This research was supported by Advanced Research Projects Agency - Energy (ARPA-E) Award No. DE-AR0000313.

1.悬浮培养的建立<ol><li> BY-2介质通过加入4.43克Linsmaier＆Skoog基础介质30克蔗糖，200毫克KH 2 PO 4制备的液体，和200微克至900的2,4-二氯苯氧乙酸（2,4-D）毫升蒸馏水水和pH至5.8，用0.1M的KOH。调节pH值之后，调节最终体积至1000毫升蒸馏水和高压釜中。介质可以在4℃保存最多2周。 </li><li>接种用100ml液体BY-2的媒体和单片BY-2愈伤组织（&gt; 1厘米直径）上生长固BY-2介质的250ml Erlenmeyer烧瓶（液体BY-2的媒体与添加1％琼脂）中，用铝箔密封。在28-30℃下孵育培养振摇5天。 注:BY-2愈伤组织被保持在为长期贮存的固体介质，如液体培养物将迅速长满。培养体积可以根据需要进行调整，通常的100毫升培养将能够装载三十3个6-WELL板。 </li><li>转让2 ml对数相BY-2培养到98毫升的新鲜BY-2培养基中，并在28-30℃振荡孵育5-7天。 注意:100稀释的5-7天龄培养物，而不是与愈伤接种:建立液体培养物的传代培养可以定期使用1来进行。 </li><li>调匀的培养，因为细胞将迅速沉降，并转移6毫升细胞培养物至15ml锥形底离心管，并让细胞沉降至少10分钟。通过除去上清液调节红细胞体积与总体积的50％。 </li><li>通过反相和吸管500微升成用于消化一个6孔板的每个孔中摇管中。使用宽孔移液管在此阶段转移的细胞，因为它们是致密的并且会阻塞标准枪头。 </li></ol> 2.原生质体分离<ol><li>打开所述机器人系统（ 图1A）的所有部件，并打开该板移动器任务调度软洁具。任务调度程序集成了其它设备，以使每件设备之间板的传送微板动。 </li><li>之前的实验中，定义为实验室器皿中的协议用于每个片实验室器皿，在板移动器软件的技术规范（ 例如 ，盘，盖），以及开始和结束的位置，如下: <ol><li>从板块发软件主工具栏中单击设置 ，然后选择管理容器类型命令。 </li><li>如果容器类型在现有容器类型库中列出，然后选择适当的容器中。 </li><li>如果容器类型没有在现有的容器类型库中列出然后单击添加以指定新的容器类型。 </li><li>添加新的容器类型后，插入新容器（高度，宽度，堆放尺寸和夹具偏移量）的尺寸到相应的选项卡，然后单击保存 。<ol><li>如果正确的尺寸不能从制造商，然后准确地用卡尺确定所有尺寸的精确到毫米。在测量容器尺寸误差将导致板动机故障。 </li></ol></li><li>重复步骤2.2.2至2.2.4.1对所有类型的容器，该板块发会遇到。此步骤为所有实验器皿完成后，有必要定义用于每个容器（步骤2.2.6）的开始和结束位置。 注意:对于这个协议的实验室器具包括6孔板中，深孔板，和96孔荧光筛选板。 </li><li>以限定每个容器的开始和结束位置，点击特定的协议的开始/结束标签。首先，选择每个容器的起始位置。接下来，检查在开始和结束位置都为带盖/ Unlidded的框。重复所有的容器。 注意:如果集装箱将被留在另一块的EQU双模接收机设备（而不是板发）结束位置可以留空。 </li></ol></li><li>在这个阶段所有平板手动装载到用于整个工作流程的开始位置。用BY-2细胞加载96孔荧光隔离板成酒店2，6孔板进酒店3，96深孔板将用于转化到板加热器/冷却器巢2，和50毫升的锥形含有酶溶液（见补充文件 ，第1.2.2节）到多模饮水机管。 <ol><li>如果变换将在协议来进行，以10微升每孔的质粒DNA的预装载96深孔板（1微克/微升，A 260/280&gt; 1.8）含有OFP报道构建和孵化在板加热器/冷却器在4℃。每个孔将用于单个转换，从而填充将取决于实验设置井的数量。 </li></ol></li><li>装载的自动完成所有物资和板ð液体处理平台，在指定地点，并定义每个项目中的自动化控制软件（ 图1B）的位置。 <ol><li>装载在MMG200μl的40％的PEG（0.4M甘露醇，100mM的MgCl 2的，4毫MES，pH值5.7）在第1列，将200μl碘化丙啶的预装入的96孔板（PI，1微克/毫升）在第2栏，以及200微升的在第3列（巢2）乙醇，和枪头的巢8的盒子。 </li><li>要定义实验室器皿中的自动化液体处理平台的软件，从菜单中的位置，选择工具 ，然后单击编辑实验室器皿 。 </li><li>选择实验室器皿从下拉菜单类型或定义使用新建医学实验按钮，一个新的实验器具类型。 </li><li>定义选择的主要协议选项卡，然后单击配置选择实验室器皿每件实验室器具的位置。确保放置在自动化液体处理平台上的每一块实验室器皿是前运行协议定义。 </li><li>要触发自动协议，单击配置文件资源管理器窗口，然后选择要运行的协议（ 原生质体分离 ， 细胞计数和PEG介导的转化 ）的名称。 </li><li>单击运行初始化将在方案中使用的所有设备。 </li><li>最后，在工作资源管理器窗口中，单击添加工作单位 ，以验证系统中的所有容器/实验室器材，并开始自动协议。 注:的自动化协议完整的描述包含在补充文件。 </li></ol></li></ol> 3.建立标准曲线用于细胞计数<ol><li>在1毫升体积的1×10 6原生质体/ ml的浓度手动集中原生质体。原生质体离心在1000 XG 10分钟，取出上清液。 </li><li>手动添加900微升70％的乙醇（保持在4℃）的原生质体沉淀并重新悬浮沉淀。孵育10分钟在冰上固定细胞。 </li><li>添加的PI 100微升（1微克/毫升）的原生质体来标记细胞核，并允许检测由板阅读器。 </li><li>负载80微升液体BY-2介质插入96孔荧光筛板起始于第2列的各列和行。 </li><li>在96孔荧光屏蔽板的第1列，加入原生质体的70微升和50微升BY-2介质到每个孔中的柱。 </li><li>放置板的自动化液体处理平台的巢6，并运行一个2倍连续稀释。 <ol><li>要进行自动化液体处理平台上的连续稀释，单击启动连续稀释向导并调整传输量（60微升），混合和体积（2，70微升），初始井数（第1列，行AF），稀释井（列2-11，行AF），以及吸入和排出性能（从井底0.5mm）的。 </li><li>设置参数后，保存d运行的协议。 注:由于所有原生质体用的PI标记（由于细胞的固定），荧光正比于原生质体的浓度。 </li><li>连续稀释完成后，从窝9取下板并插入板读数器，并运行在平板阅读器软件的标准曲线协议。 注意:标准曲线将随后由荧光从PI（激发536纳米/发射620毫微米）各孔与已知原生质体浓度进行比较来生成的。 </li></ol></li><li>一旦酶标仪协议完成后，取出板和收集高压灭菌或在生物安全议定书规定"等去振兴程序的任何转基因细胞。 </li></ol> 4.转型的微观分析<ol><li>与转化的原生质体从机器人系统的酒店1（自动协议3， 补充文件的产品）取下板。 </li><li>转在倒置显微镜，照相机和荧光灯。 </li><li>选择用于起始聚焦的原生质体的10X物镜，打开卤素灯，并关闭快门的荧光灯。 </li><li>压板到微观制度和注重利用明原生质体。 </li><li>着眼于原生质体后，关闭卤素灯泡和打开快门的荧光灯。 </li><li>选择CY3 / TRITC过滤器由转基因原生质体表达的蛋白质pporRFP可视化设置。 </li><li>扫描每个孔，以确定原生质体表达pporRFP荧光标记物的数目。 </li><li>计算转化效率原生质体的表达荧光标记物的原生质体的总数量的总数。 </li><li>收集含有生物安全认可袋高压灭菌或在生物安全议定书规定"等去振兴程序转基因细胞的板块。 </li></ol>

作者宣称，他们没有竞争的经济利益。

上述的协议已被成功验证为原生质体分离，计数，以及使用该BY-2烟草悬浮细胞培养物转化;然而，该协议可以很容易地扩展到任何植物悬浮培养。目前，原生质体分离和改造已经在众多的植物，包括玉米（ 玉米 ）10，红萝卜（ 胡萝卜 ）32，杨树（ 胡杨 ）33，葡萄（ 葡萄 ）34，油棕（ 油棕 ）35实现，生菜（ 莴苣 <su...

