我们感谢意见和有益的讨论十字架实验室成员。这项工作是由PHS 5RO1-GM078153和支持 从西蒙斯基金会米哈尔Breker的一个初级研究员奖。

1.紫外诱变<ol><li>制备一批100 - 200用Tris-乙酸盐-磷酸盐（TAP）介质9,10-矩形琼脂平板上。提前几天准备这些板块，并保持他们在板凳上干，以确保在接下来的步骤中悬浮细胞的快速吸收。 </li><li>在光下100毫升的液体的TAP的，在25℃和100rpm振荡下; -培养衣藻细胞高达0.2 0.5光密度（〜2天OD 750）。 注:Mat-湿度计R（赋予潮霉素B抗性）和Mat +帕罗R（赋予巴龙霉素抗性）:紫外诱变是在两个遗传背景下独立完成。抗生素的选择是任意的，所提供的两种交配型具有互补的药物抗性。 </li><li>检查每个培养物的样品，在显微镜下，以确保细胞是活的，健康（游泳和完整），和没有污染。 注:长满文化"撞车"和丧失活力，表现为相衬显微镜"鬼"。不要让摇去的文化一旦达到饱和。 "鬼"现象是在步骤1.3不易察觉。 </li><li>稀释文化0.003 OD 750。包住瓶用铝箔，以确保均匀的密度，因为应变是能动并响应于光定向游。 <ol><li>调整基于计划UV剂量的悬浮液的密度，从而使占细胞杀伤，200 - 600幸存者菌落将形成在平板上（ 图1）。请参阅表1的紫外线照射时间的信息。 </li></ol></li><li>附上适合的液体分配器的小管盒，并执行一系列的洗涤消毒，根据制造商的说明，以防止污染。 </li><li>使用8×12液体分配器，分配各2微升4×96滴于矩形板（ 图1 </strong&gt;）。在该板的边缘轻度挖掘，以确保所有液滴的合并成的液体薄片，并立即覆盖板，以防止暴露于光。 <ol><li>以确保甚至非常单一细胞分散，使用干板，如上所述，并迅速覆盖板，以防止细胞响应于光游泳。保持在黑暗中的覆盖板水平并直至所有液体被吸收。 </li></ol></li><li>将平板杀菌紫外灯（30瓦特紫外线杀菌管）的经验来确定给予幸存者中TS-突变体的最佳产量的时间段下。这里，可使用的0.5倍 - 1.5分钟，40的距离 - 50厘米到导致90 - 99.9％杀伤。幸存者包含100 - 1000紫外线引起的点突变，其中约10％的变化编码序列。 <ol><li>为了确保没有返UV照射的最大效力由于光依赖性DNA修复11,12，在这一步在黑暗中工作，并立即包装板在照射后暗箱。 </li></ol></li><li>保持在黑暗中板8 - 在室温下24小时。 </li><li>发生在21°C恒温箱板与照明形成集落。集落形成，大约需要10天。确保用塑料袋包住板和内添加吸油纸吸干了液体凝结。蒸发和冷凝循环，否则可以提供液膜路线污染物进入板。 </li><li>装载板中的相应堆栈作为来源机器人菌落拾取（ 图2）。挑选菌落矩形板384阵列，并在21°C与照明（〜1周）增加他们。 </li><li>冷凝384阵列成1536阵列（4:1），使用一个副本镀机器人（ 图3），并允许所述板在21℃培养箱生长〜3天。 </li><li>复制1536阵列两个板块每放置一个在21℃培养箱（许可温度），另一个在33℃培养箱（限制性温度）。以下24个小时，复制板在33°C到一组新的预热板，并将其放置在33℃培养箱。 注意:这样做的原因二次镀覆是TS致死突变体可以在某些情况下，积累大量的生物量，即使将细胞接种后在第一个细胞周期被捕。辅助副本消除了这一信号，并极大地提高灵敏度。 </li><li>拍摄用以下在33℃生长3天并在21℃生长5天后的一个数字照相机的板。在固定帧持有板。使用一个"网板"标有与该培养板（ 图4）一起拍照9对准的指标。照片培养皿的交替搭配21°C和33°C（21/33）图像。 注:不同的培养时间用来均衡增长，因为野生型显著增长在33℃的速度比在21°C。 </li></ol> 2.识别用TS突变考生:一是屏幕<ol><li>处理带有自定义Matlab的图像分析软件配对21/33板的图像，以消除背景和分割图像到1536阵列。该程序将确定在每个位置（ 图5）检测到的生物量（总像素强度）。 注:软件（的SI与说明书一起提供）将使用网板图像，以确定细胞的（个人的条目）在1536阵列在所用的放大率和板对准的位置，并在每个小区计算总像素强度。对于每个突变体，这些值随后针对可调参数进行比较，以确定位于C相对于TS +标准和温度敏感性的程度21°所需的生长，其定义为:S（MUT 33）/ S（WT 33）/ S（MUT 21）/ S（WT 21），其中S是信号（象素在经过背景减除张力），物是一个单独的突变体，以及"WT"是一个随机选择非温度敏感的殖民地（诱变菌株有TS +表型）。生长在21℃被定义为S（MUT 21）/ S（WT 21）。在这第一个屏幕，应用轻松的选择标准（允许在21°C相对低增长和相对较低度的温度敏感性），以确保假阴性率较低。 </li><li>装载由软件作为单一菌落拾取机器人（通常到384阵列）的指令文件生成所选菌落的列表。