我们感谢 Caenorhabditis 遗传学中心提供本研究中使用的菌株。本出版物得到了 JSPS KAKENHI 科学研究补助金 （B）（资助号 JP21H02532）、JSPS KAKENHI 创新领域“软机器人科学”项目补助金（资助号JP18H05474）、JSPS KAKENHI 变革性研究领域 B 补助金（资助号JP23H03845）、日本医学研究开发机构 PRIME（资助号 JP22gm6110022h9904）、JST-Mirai 计划（资助号 JPMJMI22G3）、 和 JST-FOREST 计划（批准号 JPMJFR214R）。

1.蠕虫的制备
注：制备野生型N2布里斯托尔菌株12 和ZX899菌株（lite-1（ce314）;ljIs123[mec-4p：：ChR2，unc-122p：：RFP]）13 分别用于观察集体行为和光遗传学实验。在黑暗条件下保持 ZX899 菌株。
<ol><li>将四条喂养良好的成虫沉积在含有14mL线虫生长培养基（NGM）的60mm板上，并接种大 肠杆菌 OP5012。</li>
	<li>在23°C的NGM板中将F1蠕虫培养至饥饿7天。此时，F1 蜗杆的产量约为 100 只蜗杆/板。蠕虫的各个阶段包括 dauer 和饥饿的 L1 幼虫的混合种群。</li>
</ol>2.狗粮琼脂（DFA）培养基板的制备
<ol><li>高压灭菌含有2g狗粮粉和5mL 1%琼脂培养基的玻璃瓶，并将其冷却至室温（图1A）。 注意：本实验可以使用来自不同制造商的其他狗粮。</li>
</ol>3. 将蠕虫接种到 DFA 培养基板上
<ol><li>将少量（约0.5g）DFA培养基转移到接种有 大肠杆 菌OP50的NGM板的中心（图1B）。对于光遗传学实验，在接种蠕虫之前，将 40 μL 50 μM 全反式视网膜醛（通道视紫红质 2 的辅助因子）倒入 DFA 上。</li>
	<li>使用高压灭菌水从四个NGM板中收集饥饿的蠕虫。</li>
	<li>将一小块狗粮（约0.5g）放在DFA培养基上，距离板盖约2mm。</li>
	<li>在洁净的工作台内用紫外线照射 NGM 板 15 分钟，以防止污染。</li>
	<li>将收集的蠕虫（约400条蠕虫）接种到NGM板上的DFA培养基上。不要用封口膜密封板，以免增加培养皿内的湿度并产生水滴，将蠕虫困在盖子上。</li>
	<li>在23°C下繁殖蠕虫，并让它们爬到板盖上约10-14天。 注意：由于盖子上的蠕虫数量在 10-14 天后几乎没有增加，因此推测蠕虫可能已经耗尽了食物。</li>
</ol>4. 集体行为的观察
<ol><li>在实验当天，将不包括 大肠杆菌 和狗粮琼脂培养基的新NGM板放在微距变焦显微镜载物台上的铝板上（图2A）。使用Peltier温度控制器将这种新的NGM板的底部保持在23°C至少5分钟（图2B）。然后，将这个新的NGM板的盖子换成蠕虫爬上去的板盖。使用物镜（x2，NA = 0.5）作为低倍率物镜（图2A）。</li>
	<li>将培养皿底部的温度从23°C提高到26°C，以改变该板内的湿度（图2）。</li>
	<li>使用相机以 20 帧 s-1 采集板盖内表面的图像（补充视频 S1）。</li>
	<li>将采集的图像保存为标记图像文件格式。</li>
</ol>5. 光遗传学实验
<ol><li>使用 100 W 汞灯提供蓝光，并用滤光片组过滤。使用电磁快门系统精确控制照明时间（图2B）。</li>
	<li>在这些条件下将 ZX899 在 DFA 上保持 5 分钟，然后再亮起蓝光。</li>
	<li>在保持在23°C的显微镜载物台上照亮附着在培养皿盖上的ZX899蠕虫。</li>
	<li>使用相机以 20 帧 s-1 采集板盖内表面的图像（补充视频 S2）。</li>
</ol>