近年来，一直放置在转基因作物的设计克服了各种疾病1，赋予抗除草剂2，赋予干旱3,4和耐盐5显著动力，防止虫食6，提高生物产量7，降低细胞壁顽抗8。这种趋势已经通过新的分子工具的发展，用于产生转基因植物，包括使用CRISPR和TALENS 9基因组编辑，基因通过的dsRNA 10，miRNA的11，和siRNA 12沉默辅助。虽然这些技术简化了转基因植物的一代，他们还创建了一个瓶颈，其中产生的转基因植物的数量之多，无法使用依赖于植株再生的传统系统进行筛选。与此相关的瓶颈，而沉默和基因组编辑构建体能够迅速地插进植物，许多目标性状不能产生所期望的效果，这通常没有发现直到植物在温室中进行分析。在这项工作中，我们开发了用于植物原生质体的快速，自动化，高通量筛选的方法，具体而言，以解决在大量的基因组编辑和基因沉默靶的早期筛查的当前瓶颈。 使用原生质体的，相对于完整的植物细胞，具有几个优点对自动化平台的开发。首先，原生质体的植物细胞壁的消化后分离，以及与此屏障不再存在，转化效率提高13。在完整的植物细胞，只有两种用于转化很好地建立的方法，基因枪法14和农杆菌介导的转化15。这些方法都不能够容易地转化为液体处理平台，生物射弹需要TRANSFO专门的设备细则第十五，而农杆菌介导转化需要共培养和随后的去除细菌。无论是适合于高通量方法。在原生质体的情况下，转换是经常使用聚乙二醇（PEG）介导的转染16，其仅需要几个溶液的交流，并非常适合用于液体处理平台进行。第二，原生质体，顾名思义，是单细胞培养物，从而与在植物细胞培养物结块和链的形成相关的问题，没有在原生质体观察。在使用基于平板分光光度计，细胞凝集快速筛选而言，或细胞在多个平面将导致难以取得一致的测量。因为原生质体也比其培养基稠密，它们沉淀到孔的底部，形成一单层，这有利于对基于板状光度法。最后，尽管植物细胞悬浮培养物是PRIMAR随手从愈伤组织17衍生的原生质体可以从多种植物组织的收获，从而导致识别组织特异性表达的能力。例如，能够分析根级或一个基因的叶特异性表达可以是表型的预测很重要的。由于这些原因，在该工作开发的协议使用从广泛使用的烟草（ 烟草属）"亮黄色&#39;2（BY-2）悬浮培养物分离的原生质体进行了验证。 在BY-2悬浮培养已被描述为高等植物的"的HeLa"细胞，因为在植物细胞中18的分子分析其无处不用途。最近，BY-2细胞已被用于研究植物的影响压力19-22，细胞内蛋白质定位23,24，以及基本的细胞生物学25-27表明在植物生物学这些培养物的广泛的实用性。 BY-2培养物的一个附加的优点是到培养物与阿非迪霉素同步，这可导致增强的基因表达的重现能力研究28。此外，已开发了用于使用低成本酶29,30 BY-2原生质体的提取，如传统上用于产生原生质体的酶是成本过高的高通量系统。因此，下面描述的协议已被使用BY-2悬浮培养验证，但它应该是易于进行的任何植物细胞悬浮培养。证明的概念实验使用CaMV 35S启动子的控制下，从硬盘珊瑚滨橙色荧光蛋白（OFP）报告基因（pporRFP） 滨 31进行。