根据机器人说明准备源和目标板，并挑选菌落阵列（ 图6）。 注:常规采摘需要一个指令文件中的某些格式，如为.csv，.txt或.xls的。所有采摘将必须是文件驱动的能力;不同的格式将需要MATLAB代码（提供源代码）进行较小的编辑。 </li&gt; <li>将目标板在21°C培养箱〜5天，长出股板块。 </li></ol> 3.确定TS突变体:第二个屏幕<ol><li>重复步骤2.1复试采摘的殖民地。通常为30 - 菌落的50％将重新测试清楚TS致死，为2％的产率 - 相对于初始菌落存活紫外诱变5％TS致死。 </li><li>重复步骤2.2采摘两次筛选TS致死，但设计阵列殖民地插入矩形板100菌落块在384密度修正的指令文件。这是为在下一步骤方便镜检。指令文件格式由MATLAB代码在运行时指定。 </li><li>确保包括几个WT菌落作为对照，并将板在21℃下〜5天。 </li></ol> 4.初始表型测定<ol><li>复制在步骤3.2排列在100块板，并将其放置在21°C INCU为乌兰巴托〜2天，以获得新鲜生长的菌落。 </li><li>复制100块板（4.1）的新鲜副本三份。放置在21℃培养箱一个副本，一个在33℃培养箱作为对照。 </li><li>将第三个副本（"筛选板块"）的机器人安装，并当场殖民地长销感动与无菌水。 注:固定到琼脂衣藻细胞的机器人往往最初土地作为细胞的一个相当密集的地方，以供显微镜检查几单细胞。要优化显微镜检查的殖民地，用一滴水发现最初转移细胞（由机器人长销转移水量为100〜NL）。这导致细胞分散在约初始钉扎中心半径小（〜1毫米），与许多分离的单个细胞。 </li><li>取隔离板的每个点的区域的显微照片在时间0（同时仍单个，未分开的细胞）（图7），然后将板在33℃下温育。通过设定给在4.5毫米的中心到中心的间距384密度阵列的停止的改性四分体解剖显微镜的手动阶段控制确定图像位置。 </li><li>除去从33℃培养箱的隔离板，并采取显微照片，如在步骤4.4，在不同的时间点（10小时，20小时，48小时）。确保阶段，板夹持器，和级控制器被精确地校准，以获得相同的细胞的图像在每个时间点。进行快速显微照片采集（约10分钟）。 </li><li>使用第二个副本中的33℃培养箱，以验证该TS的表型，并确保在图像采集期间温度波动对TS的表型没有重大影响。 <ol><li>分析显微图像，并选择基于期望的标准（ 图8）的突变体。发现在96排列琼脂板中的最终选择的一组。确保每个板包含相同交配型和耐药性的突变体。 </li><li>对于每块板，合并的最后两列的正（查询突变）和用于互补测定法的阴性（WT）的控制。 </li></ol></li></ol> 5.新的突变体的互补和联动测试，以"空中飞人" <ol><li>传送大量的排列菌落到无氮配子诱导培养基10在96孔微孔板（各菌落的密度应该是周围0.2的OD）。孵育板光下（13 - 20瓦的灯泡汞），用于〜5小时，让配子。 注意:此步骤可以用一个复制的机器人安装或手动用一个简单的电镀设备中进行任一。 </li><li>暂停与相反交配型携带替代抗性盒成管与配子无氮配子诱导培养基10查询。 </li><li>混合在目标板中的样品中的配合mixt20微升（ 图9）URE音量。继〜光下10分钟，从每口井的两倍现货5微升:自来水+ 5μM帕罗+ 9μM湿度计进行互补测试一次在TAP板联动测试和一次。 </li><li>孵育在21°C培养箱中过夜联动板，然后将它们包装箔。保持它们在黑暗中5天，以允许接合孢子形成。 </li><li>在21℃孵育互补测试板〜10天的光。 注:药物量被校准，以允许双重杂合子二倍体存活，但它们仍然足以消除配合单倍体。这些金额校准在这一特定项目中使用标准电阻盒集成;与新的集成，剂量应重新校准。大多数生物质被证实是通过流式细胞术（ 图9）二倍体，虽然单倍体（可能从减数分裂和双抗性生长的可变水平分离子）通常被观察为好。 </li><li>复制互补，测试板分成两个副本TS-表型鉴定。将在21°C一个副本及复印件在33℃〜为5天。 </li><li>为TS-型测试的殖民地，如第2.1节（ 图5）描述。不与查询互补菌落可能代表相同基因的等位基因作为查询（二倍体杂等位基因的突变在相同的基因）。 </li><li>对于联动测试，接下来的步骤5.4，却将板回光，以便〜7天减数分裂和单倍体分离子的产物。 </li><li>复制链接测试板，以TAP + 10μM帕罗和10μM湿度计来选择耐双后代（预测是单倍体后代的25％，由于录音带的链接断开），并在21°C孵育一个星期。 注:为提高耐加倍单倍体的药物剂量。 </li><li>测试菌落为TS-表型，因为我n步2.1。 注:TS-型预期（和观察）为同一互补组作为查询的突变体。此外，在联动测定一个TS-表型，该补充查询的突变体，可以反映查询和新的突变之间的紧密联系。补充测试查询突变很可能新的基因，这将进入管道的致病病变的鉴定生物信息学，最终为进一步的表型分析。 </li></ol>