使用狗粮琼脂培养基研究线虫的集体行为

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作者没有利益冲突需要声明。

在这项研究中，我们展示了一种在实验室中为秀丽隐杆线虫的大规模集体行为准备系统的方案。基于DFA的方法最初是用Caenorhabditis japonica14和Neoaplectana carpocapsae Weiser15建立的，两者都是非模式动物。然而，这种方法并未应用于调查集体行为。秀丽隐杆线虫是一种遗传上可处理的模式动物11,12?...

非科学家和科学家都对动物的集体行为着迷，比如成群的鸟和鱼群。集体行为已经在广泛的领域进行了分析，包括物理学、生物学、数学和机器人技术。特别是，活性物质物理学是一个不断发展的研究领域，专注于由自走式元素组成的系统，即耗散系统，如鸟群、鱼群、运动细菌的生物膜、活性分子组成的细胞骨架和自走胶体组。活性物质物理学理论认为，无论个体的行为多么复杂，大量生物的集体运动都受到少数简单规则的支配。例如，Vicsek模型是统一描述自走式粒子集体运动的候选者，它预测运动物体的短程对准相互作用需要形成具有二维偏心波动的长程有序相位，就像在动物群中一样1。与活性物质物理学有关的自上而下的实验方法正在迅速发展。先前的实验证实了大肠杆菌2中长程有序相的形成。最近的其他工作使用了细胞3,4，细菌5，运动胶体6或移动蛋白质7,8。简单的最小模型（如Vicsek模型）成功地描述了这些真实现象。与这些单细胞实验系统相比，动物的集体行为通常是在野外观察到的，因为没有人希望对10,000只真正的鸟类或鱼类进行对照实验。
生物学家与物理学家有着相同的兴趣：个体如何相互影响，如何作为一个群体在功能上表现。分析个体行为的传统研究领域之一是神经科学，其中行为背后的机制已经在神经元和分子水平上进行了研究。到目前为止，已经开发了许多神经科学自下而上的方法。物理学中的自上而下方法和生物学中的自下而上方法可以使用模式动物（如果蝇、 蠕虫秀丽隐杆线虫和小鼠）来促进 9.然而，在实验室中，关于这些模式动物的大规模集体行为的发现很少10;在实验室中大规模制备遗传上可处理的模式动物仍然很困难。因此，在目前生物学和物理学中对集体行为的研究中，通常在实验室进行研究的科学家很难研究动物的集体行为。
在这项研究中，我们建立了一种大规模培养线虫的方法，以研究它们的集体行为。该系统使我们能够改变环境条件，并使用突变体10 检查个体水平运动对集体行为的影响。在活性物质物理学中，数学模型的参数可以在实验和模拟中控制，从而可以验证该模型以识别统一描述。遗传学用于理解集体行为背后的神经回路机制11.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Escherichia coli and C. elegans strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli OP50</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>OP50</td>
			<td>Food for C. elegans. Uracil auxotroph. E. coli B.</td>
		</tr>
		<tr>
			<td>lite-1(ce314); ljIs123[mec-4p::ChR2, unc-122p::RFP]</td>
			<td>author</td>
			<td>ZX899</td>
			<td>lite-1(ce314) mutant carrying the genes expressing ChR2 and RFP under the control of the mec-4 and unc-122 promoter, respectively</td>
		</tr>
		<tr>
			<td>N2 Bristrol</td>
			<td>Caenorhabditis Genetics Center</td>
			<td></td>
			<td>Wild-type C. elegans strain</td>
		</tr>
		<tr>
			<td>For worm cultivation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agar purified, powder</td>
			<td>Nakarai tesque</td>
			<td>01162-15</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>All-trans retinal</td>
			<td>Sigma-Aldrich</td>
			<td>R2500</td>
			<td>For optogenetics</td>
		</tr>
		<tr>
			<td>Bacto pepton</td>
			<td>Becton Dickinson</td>
			<td>211677</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Wako</td>
			<td>036-00485</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Wako</td>
			<td>034-03002</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>di-Photassium hydrogenphosphate</td>
			<td>Nakarai tesque</td>
