细菌和古细菌的适应性免疫系统组成的 C上光ŕegularly，我 nterspaced 短p alindromicŕEPEAT（CRISPR）序列和的 C RISPR数sociated（CAS）的基因（图1）1-3。 CAS蛋白质和重复套不同的定义至少有三大不同类型的CRISPR / CAS系统4。通用的蛋白质CAS1，CAS2，提出要参与病毒DNA的摄取将生成一个新的间隔件元件之间延伸CRISPR簇5的5&#39;末端的两个重复。整个群集被转录到的前体crRNA的含有所有的隔板和重复序列，并随后通过酶处理成更小的crRNAs 6-8多样化Cas6系列。这些crRNAs组成的间隔区序列的5&#39;末端（8个核苷酸）和3&#39;末端标记的重复序列9来自两侧。一个重复感染的病毒现在被封锁的蛋白复合物（级联）作为新的crRNA会被定向到病毒的DNA，将其识别为通过互补碱基10。最后，CRISPR / CAS 1型系统，核酸CAS3会破坏检测到入侵者的DNA 11,12。 这些过程定义CRISPR / CAS的适应性免疫系统的原核生物，并开了一个迷人的研究领域所涉及的CAS蛋白的研究。许多CA蛋白的功能仍然是难以捉摸和明显的多样性的CRISPR / CAS系统的原因仍然被照亮。通过详细的计算分析，大多数CA蛋白的潜在活动进行了预测。一个主要部分的CAS蛋白或建议作为核酸内切酶4。 这里，我们提出的方法，产生crRNAs和前体的cRNA CAS号核糖核酸内切酶的研究。不同的核酸内切酶的测定需要，可以直接合成的RNA寡核苷酸或不再crRNA和预crRNA的序列，该序列通过在体外 T7 RNA聚合酶转录运行产生的任一短的重复序列。这种方法允许将用于生成内部标记的核酸内切酶基板和的合成或突变crRNAs的创建的放射性核苷酸。 cas6核酸内切酶活性是利用成熟预crRNAs成crRNAs与5&#39;-hydr氧基和2&#39;，3&#39;-环磷酸末端。

Watch this Scientific Journal Video about Substrate Generation for Endonucleases of CRISPR/Cas Systems at JoVE.com

Substrate Generation for Endonucleases of CRISPR/Cas Systems

CRISPR / CAS系统介导的适应性免疫中的细菌和古细菌。提出了许多CA蛋白作为核糖核酸内切酶作用于不同长度的crRNA前体。在这里，我们说明了三种不同的方法，CAS核酸内切酶活性的生化分析以产生前crRNA的底物。

CRISPR / CAS系统内切酶的底物生成

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

substrate-generation-for-endonucleases-of-crisprcas-systems

Research

JoVE Journal

Biology

27.2K 次观看.  Max-Planck-Institute for Terrestrial Microbiology. CRISPR / CAS系统介导的适应性免疫中的细菌和古细菌。提出了许多CA蛋白作为核糖核酸内切酶作用于不同长度的crRNA前体。在这里，我们说明了三种不同的方法，CAS核酸内切酶活性的生化分析以产生前crRNA的底物。

视频: Substrate Generation for Endonucleases of CRISPR/Cas Systems

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

使用微机电系统（MEMS）开发诊断工具

科研

生物学

Cancer Borealis Stomatogastric Nervous System Dissection

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of cells (approximately 25 in C. borealis) which are well characterized. The cells in the STG exhibit a broad range of outputs and are responsible for the motor actions of the stomach. The stomach contains the gastric mill which breaks down food with three internal teeth, and the pylorus which filters the food before it reaches the midgut. The STG produces two rhythmic outputs to control the gastric mill and pylorus known as central pattern generators (CPGs). Each cell in the STG can participate in one or both of these rhythms. These CPGs allow for the study of neuromodulation, homeostasis, cellular and network variability, network development, and network recovery.
The dissection of the stomatogastric nervous system (STNS) from the Jonah crab (Cancer borealis) is done in two parts; the gross and fine dissection. In the gross dissection the entire stomach is dissected from the crab. During the fine dissection the STNS is extracted from the stomach using a dissection microscope and micro-dissection tools (see figure 1). The STNS includes the STG, the oesophageal ganglion (OG), and the commissural ganglia (CoG) as well as the nerves that innervate the stomach muscles. Here, we show how to perform a complete dissection of the STNS in preparation for an electrophysiology experiment where the cells in the STG would be recorded from intracellularly and the peripheral nerves would be used for extracellular recordings. The proper technique for finding the desired nerves is shown as well as our technique of desheathing the ganglion to reveal the somata and neuropil.

Cancer Borealis Stomatogastric Nervous System Dissection

The stomatogastric nervous system (STNS) of the Jonah crab (C. borealis) can be used for electrophysiology, immunohistochemistry, and cell culture studies. The STNS extraction is done in two parts: the gross and fine dissection.

癌症北欧化工 Stomatogastric神经系统解剖

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of ...

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of the stomach is defined by the mouth opening and the posterior end by the bottom of the pylorus. Along the dorsal side of the stomach lies the stomatogastric nervous system (STNS). This nervous system, which contains rhythmic networks that underlie feeding behavior, is an established model system for studying rhythm generating networks and neuromodulation 1,2. While it is possible to study this system in vivo 3, the STNS continues to produce its rhythmic activity when isolated in vitro. In order to study this system in vitro the stomach must be removed from the animal. This video article describes how the stomach can be dissected from the American lobster. In an accompanying video article4 we demonstrate how the STNS can be isolated from the stomach.

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

We describe the gross dissection of the stomach of the American lobster (Homarus americanus).

肚子里的龙虾总夹层， Homarus Americanus</em

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

人CD40激活的B细胞的生成

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

脂肪酸定量磷酸化刺激酿酒酵母</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

植物原生质体的荧光激活细胞分选

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood or interstitial fluid); 2) the object is illuminated with light of appropriate wavelength in order to excite the probe into its triplet state; 3) the emitted phosphorescence is collected, and its time course is analyzed to yield the phosphorescence lifetime, which is converted into the oxygen concentration (or partial pressure, pO2). The probe must not interact with the biological environment and in some cases to be 4) excreted from the medium upon the measurement completion. Each of these steps imposes requirements on the molecular design of the phosphorescent probes, which constitute the only invasive component of the measurement protocol. Here we review the design of dendritic phosphorescent nanosensors for oxygen measurements in biological systems. The probes consist of Pt or Pd porphyrin-based polyarylglycine (AG) dendrimers, modified peripherally with polyethylene glycol (PEG's) residues. For effective two-photon excitation, termini of the dendrimers may be modified with two-photon antenna chromophores, which capture the excitation energy and channel it to the triplet cores of the probes via intramolecular FRET (F&ouml;rster Resonance Energy Transfer). We describe the key photophysical properties of the probes and present detailed calibration protocols.

We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.

氧成像在生物系统中的磷光纳米探针的合成和校准

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

生物物理检测探头相间细胞核的力学性能：基材应变的应用和微针操纵

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

恒化器中微生物系统生物学中的应用

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Myospheres的产生从人类胚胎干细胞通过表观遗传修饰

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...