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作者宣称没有显著财务权益。

这里描述为TS致死突变高产隔离管道确保想必衣藻基因组的所有蜂窝必要的途径来表示。潜在的细胞周期基因的高效收集和消除重复的"常客"等位基因的两个最关键的步骤是:不完整的细胞周期; 2）对已确定的并行互补法逮捕表型特征1）相干定义查询基因放大与新分离出的那些集合。 当明暗周期同步，衣长在白天和单元尺寸&gt; 10倍的增加光合没有任何DNA复制和细胞分裂13。夜间发作大致一致，将细胞再经历交替DNA复制，有丝分裂和细胞分裂（ 图8）的多次循环。这种监管舍姆E提供主要需要用于细胞生长和完整性以及专门为细胞分裂周期所需的基因的基因之间的自然区分。我们发现，在10小时和20小时的时间点是非常丰富的用于初始粗糙表型切1。我们目前认识到TS致死突变，基于这些图像的大类（见SI的图林和跨，2014年）1，分别是:缺口，爆米花，圆形，小，中，早期的裂解和多周期（ 图8 ）。 三个最相关的类别，我们重点关注的缺口，爆米花，而圆。的"缺口"和"爆米花"表型以前证明是最细胞周期特异性的病变的特征（ 例如，有丝分裂细胞周期蛋白依赖性激酶，DNA复制机械，和拓扑异构酶II）的1。一（缺口）或多个（爆米花）早期，但不成功的细胞分裂的视面的外观是一个方便的吗啉细胞周期起始ogical指标。这些突变体通常表现出很少或没有生长缺陷，并在在10小时标记类似于WT细胞体积增大。该Notch和爆米花的表型是在10小时明显和充分开发（常与细胞裂解有关）20小时。 "圆"类似的细胞长到WT但备受产量减少明显的初始分割平面，从而产生大而圆被捕细胞。此类别中以前突变落入为微管功能（微管蛋白折叠辅助因子，γ-微管蛋白环络合物）1所需的后期促进复合物14或在基因的组件。在稍后的时间，这些细胞经常表现出明显的细胞溶解。 "小"和"中"细胞的生长或者可以忽略不计（小）或显著小于WT（中）。许多这些突变体确定日期的有在基因，其注释表明在基本cellula角色病变- [R生长过程（翻译或膜生物合成）。中型和圆之间的主要微观歧视依赖于量的增长在10小时（回合:像WT;介质:减少）。由于中小企业的类别非常大，可能反映在一个伟大的范围内细胞通路的病变，我们并不试图饱和这些类别;但是，我们希望分子标识类的代表，了解损失的表型多样化的途径。两个研究忽略类别是:1）早期裂解，通过10小时的标记失去完整性（损失绿色的，refractility的损失），与现有的细胞生长的迹象和2）的多次循环的突变体。细胞增殖同样在10和20小时，WT，尽管它们表现出完全不能进行长期增殖。 我们大多表征"缺口"，"爆米花"和"圆"和排除中小型圆形细胞，以及因为漏水突变体完整的少数细胞分裂。这主要是保证基本细胞功能，如生长及膜的完整性，是功能性和富集分裂相关基因的概率。这种方法被发现是凭经验高效;但是，它可能是一个细胞周期的基因多效性的是，具有早些时候在G1其他角色，之前的实际分工。这种情况下，我们预计是罕见的，被错过。更一般地，我们的目标是同质逮捕，这在高概率是由于一个致病突变是一个完全不正常的蛋白质。然而，对于如刚刚所描述的相同的原因，可能有几种停滞点，因此，一定程度的灵活性是在选择候选为宜。 为了丰富与新发现的基因集合，所选择的候选人测定互补。我们要求TS-在阳性对照阴性对照（针对查询突变查询）和TS +（曲儿Ÿ对WT）。同一互补组的查询在新突变体是TS-。