在实验室的工作是由NSF（MCB0919933）和国立卫生研究院（GM088790）的支持。

1。中继片段再生实验<ol><li>再生实验前至少5天停喂的蠕虫病毒的队列，以确保动物废物和摄取食物（30〜完整的蠕虫，每个蠕虫〜8-10毫米长的饥饿后）的。 </li><li>在检测的一天，冲洗与水预先冷冻的平整的冰盘，并用保鲜膜覆盖平的冰面。使用镊子，一个滤纸上的保鲜膜。 </li><li>几滴泉水浸湿的滤纸。转移到过滤器使用的移液管（&lt;20％过滤蠕虫）涡虫。蠕虫过滤器，如果有必要可以重新定位，通过与更多的泉水repipetting。删除多余的液体。 </li><li>使用手术刀，截肢动物的前尖，咽（ 图1A）前结束之间约中途一个单切头。 </li><li>使用手术刀，截去尾部区域，由一个单一的削减约中间动物的尾部和后咽（ 图 1A）结束。删除多余的粘液，用手术刀切割后蘸用70％乙醇的纸巾。用手术刀多余的乙醇。 </li><li>转移到培养皿中（100 x 25毫米深）通过含有泉水钳过滤器。等待〜3分钟（伤口缝合，观察一个捏和伤口变黑）。 </li><li>冲洗干净，滤纸的Petri菜含有所需再生法和转移到恒温培养箱（24℃）介质的主干片段。 </li><li>分数再生表型〜1周后，当再生结构识别（ 图 1A） 。 </li></ol> 2。吡喹酮诱导再生：两极药理操纵<ol><li>准备溶药物在DMSO（62.4mg 1ML PZQ 200MM股票）吡喹酮（PZQ）的股票。当天使用。 </li><li>设为50毫升含药溶液中缓冲Montjuch盐（PZQ 90微米）。涡〜1分钟，以确保分散。 </li><li>执行主干片段[＃1]，露出的树干碎片队列的最后一步[1.7] PZQ协议的详细以上的再生法。 </li><li> 24-48小时后，交换PZQ含有温泉水的媒体，洗衣机蠕虫（&gt; 3）次。 </li><li>返回蠕虫孵化器。分数两极（两个头， 图1B）切割后的一周。 </li></ol> 3。再生的基因操作：在体内RNAi的喂养协议<ol><li>检测前，准备鸡肝匀浆。丢弃从foodstock脂肪，黄颜色的部分，菜泥其余肝。过滤菜泥通过滤网和存储等分悬浮液（1.5ml离心管，-20℃）。 </li><li>检测之前，准备aliquoting到1ML样品供应，储存于-20 ° C牛的红血细胞（红细胞） </li><li>选择用于RNAi的蠕虫病毒。三个同伙：感兴趣的基因，阳性对照组（基因与已知的RNA干扰表型;图1C，D和 E）和负控制（无RNAi的表型的基因，也有用的评估击倒水平相比，实验队列）。对于每个队列，使用〜250的蠕虫（〜8-10毫米长至少5天的饥饿后）。存放在塑料桶泉水。 </li><li>对于每个RNAi的队列中，大肠杆菌表达dsRNA的目标感兴趣的基因大肠杆菌解冻股票[8]，随着鸡肝匀浆管[3.1]和红细胞管[3.2]。 </li><li>佩莱细菌（13000克，1分钟）。移除上清液2xYT媒体（〜700μl）和重新悬浮颗粒。 Recentrifuge，在冰上，丢弃上清液，沉淀的地方。 </li><li>创建大缸径P200的枪头，切断提示结束。在鸡肝匀浆（150μL）和红细胞（50μL）的混合物创建RNAi的喂养混合彻底重悬细菌沉淀。取出离心中的气泡。 </li><li>觅食，删除包含蠕虫的塑料浴盆的水多数（离开〜1英寸深入）。涡流浴缸集中在这个容器中的中心的蠕虫，和吸管RNAi在一个网罗蠕虫圈饲养的组合。请使用移液管，仔细同轴电缆上的RNAi喂养混合逃出，没有其他食客的干扰！ </li><li>喂奶后（约1小时），确定，美联储以及涡虫。这些蠕虫从摄入的红细胞深红色着色。丢弃他人。 </li><li>小心地更换新鲜的泉水喂养的解决方案，最大限度地减少干扰，防止食物的排泄。要做到这一点，轻轻地本地化蠕虫使用移液管的管的一侧，倒入水混浊，并仔细地用清水取代倾盆而下的浴缸对面的墙上。长时间暴露在空气Resubmerge涡虫很快也会导致食物排泄。 </li><li>松散SE铝容器的盖子，并返回到培养箱（24℃） </li><li>重复RNAi在多个周期中喂养协议（〜2-3天），穿插协议＃1]屏幕RNAi的表型再生周期。许多基因的有效供料和再生的一个标准协议显示在图2〜1个月，总工期。 </li></ol>修改这个计划，针对不同的基因作为最佳的协议将取决于mRNA的稳定性，组织定位，蛋白质perdurance，或排除多个喂养周期后再生一个表型的发展等因素。评估只是表型的显性筛选（明显），或定量PCR方法，有针对性的mRNA水平与阴性对照队列击倒水平。 4。代表性的成果： 主干片段再生实验是健壮的所有蠕虫应重新正常前后（“头到尾”）极性。这可以切割后的5天，前眼点（asterixed图1A）的出现，促成尽快拿下。然而，一周后完成恢复失去结构的形态发生。药理操纵主干片段再生PZQ产量双头蠕虫（ 图1B）也很强大（欧共体50 = 87（+ - ）11％的双极片段，70μM为48 小时 20 PZQ） 。两极发生在一个单一的再生周期。下在这些实验的疗效可能与药物的溶解度和/或存储，或使用不同的扁虫种PZQ是无效的问题。对于外显率较 ​​低的药物，通常用于anteriorization指数得分从 22完整的两极的中间表型搁置。表型从积极的控制结构的RNAi， 如图1所示：RNAi 的日本三角涡虫6 - 1（D J六个- 1）的结果，在无眼表型23（ 图 1C），日本三角涡虫的PC2的RNAi（DJ - PC2）在损失厌恶的光响应24（ 图1D）和RNAi的结果， 日本三角涡虫βcatenin- 1（βcateninDJ - 1）的结果在两个为首的表型16-19日，从主干片段（ 图1E）21。这些表型，可以实现用下面这个简单的RNA干扰协议：DJ -六（FFxFx），DJ - PC2（FFFx），DJ -βcatenin- 1（FFxFx），其中F =饲养周期和x =分别再生周期。 <img src="/files/ftp_upload/3058/3058fig1.jpg" alt="图1"/> 图1：主干片段再生检测涡虫（上）和原理图（中） 左 ，形象地展现切除主干片段的位置。右键，躯干片段再生timecourse显示外观再生片段在指定的时间（天）乙PZQ曝光产生。双头涡虫的图像C“无眼”DJ -六- 1 RNA干扰，D动的DJ - PC2的RNAi蠕虫（染成红色）轻厌恶反应，亏损相比，移动控制蠕虫（染色产生的蠕虫绿色），E双极型涡虫的DJ -βcatenin1的RNAi 。在Be，原来的RNAi蠕虫前到底是面向左侧。 <img src="/files/ftp_upload/3058/3058fig2.jpg" alt="图2"/> 图2：典型RNAi的协议包括多个喂养（F）和再生周期（X）是有效的 D.许多基因敲除喂养和切割周期序列粳稻 。本议定书的个体基因可能需要修改，​​以产生最佳的结果，使用例如一个较少（括号）或更大数量的喂养和再生周期。