<table><tbody><tr><td>Orbitor RS Microplate mover</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Bravo Liquid Handler</td><td>Agilent</td><td></td><td></td></tr><tr><td>Synergy H1 Multi-mode Reader</td><td>BioTek</td><td></td><td></td></tr><tr><td>MultiFlo FX Multi-mode Dispenser</td><td>BioTek</td><td></td><td></td></tr><tr><td>Teleshake</td><td>Inheco</td><td>3800048</td><td></td></tr><tr><td>CPAC Ultraflat Heater/cooler</td><td>Inheco</td><td>7000190</td><td></td></tr><tr><td>Vworks Automation Software</td><td>Agilent</td><td></td><td>Software used to control and write protocols for Agilent Bravo</td></tr><tr><td>Momentum Software</td><td>Thermo Scientific</td><td></td><td>Task scheduling software for controlling Orbiter RS</td></tr><tr><td>Liquid Handling Control 2.17 Software</td><td>Biotek</td><td></td><td>Software used to control and write protocols for MultiFlo FX</td></tr><tr><td>IX81 Inverted Microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Zyla 3-Tap microscope camera</td><td>Andor</td><td></td><td></td></tr><tr><td>ET-CY3/TRITC Filter Set</td><td>Chroma Technology Corp</td><td>49004</td><td></td></tr><tr><td>Rohament CL</td><td>AB Enzymes</td><td>sample bottle</td><td>low-cost cellulase</td></tr><tr><td>Rohapect UF</td><td>AB Enzymes</td><td>sample bottle</td><td>low-cost pectinase</td></tr><tr><td>Rohapect 10L</td><td>AB Enzymes</td><td>sample bottle</td><td>low-cost pectinase/arabinase</td></tr><tr><td>Linsmaier &amp;amp; Skoog Basal Medium</td><td>Phytotechnology Laboratories</td><td>L689</td><td></td></tr><tr><td>2,4-dichlorophenoxyacetic acid</td><td>Phytotechnology Laboratories</td><td>D295</td><td></td></tr><tr><td>propidium iodide</td><td>Sigma Aldrich</td><td>P4170</td><td></td></tr><tr><td>Poly(ethylene glycol) 4000</td><td>Sigma Aldrich</td><td>95904-250G-F</td><td>Formerly Fluka PEG</td></tr><tr><td>Propidium Iodide</td><td>Fisher Scientific</td><td>25535-16-4</td><td>Acros Organics</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>C7902-1KG</td><td></td></tr><tr><td>Sodium Acetate</td><td>Fisher Scientific</td><td>BP333-500</td><td></td></tr><tr><td>Mannitol</td><td>Sigma Aldrich</td><td>M1902-1KG</td><td></td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S5-3</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>AC424205000</td><td></td></tr><tr><td>KOH</td><td>Sigma Aldrich</td><td>P1767</td><td></td></tr><tr><td>Gelzan CM</td><td>Sigma Aldrich</td><td>G1910-250G</td><td></td></tr><tr><td>6-well plate</td><td>Thermo Scientific</td><td>103184</td><td></td></tr><tr><td>96-well 1.2 ml deep well plate</td><td>Thermo Scientific</td><td>AB-0564</td><td></td></tr><tr><td>96 well optical bottom plate</td><td>Thermo Scientific</td><td>165305</td><td></td></tr><tr><td>Finntip 1000 Wide bore Pipet tips</td><td>Thermo Scientific</td><td>9405 163</td><td></td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>BP358-10</td><td></td></tr><tr><td>KCl</td><td>Sigma Aldrich</td><td>P4504-1KG</td><td></td></tr><tr><td>MES</td><td>Fisher Scientific</td><td>AC17259-5000</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific</td><td>M33-500</td><td></td></tr></tbody></table>