会员在同一互补组中几乎无一例外地反映了相同基因的分子损伤（这一直是我们测试过每一个这样的基因的情况下）。因此，对于"空中飞人"，这个标准是排除作进一步鉴定。这还以相同的互补基团为试查询突变体是新的基因的候选并通过生物信息学和实验工具被进一步表征。 TS-等位基因为不同互补群的高度可变的恢复是一个众所周知的现象，也就是说，变化是明显高于泊松噪声较大，由于可变性不同基因之间TS-的巨大内在的变异。原因可能包括内在热敏性差异;不同的蛋白质大小;的蛋白质的存在，作为一个单体与作为大的，稳定的复合物;和致突变热点。这几乎是一个纯nuisaNCE。然而，一个所得有利的结果是，"飞行常客"名单不长（只有几个目标占据了大部分的列表），所以劳动密集型互补测试不是一个艰巨的任务，直到项目的后期阶段。 作为一个补充的方法，我们进行了联动试验。在该测定中，抗双后代被选择并且用于TS-表型进行测试。一个TS-型预期（和观察）相同互补组在突变体作为查询或紧密连锁的突变。对于每个被测试的基因中，WT子代预计出现（TS +表型）中以一定的概率取决于遗传距离。我们估计，大约有100接合孢子在这些斑点每个交配。假设100％减数分裂效率，这将导致从取消关联药物抗性盒大约100双药物抗性后代（减数分裂后代的25％，四每减数分裂，由于至Mendelian继承）。这也将是TS-突变，其中所述后代的25％将是双突变的情况下，与25％将是WT，如果查询和测试的突变是无关联。因此，出了双药物抗性子代，25％将是WT（约25个细胞）。这是完全无关联突变的情况;然而，中度连锁（约20厘米，约2兆，或基因组115的2％以内）将强有力地减少或消除的TS +信号用。在被测试的突变的联系的对抗生素盒的情况下，即是双药物抗性TS +单倍体存在于非常低量。这表现为明显的故障与测试的所有突变体重组，尽管所有的补充测试的突变体，异常的结果，很容易注意;在这种情况下，回交将解决这个问题。 无论从先前的知识和序列分析，我们预计在衣藻细胞周期基因是大约500个基因2，虽然莫ST，但可能不是全部，是必不可少的。我们将评估为更多的突变体收集额外诱变轮的必要性和饱和升高的水平。 这个过程的独特设计用于研究基本生物过程和实施它们的基因和蛋白质。其他方法来产生必需基因扰动存在（ 例如，随机诱变的等位基因16，有条件地转录等位基因17，或亚效等位基因等位基因18的变换）。然而，它们都需要同源重组，其在营养衣强烈抑制。群集，定期相互间隔短回文重复（CRISPR）/ Cas9系统已经被确立为基因修饰19的有力工具;但是，它是尚未在衣20有效地工作。关键的是，所有这些方法要求目标的先验知识。这是一个严重的限制如果希望有学习新东西的可能性！我们的做法会产生突变鉴定必需基因，独立于任何先验知识。因此，在技术的现有水平，随机TS突变随后通过深度测序基因鉴定的隔离可能是获得快速进入微生物细胞生物学在植物superkingdom的最有效的方法。 致病突变的鉴定（选自〜100的编码序列变化的突变每个克隆）是超出了本文的范围。分群池1的深度测序是有效的，但劳动密集型的。一种用于在大量的菌株的所有突变的一种测定组合池策略，测序少数池后，是非常成本和劳动高效。对于组合分群测序的新战略正在开发中，将允许致病突变的鉴定几十个突变体的SIM卡ultaneously单个测序运行（筹备中）。这些效率是非常重要的，以允许关键基因鉴定的步骤，以保持与由此处描述的过程成为可能的突变体的非常迅速积累速度。