			<td>28727-95</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>Dog food</td>
			<td>Nihon Pet Food</td>
			<td>VITA-ONE</td>
			<td>For preparation of dog food agar medium</td>
		</tr>
		<tr>
			<td>LB broth, Lennox</td>
			<td>Nakarai tesque</td>
			<td>20066-95</td>
			<td>For culture of E. coli OP50</td>
		</tr>
		<tr>
			<td>Magnesium sulfate anhydrous</td>
			<td>TGI</td>
			<td>M1890</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>Petri dishes (60 mm)</td>
			<td>Nunc</td>
			<td>150270</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>Potassium Dihydrogenphosphate</td>
			<td>Nakarai tesque</td>
			<td>28720-65</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Nakarai tesque</td>
			<td>31320-05</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>Observation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>CT solution</td>
			<td>CS6229</td>
			<td>Windows10 Pro with Intel Xeon Gold 6238R CPU and 768 GB of RAM</td>
		</tr>
		<tr>
			<td>CMOS Camera</td>
			<td>Hamamatsu photonics&#160;</td>
			<td>ORCA-Lightning C14120-20P</td>
			<td>For data acquisition</td>
		</tr>
		<tr>
			<td>CMOS Camera</td>
			<td>Olympus</td>
			<td>DP74</td>
			<td>For data acquisition</td>
		</tr>
		<tr>
			<td>Microscope with SZX-MGFP set</td>
			<td>Olympus</td>
			<td>MVX10</td>
			<td>For data acquisition</td>
		</tr>
		<tr>
			<td>x2 Objective lens</td>
			<td>Olympus</td>
			<td>MV PLAPO 2XC</td>
			<td>Working distance 20 mm and numerical aperture 0.5</td>
		</tr>
		<tr>
			<td>Shutter control</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Shutter</td>
			<td>OptoSigma</td>
			<td>BSH2-RIX</td>
			<td>For controlling temporal pattern of&#160; light illumination</td>
		</tr>
		<tr>
			<td>Shutter controller</td>
			<td>OptoSigma</td>
			<td>SSH-C2B-A</td>
			<td>For controlling temporal pattern of&#160; light illumination</td>
		</tr>
		<tr>
			<td>Temperature control</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Peltier temperature controller unit</td>
			<td>VICS</td>
			<td>WLVPU-30</td>
			<td>For controlling humidity inside a Petri plate</td>
		</tr>
		<tr>
			<td>UNI-THEMO CONTROLLER</td>
			<td>Ampere</td>
			<td>UTC-100</td>
			<td>For controlling humidity inside a Petri plate</td>
		</tr>
		<tr>
			<td>Data acquisition software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HCImage</td>
			<td>Hamamatsu photonics</td>
			<td></td>
			<td>For data acquisition</td>
		</tr>
	</tbody>
</table>

