我们感谢圣地亚哥苏亚雷斯的手稿和贝蒂Cowgill援助在摄影的批判性阅读。我们也感谢匿名审稿人的批评性意见。这项工作是由特拉华大学的研究基金的支持。

1。硫酸鱼精蛋白的制备（PS）包被的培养皿<ol><li>通过在50ml锥形管中加入0.5 G PS的50毫升去离子蒸馏水（双蒸2 O）制备硫酸鱼精蛋白（PS）的1％溶液。摇匀在一条长凳上振动筛在室温下1-2小时高速，确保PS完全溶解。此溶液可以储存在4℃下至少3个月（确保在每次使用前，以完全溶解的凝胶样沉淀物）3。 </li><li>取为60毫米×15毫米的聚苯乙烯培养皿套筒，奠定了两个盖子和底部的板凳上。 </li><li>倒在每一道菜1％的PS溶液（两者底部和盖子都可以使用）就足以覆盖表面，留下至少2分钟。剩的PS溶液可在3个月内，贮存于4℃。当多次重复使用</li><li>在盛有蒸馏水（DH 2 O）的烧杯代替PS处理过的菜。离开下运行DH 2 O的烧杯中至少10分钟。 </li><li> PS涂层的菜肴可以立即或风干用于存储。掩护他们，以防止灰尘堆积。它们可以在室温下保存1个月3。 </li></ol> 2。获得海胆配子及固定的鸡蛋在PS上涂盘的显微注射<ol><li>产卵海胆通过摇动或intracoelomic注射高达至1ml 0.5％的KCl诱发的平滑肌收缩，释放配子3（ 图2）。 </li><li>如果动物是雄性，由白色精子所示，收集精子干从动物的表面至1.5 ml管。干燥的精子可以保存在4℃下一个星期没有显著亏损生物学功能。 </li><li>如果动物是雌性，由黄色彩色由于蛋黄含量所指示的，通过浸渍生殖孔在一个充满海水烧杯中收集其配子。这些卵会从生殖孔释放，通过重力沉淀下来。&lt;/ LI&gt; <li>筛选蛋碎片，如使用80微米的尼龙过滤网动物棘。最好的结果是，如果卵产卵后的5-7小时，用于达到。 </li><li> Dejelly鸡蛋： <ol><li>制备100ml的酸性海水约1毫升蛋。用0.5M的柠檬酸在pH 8.0调节pH海水到5.1-5.2。太低的pH值将减少施肥和过高的pH值不会允许卵附着到PS包被的板。 </li><li>加入鸡蛋（不超过1毫升）中，以酸性海水和允许卵定居于冰上10分钟。可选地，除去透明带的可通过蛋轻轻旋转在治疗过程中提供便利。一方面可以有效dejelly S.紫石鸡蛋3-4分钟这样。 </li><li>小心倒出酸性海水，补充新鲜海水，让鸡蛋落户在冰上了。 Dejellied鸡蛋可以储存在冰上直至6小时没有受精的问题</li></ol></LI&gt; </ol> 3。行的蛋<ol><li>通过在10毫升双蒸2 O溶解0.84克3-AT的准备3 -氨基三唑（3-AT，MW = 84.08）1米原液这种解决方案可以被存储在4℃下进行长达6个月。 </li><li>制备3 - 以1mm工作液通过加入50μl的1M储备溶液至50ml预冷的海水。置于冰上的解决方案。 </li><li>准备口吸管（ 图3）鸡蛋的柔和处理： <ol><li>吸头从玻璃微（100×外径1.09毫米）手动拉。握住微量用双手在明火。作为玻璃开始融化，小心地将吸液管的端部在相对的方向。 </li><li>连接一个上拉移液管的塑料管（编号1.14毫米），用封口膜封紧。管子的长度应为约50-70厘米。 </li><li>插入无菌过滤P20或P200的尖管对面的结束玻璃管到用作口片。 </li><li>夹用剪刀来调整前端部的直径的玻璃微量的末端。的最佳直径略微超过蛋（80微米）的直径。 </li></ol></li><li>倒入加入4ml 1毫米3-AT的海水到每个PS-涂层菜（底部或盖）。 </li><li>搭口吸管，把P20/P200尖的嘴和牙齿固定。抽吸鸡蛋，先吸出少量的海水。通过装载的鸡蛋之前有液体柱，人们将有极大的控制口移液技术。 </li><li>定位微量移液器尖端附近的鸡蛋和几百鸡蛋中的吸管轻轻吸出。玻璃微吸管内限制了鸡蛋。 </li><li>排轻轻吹出来的吸管口在移动笔尖在一条直线上dejellied鸡蛋的菜一条直线。排鸡蛋打针之前正确的，因为可能会因为p发生受精的问题rolonged暴露在3-AT的海水3。此外，阳离子性胶粘剂像PS可能是有毒的鸡蛋，这是不可取的，以暴露胚到这些试剂很长时间，特别是如果受精信封已被删除。 </li><li>就用玻璃吸管划鸡蛋的线附近的菜划痕。此刮将是重要的突破注射针根据需要调整溶液的流动。或者，可以从头浇3-AT海水的菜前进行。 </li></ol> 4。海胆受精卵显微注射<ol><li>开启显微注射站。在这里，我们描述了使用FemtoJet喷射系统。 </li><li>用针拉马拉注射毛细血管。一个好的针的一个例子示于图4。注意：对于RNA注射，注射毛细血管应进行预处理，以避免RNA酶污染31： <ol><li>将注射毛细血管进入50ml锥形管和加入50 mL过滤甲醇/盐酸（9:1）的。摇匀并留下过夜不超过10小时以上。 </li><li>倾掉甲醇/ HCl和冲洗毛细管2倍与过滤甲醇在同一管中。 </li><li>漏甲醇尽可能和冻干毛细血管过夜以确保完全除去甲醇。 </li></ol></li><li>小心装入毛细管针上的针保持器加载并以&lt;0.5微升的样品溶液使用微加载提示加载。样品溶液一般含有20％甘油，取决于注射材料3。 </li><li>将菜划鸡蛋放在显微镜阶段（鸡蛋应垂直对齐，通过目镜观察时），并专注于鸡蛋。 </li><li>小心装入装入针到针质注射支架和调整针的位置有一个完全集中在尖端的场的中间。 </li><li>压下按钮应用FO的最大压力的R针清洗。解决方案应该出来了针。如有必要，调整的压力：注射压力应在90-360百帕的范围内，补偿压力大约为注射压力的1/3。 </li><li>通过注入未受精的卵子调节溶液流。使用操纵杆显微操作，直接注射针的未受精的卵的小费。为了便于落针，用硬物如螺丝刀舞台轻拍创建一个轻微的颤抖。一个注射小丸形成蛋内应该被看作（ 图5）。 </li><li>如果注射小丸是不可见的，稍微抬高以上鸡蛋的针尖，找到划痕上的菜，并调整它被放置在场地中央。轻轻拍打针尖划伤大关注射针的尖端。调整注入和补偿压力，以确保注射小丸不超过的1/5吨的他径蛋（下午1-2点）的。 </li><li>一旦注入丸校准，加入稀释的精液（1:1000）或1-2微升精子受精的卵子。拌匀小心，以防止撞出鸡蛋的行。 </li><li>开始注射后立即受精信封变得可见（ 图5）。待着使用阶段控制器鸡蛋划的线，并通过由操纵杆控制的显微操作针戳他们注入许多受精卵越好。 </li><li>后10〜15分钟后，将新的受精卵将变得硬化并不能注射任何更长的时间。从舞台中删除的菜，仔细用塑料巴斯德吸管吸出精子在3-AT的海水。 </li><li>不要让受精卵干。迅速补充新鲜预冷的海水覆盖的菜。孵化的胚胎在15℃。 </li></ol>