模式生物的系统性突变集合的表型特征是细胞解剖复杂一个行之有效的方法。单倍体单细胞绿藻莱茵衣藻具有类似植物的基因组，但它会从陆地上的植物前的土地厂房血统2多个基因组复制分歧。在原则上，缺乏基因复制和一个主要单倍体生命周期大大方便失功能遗传方法。然而，感兴趣的基因的靶向破坏几乎是不可能的，由于缺乏有效的同源基因组的整合。随机插入中断库正在建设中，结合打乱现场鉴定，到目前为止产生的阵列组代表1562个基因3 1,935映射中断。但是，这种方法（预期一般产生无效突变）并不适用于必需基因。温度敏感（TS）的突变可以recovered在必需基因，以及最近的方法允许突变基因和致病病变的有效识别。在高温下的表型分析，然后提供关于突变基因的功能即时信息。我们报道了在衣 〜70必需基因TS致死突变的分离和表征，特别是着眼于参与细胞周期进展的基因和控制1,4。 TS致命的屏幕已经遗传分析的支柱微生物几十年5,6。原则上，一个期望的特征是接近"饱和"，这意味着能够突变至TS杀伤力的所有基因通过至少一个突变鉴定，允许一个完整的分析。然而，在实践中，有几个因素限制了方法来饱和。首先，虽然几乎所有的基因可以被突变，以在高温下活性的损失，这种突变体的回收效率至少上变化7,8级的顺序。因此，随机屏幕开始回升复发安打饱和度接近很久以前"常客"。其次，虽然TS突变通常导致减少的功能，他们可能不以限制性的温度真空值（而相反地，常常没有充分发挥作用以允许温度）。这个问题可以通过比较多个等位基因处理到一定程度;如果它们都有一个共同的表型，这是更可能反映基因的简单失活的结果。多重等位基因也是致病病变1确切分子鉴定非常有帮助。然而，"常客"的问题意味着很少打基因复等位基因可能难以恢复。 由于这些原因，我们一直在开发一种增强管线来分离和表型表征TS突变体。我们收集了超过3000 TS突变体到目前为止，我ncluding约200新的候选细胞周期突变。这个集合，它可能已经包含大多数或所有细胞的必需途径突变的分子和表型分析应提供在植物细胞生物学新的见解和假设。重要的是，这条管道可以应用于生长在琼脂上以有效地构造TS突变体集合的任何微生物。 在设备上的说明:两个机器人是在此过程中（殖民地选择器和组合副本镀覆/樱桃采摘）的效率非常重要。采摘通常有金属针。挑菌落保持在空气中对这些引脚一段时间（数秒至数分钟，视型号而定）。衣死在约20秒在空气中的金属引脚上。这限制模式选择有机体。精度问题。我们的殖民地选择器是相当准确;但是，它是在它的动作有所暴力并且具有用于基于喷雾交叉污染一些可能性。它不是在高使用尔比384密度（4.5毫米中心 - 中心间距）;在此密度，精度是完全可以接受的。副本电镀/樱桃采摘机器人（用于这些应用不同的附件）是采摘慢得多，但是对于1536密度足够准确的（6,144是一个挑战，即使是这种非常准确机器人;我们已评估了此密度，但决定不使用它，因为它的各种困难）。如果板装载不均匀等机器人将不能很好地工作。以抽查，以确保正确的事情正在发生是很重要的;当然，机器人会自动运行，并在一般情况下，一切都会顺利，如果前几个板块是正确的。