a robotic platform for high-throughput protoplast isolation and transformation

在过去的十年里，已经在使用植物原生质体，范围从模型物种作物物种，信号传导途径，转录调控网络，基因表达，基因组编辑，和基因沉默的分析的回潮。此外，显著进展已在植物的原生质体的再生，这已产生在使用这些系统用于植物基因组的甚至更多的利益作出。在这项工作中，一个协议已使用机器人平台已经开发了用于原生质体分离和转化的自动化从&#39;亮黄色&#39;2（BY-2）烟草悬浮培养。使用花椰菜花叶病毒35S启动子（35S）的控制下，为橙色荧光蛋白（OFP）报告基因（pporRFP）的转化方法进行了验证。原生质体OFP表达，荧光显微镜证实。分析还包括使用propidiu原生质体生产效率的方法米碘。最后，被用于原生质体分离过程的低成本食品级酶，绕过用于实验室级的酶，它们成本昂贵的自动化原生质体分离和分析的高通量的需求。根据此工作开发的协议，从原生质体分离到转化完成过程可以在4小时进行中，在不脱离操作者的任何输入。而在这一工作开发的协议是与BY-2细胞培养物中证实，程序和方法应该是可平移的任何植物悬浮培养/原生质体系统，该系统应使作物基因组学研究的加速度。

在目前的研究中，BY-2的倍增速率从14-18小时取决于在该培养物温育的温度，用15小时的平均细胞周期长度的先前的报告一致变化。与此倍增速率，以1:100起始接种物用于启动培养，导致培养物在5-7天的50％的收集细胞体积（PCV）。在当前的协议，其中培养物在200ml培养基中生长，在7天，这提供了足够的细胞以填充33 6孔板生成100ml的PCV。在原生质体产率而言，产生在协议中规?...

Watch this Scientific Journal Video about A Robotic Platform for High-throughput Protoplast Isolation and Transformation at JoVE.com

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

高通量，自动化，烟草原生质体的生产和改造的方法进行描述。该机器人系统可以在模型BY-2系统应该是翻译到非模式作物大规模并行基因表达和发现。

机器人平台的高通量原生质体分离和转型

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

a-robotic-platform-for-high-throughput-protoplast-isolation

Universidad San Sebastian

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JoVE Journal

Genetics

14.2K 次观看.  University of Tennessee, Knoxville. 高通量，自动化，烟草原生质体的生产和改造的方法进行描述。该机器人系统可以在模型BY-2系统应该是翻译到非模式作物大规模并行基因表达和发现。 

视频: A Robotic Platform for High-throughput Protoplast Isolation and Transformation

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the conventional approaches of permanent gene deletion cannot be applied to essential genes. We have pioneered a unique collection of ~70 temperature-sensitive (ts) lethal mutants for studying cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii1. These mutations identify essential genes, and the ts alleles can be conditionally inactivated by temperature shift, providing valuable tools to identify and analyze essential functions. Mutant collections are much more valuable if they are close to comprehensive, since scattershot collections can miss important components. However, this requires the efficient collection of a large number of mutants, especially in a wide-target screen. Here, we describe a robotics-based pipeline for generating ts lethal mutants and analyzing their phenotype in Chlamydomonas. This technique can be applied to any microorganism that grows on agar. We have collected over 3000 ts mutants, probably including mutations in most or all cell-essential pathways, including about 200 new candidate cell cycle mutations. Subsequent molecular and cellular characterization of these mutants should provide new insights in plant cell biology; a comprehensive mutant collection is an essential prerequisite to ensure coverage of a broad range of biological pathways. These methods are integrated with downstream genetics and bioinformatics procedures for efficient mapping and identification of the causative mutations that are beyond the scope of this manuscript.

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Temperature-sensitive (ts) lethal mutants are valuable tools to identify and analyze essential functions. Here we describe methods to generate and classify ts lethal mutants in high throughput.

温度敏感致死突变体的高通量机器人辅助隔离<em&gt;莱茵衣藻</em

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the ...

科研

遗传学

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive, low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics.
The combination of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a high-throughput and more efficient cell line development process. In this operation, production host cells are first transfected with an expression vector carrying the gene of interest 1, followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody, and are subsequently subject to flow cytometry analysis 2-4. Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed, these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 - 10,000 clones can be screened per operation cycle with the current system setup.
This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes, the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure, this automated platform demonstrated advantages of significantly increased capacity, ensured clonality, traceability in cell line history with electronic documentation and much reduced opportunity in operator error.

A high-throughput, automated platform of manufacturing cell line development for producing protein therapeutics is described. Implementation of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has demonstrated significantly increased processing capacity in cell line development with improved cell line quality and high reproducibility.

一种高通量自动化的平台，制造蛋白质治疗的细胞系发展

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing ...