the large-scale cultivation of nematodes to study their collective behaviors

动物表现出动态的集体行为，如在成群的鸟类、鱼群和成群的人类中观察到的那样。动物的集体行为已经在生物学和物理学领域进行了研究。在实验室中，研究人员使用各种模式动物（如果蝇和斑马鱼）大约一个世纪，但研究这些基因可处理的模式动物精心策划的大规模复杂集体行为仍然是一个重大挑战。本文提出了一种在 秀丽隐杆线虫中创建集体行为实验系统的协议。繁殖的蠕虫爬上培养皿的盖子，并表现出集体蜂拥行为。该系统还通过改变湿度和光刺激来控制蠕虫的相互作用和行为。该系统使我们能够通过改变环境条件和使用突变体检查个体水平运动对集体行为的影响来检查集体行为背后的机制。因此，该系统对物理学和生物学的未来研究都很有用。

在这里，野生型dauer蠕虫被用于集体行为观察。将蠕虫在23°C下培养约10-14天，并爬到DFA培养基板盖的内表面。在实验当天，仅将盖子转移到没有 大肠杆菌 和DFA培养基的新NGM板上。该培养皿的底部最初使用帕尔贴系统保持在23°C，然后将其温度提高到26°C。 在显微镜下拍摄了一部电影。 图 3 显示了影片的快照。蠕虫在湿度变化期间动态重塑了它们的网络模式。随着湿?...

Watch this Scientific Journal Video about The Large-Scale Cultivation of Nematodes to Study Their Collective Behaviors at JoVE.com

作者聚焦：线虫 Caenorhabditis elegans 的集体行为分析

在这里，报道了一种通过使用狗粮琼脂培养基大量培养线虫来研究线虫集体行为的系统。该系统允许研究人员繁殖大量的dauer蠕虫，并可应用于 秀丽隐杆线虫 和其他相关物种。

大规模培养线虫以研究其集体行为

Animals exhibit dynamic collective behaviors, as observed in flocks of birds, schools of fish, and crowds of humans. The collective behaviors of animals ...

我们正在从活跃的形而上学和神经科学的角度研究集体行为背后的机制。在集体行为的研究领域，已经建立了使用非生物和细胞水平的实验系统。然而，设计动物水平的实验系统来研究实验室中的集体行为仍然具有挑战性。我们成功地用传统使用的模式动物线虫秀丽隐杆线虫重建了集体行为。该系统可以利用动物集体行为的数学模型控制参数，并利用分子遗传学研究集体行为背后的神经机制。

the-large-scale-cultivation-nematodes-to-study-their-collective

Research

JoVE Journal

Biology

The Large-Scale Cultivation of Nematodes to Study Their Collective Behaviors

594 次观看.  Hiroshima University. 我们正在从活跃的形而上学和神经科学的角度研究集体行为背后的机制。在集体行为的研究领域，已经建立了使用非生物和细胞水平的实验系统。然而，设计动物水平的实验系统来研究实验室中的集体行为仍然具有挑战性。我们成功地用传统使用的模式动物线虫秀丽隐杆线虫重建了集体行为。该系统可以利用动物集体行为的数学模型控制参数，并利用分子遗传学研究集?...

视频: 作者聚焦：线虫 Caenorhabditis elegans 的集体行为分析

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

宏基因组库的大型屏幕

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

计算机生成的动物模型的刺激

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the expression pattern of a target protein across the entire brain are relatively easy to answer when using IHC in small brains, such as those of rodents. However, answering the same questions in large and convoluted brains, such as those of primates presents a number of challenges. Here we present a systematic approach for immunodetection of target proteins in an adult monkey brain. This approach relies on the tissue embedding and sectioning methodology of NeuroScience Associates (NSA) as well as tools developed specifically for batch-staining of free-floating sections. It results in uniform staining of a set of sections which, at a particular interval, represents the entire brain. The resulting stained sections can be subjected to a wide variety of analytical procedures in order to measure protein levels, the population of neurons expressing a certain protein.

Large-scale immunodetection of target proteins across the entire primate brain is possible by employing novel tissue embedding and sectioning methods combined with the use of creative apparatus for batch staining of multiple free-floating sections at a given time.

在整个猴脑进行大规模蛋白质检测的批次免疫染色

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to mammals, zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

斑马鱼为基础的大型在体内小分子屏幕

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

Infinium检测的大型SNP基因分型应用

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

在大规模的基因敲除<em&gt; C。线虫</em&gt;使用双链RNA料库产生失去功能的鲁棒表型

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

敏感，大规模定量代谢组学的策略

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

ODELAY:用于酵母生长多参数定量的大规模方法

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...