<table border="0" cellpadding="0" cellspacing="0" width="672">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:293px;">Equipment:</td>
			<td style="width:144px;"></td>
			<td style="width:147px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hudson RapidPick colony picker</td>
			<td>Hudson Robotics</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MultiDrop Combi Reagent Dispenser</td>
			<td>Thermo Scientific</td>
			<td>5840300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small tube metal tip cassette</td>
			<td>Thermo Scientific</td>
			<td>24073295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Singer RotoR replica-plating robot</td>
			<td>Singer Instruments</td>
			<td></td>
			<td>Very essential for the process. For lower scale screenings you can use an in-house manual tool</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Singer single-colony picking attachment (&#8216;Stinger&#8217;)</td>
			<td>Singer Instruments</td>
			<td></td>
			<td>Can be picked manually, however for large scales it is nearly impossible</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Materials:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYTOX Green Nucleic Acid Stain</td>
			<td>ThermoFisher Scientific</td>
			<td>S7020</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

我们发现在衣隔离TS突变体加速管道。将细胞分配在琼脂平板上，并且不久后在显微镜单细胞密度下的快速确认，板是经UV照射（ 图1）。典型辐照菌落鉴定并在许可温度（ 图2）挑入的阵列格式后生长10天。在384格式所得板被合并到一个1536阵列（ 图3）。从收集照射菌落这些第一步，我们迄今摆着〜20万的殖民地。 600幸存者菌落将形成在平板上 - 的悬浮液的密度是根据计划UV剂量，使得，占死亡，200调整。三紫外线照射时间（1.5分钟，1分钟，0.5分钟）和三个密度进行了相应的测试（ 表1）。根据经验，在1.5分钟的曝光时间取得了大部分的TS- 突变体（〜50％）;然而，到目前为止，1分钟，得到最细胞周期的候选人。因此，未来的紫外诱变轮用仅1分钟完成。一个重要的问题是初始采摘和交叉污染（特别是在高密度格式）中的飞溅。这可能导致在重复和相同的突变体的进一步表型的两倍。为了尽量减少这种情况的概率，照顾在最佳尺寸收集的殖民地，并确保相邻的殖民地没有在最初的试验采摘。 接着，两个连续的TS-表型分析（ 图5）进行，并在3000 TS-突变体分离并通过时间推移显微镜（ 图8）表型特征。由于学习兴趣细胞周期中符合特定标准的表型的生物学重点，我们没有兴趣在每个目标根收集两个以上的等位基因即因此，我们进行互补和联动与检测特点已与基因两个以上的等位基因（查询）针对新收集的考生请从下游管道（ 图9）极易复发的基因。 <img alt="图1" src="/files/ftp_upload/54831/54831fig1.jpg" /> 图1: 未诱变细胞与诱变的统一传播。 （一）384×2微升液滴分配在使用试剂分配器的琼脂平板。 （二）随机板显微镜验证单细胞密度下测试，并且以1分钟的曝光照射紫外线。比例尺= 50微米。 <a href="http://ecsource.jove.com/files/ftp_upload/54831/54831fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2"SRC ="/文件/ ftp_upload / 54831 / 54831fig2.jpg"/&gt; 图2.紫外诱变菌落钦点为384阵列。 （一）紫外线照射单细胞生长为约10天进可见菌落。比例尺= 2厘米。 （二）图像分析程序识别按照一定参数可分离的殖民地和机器人阵列它们变成384板。 <a href="http://ecsource.jove.com/files/ftp_upload/54831/54831fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54831/54831fig3.jpg" /> 图3. 合并到1536阵列。四384格式板合并成一个1536板;一旦生长，它们被再次复制到测试对于TS-表型。 <a href="http://ecsource.jove.com/files/ftp_upload/54831/54831fig3large.jpg" target="_blank">请点击这里查看</a>更大的版本这个数字。 <img alt="图4" src="/files/ftp_upload/54831/54831fig4.jpg" /> 图4.成像的定量。 （一）将板在一帧的文档摄影待机下保持，使得在一个系列中的所有板在相同的放大倍率和定位拍照。 （二）第一图像是与位于角部和中点红色点的1536孔微量滴定板的。这个"网板"图像定义了阵列的位置。 （三）孵育后一个1536阵列的实施例。 <a href="http://ecsource.jove.com/files/ftp_upload/54831/54831fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54831/54831fig5.jpg" /> 图5.