医学

Studying Protein Import into Chloroplasts Using Protoplasts

The chloroplast is an essential organelle that is responsible for various cellular processes in plants, such as photosynthesis and the production of many secondary metabolites and lipids. Chloroplasts require a large number of proteins for these various physiological processes. Over 95% of chloroplast proteins are nucleus-encoded and imported into chloroplasts from the cytosol after translation on cytosolic ribosomes. Thus, the proper import or targeting of these nucleus-encoded chloroplast proteins to chloroplasts is essential for the proper functioning of chloroplasts as well as the plant cell. Nucleus-encoded chloroplast proteins contain signal sequences for specific targeting to chloroplasts. Molecular machinery localized to the chloroplast or cytosol recognize these signals and carry out the import process. To investigate the mechanisms of protein import or targeting to chloroplasts in vivo, we developed a rapid, efficient protoplast-based method to analyze protein import into chloroplasts of Arabidopsis. In this method, we use protoplasts isolated from leaf tissues of Arabidopsis. Here, we provide a detailed protocol for using protoplasts to investigate the mechanism by which proteins are imported into chloroplasts.

Here we describe a protocol to express proteins into protoplasts by using PEG-mediated transformation method. The method provides easy expression of proteins of interest, and efficient investigation of protein localization and the import process for various experimental conditions in vivo.

原生质体在叶绿体中导入蛋白质的研究

The chloroplast is an essential organelle that is responsible for various cellular processes in plants, such as photosynthesis and the production of many ...

生物化学

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 ...

生物学

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

生物工程

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

模块化 DNA 设备的自动机器人液体处理组件

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella protonemal protoplast and Arabidopsis mesophyll protoplast transformation1. Due to its capacity to undergo efficient mitotic homologous recombination, Physcomitrella patens has emerged as an important model system in recent years2. This capacity allows high frequencies of gene targeting3-9, which is not seen in other model plants such as Arabidopsis. To take full advantage of this system, we need an effective and easy method to deliver DNA into moss cells. The most common ways to transform this moss are particle bombardment10 and PEG-mediated DNA uptake11. Although particle bombardment can produce a high transformation efficiency12, gene guns are not readily available to many laboratories and the protocol is difficult to standardize. On the other hand, PEG mediated transformation does not require specialized equipments, and can be performed in any laboratory with a sterile hood. Here, we show a simple and highly efficient method for transformation of moss protoplasts. This method can generate more than 120 transient transformants per microgram of DNA, which is an improvement from the most efficient protocol previously reported13. Because of its simplicity, efficiency, and reproducibility, this method can be applied to projects requiring large number of transformants as well as for routine transformation.

Efficient Polyethylene Glycol PEG Mediated Transformation of the Moss Physcomitrella patens

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella protonemal protoplast and Arabidopsis mesophyll protoplast transformation1.

高效的聚乙二醇（PEG）介导的转化苔小立碗藓</em

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.

大豆原生质体分离的简易方法及其在瞬态基因表达分析中的应用

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

Scalable Transfection of Maize Mesophyll Protoplasts

The transfection of maize mesophyll cells often involves digesting the plant cell walls to create protoplasts and then inserting DNA via electroporation or polyethylene glycol (PEG). Previous methods were developed to produce tens of thousands of transfected protoplasts at once. Here,&#160;we describe a straightforward method to isolate and transfect millions of leaf mesophyll protoplasts in maize (Zea mays L.). This streamlined process removes certain common protoplasting steps, such as washing in W5. Additionally, steps such as centrifugation, PEG-mediated transfection, and incubation have been modified to work with a greater number of protoplasts. The ability to express large libraries of plasmid constructs enables genome-scale experiments, such as massively parallel reporter assays in maize.

Here, we present a high-throughput protocol to transiently transfect millions of maize protoplasts for testing large libraries of plasmids in maize mesophyll cells.

玉米叶肉原生质体的可扩展转染

The transfection of maize mesophyll cells often involves digesting the plant cell walls to create protoplasts and then inserting DNA via electroporation ...

机器人平台的高通量原生质体分离和转型

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温度敏感致死突变体的高通量机器人辅助隔离