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没有利益冲突的声明。

这里描述协议详细分析，研究和操纵涡虫三角涡虫再生的粳稻。他们很简单，不需要专用设备等，他们可以很容易地在实验室或教室进行。分析可以单独进行，或合并化学药物药效在体内的基因筛查，可以在候选基因水平上进行，或适应无偏见的，高通量筛选 8 。无论是耐人寻味的涡虫的生物学研究自己的权利，或在体内功能在哺乳动物的同源性研究组织再生的替代模型的评估，这些方法应推动多元化的研究人员的兴趣。

<table border="1"><tbody><tr><th>试剂</th><th>供应商</th><th>目录编号</th><th>评论</th></tr><tr><td>泉水</td><td> Kandiyohi。高级沃特公司明尼苏达州明尼阿波利斯</td><td> N / A </td><td>其他形式的泉水也很好。审判第一的可行性分析。 </td></tr><tr><td> 1 ×缓冲蒙特伊克（Montjuich）盐：氯化钠（1.6毫米），氯化钙（1MM），硫酸镁（1MM），氯化镁（0.1MM），氯化钾（0.1毫米），碳酸氢钠（1.2MM），HEPES（1.5）。 pH值7.4在24 ° C。 </td><td>多个供应商</td><td> N / A </td><td>人工水再生实验期间的药物治疗，以确保pH缓冲。 5 / 8 Holtfreter的解决方案是一个另类。 </td></tr><tr><td> 2xYT肉汤</td><td> Fisher Scientific则</td><td> BP2467 - 500 </td><td>媒体= 31 g / L的。高压灭菌。 </td></tr><tr><td>培养皿（100x25mm） </td><td> Fisher Scientific则</td><td> 08-757-11 </td><td>在再生周期的房屋蠕虫</td></tr><tr><td>广场菜（100x100x15mm） </td><td> Fisher Scientific则</td><td> 08 - 757 - 11A </td><td>与水，冻结填补蠕虫切割面的冰盘</td></tr><tr><td>塑料桶：密保诺拉链式扭&#39;N LOC（16盎司）。 </td><td>各种零售商</td><td> N / A </td><td>便民水密封容器内的RNAi同伙</td></tr><tr><td>鸡肝</td><td>商业杂货店</td><td> N / A </td><td>偏见，对有机用品，由于避免抗生素。 </td></tr><tr><td>手搅拌机</td><td>任何厨房供应商</td><td> N / A </td><td>使用鸡肝脏原浆</td></tr><tr><td>电线1毫米滤网</td><td>任何厨房供应商</td><td> N / A </td><td>使用使劲鸡肝脏原浆</td></tr><tr><td>牛红细胞</td><td> Lampire生物实验室</td><td> 7240807 </td><td>冲到100％，并汇集了红细胞悬浮液</td></tr><tr><td>圆形滤纸</td><td>的Whatman＃3 </td><td> 1003 055 </td><td></td></tr><tr><td>移液管</td><td> Fisher Scientific则</td><td> 13-711-41 </td><td></td></tr><tr><td>无菌，手术刀</td><td>多个供应商</td><td> N / A </td><td></td></tr><tr><td> ±吡喹酮</td><td>西格玛爱秩序</td><td> P4668 </td><td>在黑暗中商店粉末分装在4 ° C变干。 </td></tr><tr><td> DJ -六1 </td><td></td><td> GenBank中AJ557022.1 </td><td> RNAi的“眼”23表型阳性对照</td></tr><tr><td> DJ -βcatenin- 1 </td><td></td><td> GenBank中AB181913.1 </td><td> RNAi的阳性对照两个为首的表型 17 </td></tr><tr><td> DJ - PC2 </td><td></td><td></td><td> RNAi的阳性对照光厌恶表型24损失</td></tr></tbody></table>