鉴定半干型TS-的-automated图像分析。板图像被定制MATLAB软件（的SI提供）进行分析。图像被自动分割成单元阵列（96，384，或1536），并且在每个小区中的信号进行量化。在增长21°C标记为蓝色，并在33°C的增长标记为黄色。相比于33°C在21°C表现出显著较高的成长殖民地（标准化TS +）被选中并标注在黑色方块有绿点。这些选择可以手动编辑。最终的选择被转移到由集落采摘机器人使用的文件。 <a href="http://ecsource.jove.com/files/ftp_upload/54831/54831fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54831/54831fig6.jpg" /> 图6. 第一轮TS致命考生机器人采摘。樱桃，pickiNG机器人沿着从如步骤2.1中所述挑选的候选人为新阵列生成的文件的说明。顶板示出了灭菌销的加载。底部面板显示从在源板一定菌落精确采摘。 <a href="http://ecsource.jove.com/files/ftp_upload/54831/54831fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/54831/54831fig7.jpg" /> 图7. 时间推移筛选TS致死表型进行初步分类。即通过两轮在图5中示出的筛选方案的克隆在细胞密度，使得单个细胞可被微观解决复制到板中。一种改性四分体解剖显微镜被用于识别在该显微照片后0小时，10小时，和20小时孵育一个拍摄位置<html> ŧ33℃。 <a href="http://ecsource.jove.com/files/ftp_upload/54831/54831fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8" src="/files/ftp_upload/54831/54831fig8.jpg" /> 图8:按照时间推移显微镜鉴定衣藻细胞周期的突变体。 （a）该衣藻独特的细胞周期的示意图描述了长G1期特征的细胞生长，随后裂变SM周期，这结束了新生的子细胞的孵化。 （二）显微图像细胞周期和失败完成有丝分裂典型的细胞周期的突变体的鉴定过程中表现出野生型细胞。比例尺= 50微米。 <a href="http://ecsource.jove.com/files/ftp_upload/54831/54831fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> ENT"FO: - together.within页保留="1"&gt; <img alt="图9" src="/files/ftp_upload/54831/54831fig9.jpg" /> 图9:互补和联动试车。 （a）用频繁重复突变基因的抗生素抗性（ 例如，温湿度计）查询越过在一个96孔板有一组窝藏的第二抗生素抗性盒（ 例如，帕罗）相反交配型的突变体。互补板产生二倍体高馏分如通过（在左下面板）在流式细胞仪的核酸染色和分析示出。 （二）配合混合物既用于在二倍体互补并用于抗双减数分裂后代联动测试。阳性对照是查询本身在相反交配型，而且，正如预期的那样，显示了在33℃的TS-表型，而阴性对照是WT和示出了TS +表型。在盘旋的菌落显示与查询没有互补，因此ñEW TS-等位基因为查询基因。这些通常是从进一步鉴定排除在外，因为只有"空中飞人"是在互补的测试集。 <a href="http://ecsource.jove.com/files/ftp_upload/54831/54831fig9large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 时间 </td><td> OD </td><td> 菌落挑（＃） </td><td> TS-型 </td><td> 细胞周期候选人 </td></tr><tr><td> 1.5&#39; </td><td> 0.012 </td><td> 6000（平均=〜200） </td><td> 200（3.3％） </td><td> 7（3.5％） </td></tr><tr><td rowspan="2"> 1&#39; </td><td rowspan="2"> 0.003 </td><td> 11000 </td><td rowspan="2"> 131（1.19％） </td><td rowspan="2"> 15（11.4％） </td></tr><tr><td> （平均=〜360） </td></tr><tr> &lt;TD行跨度="2"&gt; 0.5&#39; </td><td rowspan="2"> 6E-04 </td><td> 9000 </td><td rowspan="2"> 40（0.45％） </td><td rowspan="2"> 1（2.5％） </td></tr><tr><td> （平均=〜300） </td></tr></tbody></table> 表1:照射时间校准，以最大限度地提高细胞周期突变考生的产量。三照射时间进行了测试（各30片）具有相应的消耗臭氧层物质保证存活菌落数（200 - 600）。

Watch this Scientific Journal Video about High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii at JoVE.com

莱茵衣藻温度敏感致死突变体的高通量机器人辅助分离遗传学 |视频

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 ...

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

Classical and Modern Genetics

SCIENTISTS IN ACTION

9.7K 次观看.  The Rockefeller University. 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. 

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

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

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 transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

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 ...

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遗传学

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3&#39; end. Reverse transcription and amplification of the transcripts are not required. Reporter probes contain a sequence of six color positions populated using a combination of four fluorescent colors. The four colors over six positions are used to construct a gene-specific color barcode sequence. Post-hybridization processing is automated on a robotic prep station. After hybridization, the excess probes are washed away, and the tripartite structures (capture probe-miRNA-reporter probe) are fixed to a streptavidin-coated slide via the biotin-labeled capture probe. Imaging and barcode counting is done using a digital analyzer. The immobilized barcoded miRNAs are visualized and imaged using a microscope and camera, and the unique barcodes are decoded and counted. Data quality control (QC), normalization, and analysis are facilitated by a custom-designed data processing and analysis software that accompanies the assay software. The assay demonstrates high linearity over a broad range of expression, as well as high sensitivity. Sample and assay preparation does not involve enzymatic reactions, reverse transcription, or amplification; has few steps; and is largely automated, reducing investigator effects and resulting in high consistency and technical reproducibility. Here, we describe the application of this technology to identifying circulating miRNA perturbations in irritable bowel syndrome.

We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.

肠易激综合征的循环miRNA的扰动检测使用复用高通量基因表达平台

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site. However, an individual guide RNA may cause undesirable off-target effects, and to rule these out, the use of multiple guide RNAs is necessary. This multiplicity of targets also means that a high-volume screening of clones is required, which in turn begs the use of an efficient high-throughput technique to genotype the knockout clones. Current genotyping techniques either suffer from inherent limitations or incur high cost, hence rendering them unsuitable for high-throughput purposes. Here, we detail the protocol for using fluorescent PCR, which uses genomic DNA from crude cell lysate as a template, and then resolving the PCR fragments via capillary gel electrophoresis. This technique is accurate enough to differentiate one base-pair difference between fragments and hence is adequate in indicating the presence or absence of a frameshift in the coding sequence of the targeted gene. This precise knowledge effectively precludes the need for a confirmatory sequencing step and allows users to save time and cost in the process. Moreover, this technique has proven to be versatile in genotyping various mammalian cells of various tissue origins targeted by guide RNAs against numerous genes, as shown here and elsewhere.

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

用荧光PCR毛细管电泳技术在基因型CRISPR / Cas9介导的敲除突变体的高通量形式

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

高脂膳食喂养和高通量三测定在果蝇中的含量

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

核密度分析的天然染色质沉淀测序文库的生成

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our entire society's ability to treat systemic infections. In the United States alone, antibiotic-resistant infections kill approximately 23,000 people a year and cost around 20 billion USD in additional healthcare. One approach to combat antimicrobial resistance is combination therapy, which is particularly useful in the critical early stage of infection, before the infecting organism and its drug resistance profile have been identified. Many antimicrobial treatments use combination therapies. However, most of these combinations are additive, meaning that the combined efficacy is the same as the sum of the individual antibiotic efficacy. Some combination therapies are synergistic: the combined efficacy is much greater than additive. Synergistic combinations are particularly useful because they can inhibit the growth of antimicrobial drug resistant strains. However, these combinations are rare and difficult to identify. This is due to the sheer number of molecules needed to be tested in a pairwise manner: a library of 1,000 molecules has 1 million potential combinations. Thus, efforts have been made to predict molecules for synergy. This article describes our high-throughput method for predicting synergistic small molecule pairs known as the Overlap2 Method (O2M). O2M uses patterns from chemical-genetic datasets to identify mutants that are hypersensitive to each molecule in a synergistic pair but not to other molecules. The Brown lab exploits this growth difference by performing a high-throughput screen for molecules that inhibit the growth of mutant but not wild-type cells. The lab's work previously identified molecules that synergize with the antibiotic trimethoprim and the antifungal drug fluconazole using this strategy. Here, the authors present a method to screen for novel synergistic combinations, which can be altered for multiple microorganisms.

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Synergistic drug combinations are difficult and time-consuming to identify empirically. Here, we describe a method for identifying and validating synergistic small molecules.

通过重叠2方法高通量识别协同药物组合

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.

The plasticity of nuclear architecture is controlled by dynamic epigenetic mechanisms including non-coding RNAs, DNA methylation, nucleosome repositioning as well as histone composition and modification. Here we describe a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) protocol which allows the determination of chromatin accessibility in a reproducible, inexpensive, and straightforward manner.

甲醛辅助分离测定哺乳动物细胞染色质可及性的调控元素

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

3dpf 斑马鱼幼虫的高通量 DNA 提取及基因分型研究

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

基于数字PCR的沙门氏菌健康高通量分析竞争指数

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...