pharmacological and functional genetic assays to manipulate regeneration of the planarian dugesia japonica

自由生活涡虫扁虫有一个历史悠久的实验使用，因为他们非凡的再生能力 1 。从这些动物切除的小片断，改革原体计划，缺少的车身结构的再生。例如，如果一个“主干”的片段是从一个完整的蠕虫切割，一个新的“头”将再生前方一个“尾巴”后方将重新恢复了原有的“头部到尾部的车身结构的极性截肢前（图1A）。 涡虫被称为“neoblasts&#39;〜30种不同类型的细胞分化，在正常的身体动态平衡和执行组织再生的干细胞，是由再生。这种再生过程是可靠和容易证明。由于几个创业实验室，许多工具和功能基因的方法的奉献精神，现在已经为这个模型系统进行了优化。因此，相当最近已经取得了进展，在理解和操作基础 2-9涡虫的发育可塑性的分子事件。 涡模型系统将会以一个科学家的广泛利益。神经学家，模型给予机会研究整个神经系统的再生，而不是简单的再生/修复单个神经细胞的过程中，通常是在许多已建立的模型研究的重点。涡虫表达的神经递质10过多，代表的一项重要制度，为研究中枢神经系统的11，12的演变，行为甄别潜在的13，14 。 再生的结果是经得起药理和遗传apparoaches操纵。例如，可以进行筛选药物对再生的影响，只需放置在身体片段的药物含截肢后在不同时间点的解决方案。使用击倒方法（ 在体内的RNAi），这可以实现，通过显微注射周期或喂食细菌表达双链RNA的结构8，9，15，可以研究单个基因的作用。这两种方法都可以产生高外显率，例如，双极动物16-21再生的视觉冲击的表型。为了便于采用这种模式和实施这种方法，我们将展示使用涡虫日本三角涡虫的药理学和基因检测（在体内RNAi技术通过喂食）的视频在这条协议。

Watch this Scientific Journal Video about Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica at JoVE.com

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

在一个活的动物研究干细胞分化的一个有吸引力的模式是涡虫扁虫。再生是由简单的截肢手术，很容易在一个基本的实验室进行的实验研究和药理学和基因（适合<em&gt;在体内</em&gt; RNAi）的操纵，在这篇文章中的协议详细。

药理和功能的遗传分析处理涡虫的再生日本三角涡虫</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

pharmacological-functional-genetic-assays-to-manipulate-regeneration

Research

JoVE Journal

Biology

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

36.7K 次观看.  University of Minnesota Medical School. 在一个活的动物研究干细胞分化的一个有吸引力的模式是涡虫扁虫。再生是由简单的截肢手术，很容易在一个基本的实验室进行的实验研究和药理学和基因（适合在体内 RNAi）的操纵，在这篇文章中的协议详细。

视频: Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

光/暗转换测试小鼠

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

科研

生物学

Cortical Neurogenesis: Transitioning from Advances in the Laboratory to Cell-Based Therapies

皮层神经再生：从实验室中的研究进展过渡到细胞疗法

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

在鸡胚神经诱导的含量为

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

对小鼠高架十字迷宫

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

从成年小鼠海马齿状回的剖析

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

一个反向遗传学方法测试胚胎发育过程中的功能冗余

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

等位基因特异性基因表达检测，测试的遗传协会的功能基础

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

涡固定，局部照射，组织移植

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

测绘细菌功能的网络和途径<em&gt;大肠杆菌</em使用合成基因阵列

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

小规模的核提取物，基因表达机具功能分析

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...