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作者宣称没有竞争的经济利益或其他的利益冲突。

显微注射法是一种强大的技术用于递送各种治疗方法，如DNA，mRNA表达，重组蛋白，功能丧失和增益的功能的试剂，染料和它们的组合成蛋，胚胎，和各种生物1-7细胞。然而，设计一个显微注射实验的时候几方面的考虑，应牢记。 这是极为重要的是要考虑的递送治疗和注射量的溶解性。如果显微注射液趋向于形成即使是轻微的沉淀，在质注射针将被堵塞。一般来说，?...

高效的和可重复的治疗分娩是为研究人员的主要方法上的挑战之一。几种方法已经被建立以瞬时传送处理溶液进入卵，胚胎，和细胞。这些方法包括电穿孔（基于使用短的电脉冲的产生瞬态毛孔中的膜）1,2，脂质转染（输送通过含有治疗的脂质体与细胞膜的融合体）1，微粒轰击1（沉淀DNA上的微米这随后被用来穿透细胞在高流速大小的金属颗粒），和转导（病毒被用作转基因的递送媒介物）。此刻，显微注射是保存提供任何溶液与100％的效率以最小的试剂的优点的唯一方法。此外，单一的注射液可组成一个处理复杂的鸡尾酒。这一技术已被用于成功LY微导的卵子和胚胎从众多种类，例如海胆3,4，斑马鱼5，鼠标器6，青蛙7，和牛8以及单个细胞在组织培养9。单个卵裂球在注射后的发育阶段也已经进行了10-12。 显微注射的当前方法是基于最初由平本10所述的压力注入法，但是，大的进展已经取得了这一过程的优化。优良的显微注射技术已在别处11描述了，这里我们介绍的是目前用于微导海胆（ 紫色球海胆 ）新受精卵的具体方法之一。一个多世纪以来，海胆是一个有价值的实验模型15,16。海胆是进化密切相关的脊索动物（包括我们），并分析它们的基因组发现，它们包含所有主要的基因家族的人17。他们生产了大量的同步开发，可以很容易地操纵胚胎透明的。利用海胆作为模式生物，海胆社会对我们的受精过程的理解18-21，细胞生物学过程22-24和基因调控网络（GRNS）25-28贡献。 显微注射到海胆受精卵需要几个步骤。首先，鸡蛋需要注射之前（在下面描述），固定。显微注射的菜肴都涂有硫酸鱼精蛋白（PS），它创建了一个带正电荷的表面到带负电荷的蛋能坚持3。将卵孵育在酸性海水（pH值5.15）10分钟dejellied，随后在天然海水或人工海水（pH8.0）中洗涤两次。该dejellied鸡蛋小心划在一条直线上在1mM的3 -氨基三唑（3-AT），这是需要抑制是从卵的皮质颗粒分泌为受精29的结果ovoperoxidase的活性存在PS涂 ​​覆盘的中间。这个步骤是重要的，以防止受精信封的硬化，并促进微注射针条目。作为替代，以1mM的3-AT，10毫对氨基苯甲酸（PABA），都可以使用。注射溶液装入用专门的微负载枪头微注射针以及安装在连接到显微操纵器和压力单元的保持器（ 图1）。每根针可以用于微导合子个体中在不同的日子多个实验。可10-15分钟进行显微注射​​，直至受精卵变硬。受精卵然后用海水和培养在15℃下当到达胚胎孵化囊胚期，它们会释放孵化酶，消化外汇储备的组成部分tilization信封30，让他们自然地从PS-涂层盘分离。如果需要的话，将胚胎可以轻轻地从用口吸移管或巴斯德吸管海水轻轻地吹入胚胎培养皿分离。所描述的方法使得能够在一个单一的盘100-400新近受精的卵有效和可靠的显微注射，用于下游分析提供了一种高通量的方法。

<table border="0" cellpadding="0" cellspacing="0" style="width:793px;" width="794">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass pasteur pipettes</td>
			<td style="width:200px;">VWR</td>
			<td style="width:211px;">14673-043</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Inverted microscope Axiovert 40C</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:211px;">4109431007990000</td>
			<td>Injection microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microloader tips</td>
			<td style="width:200px;">Eppendorf</td>
			<td style="width:211px;">5242 956.003</td>
			<td style="width:167px;">Load injection solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nylon filter mesh 80 &#956;m</td>
			<td style="width:200px;">Amazon.com</td>
			<td style="width:211px;">03-80-37</td>
			<td style="width:167px;">Filter eggs to get rid of debris</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">P20 or P200 Aerosol Barrier Pipette Tips</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:211px;">02707432 or 02707430</td>
			<td style="width:167px;">Part of a mouth pipette</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Parafilm</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:211px;">13 374 12</td>
			<td style="width:167px;">Part of a mouth pipette</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polyethylene tubing</td>
			<td style="width:200px;">Intramedic</td>
			<td style="width:211px;">PE-160</td>
			<td style="width:167px;">Part of a mouth pipette</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Protamine sulfate</td>
			<td style="width:200px;">MP Biomedicals, LLC</td>
			<td style="width:211px;">194729</td>
			<td style="width:167px;">Attach dejellied eggs to injection dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sea urchins S. purpuratus</td>
			<td>Pt. Loma Marine Invertebrate Lab</td>
			<td style="width:211px;">N/A</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sea water</td>
			<td style="width:200px;">any pet store</td>
			<td style="width:211px;">Instant Ocean</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sterile 60 mm x 15 mm Polystyrene Petri Dish</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:211px;">0875713A</td>
			<td style="width:167px;">Injection dishes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Three-Axis Coarse Positioning Micromanipulator MMN-1</td>
			<td style="width:200px;">Narishige</td>
			<td style="width:211px;">9124</td>
			<td>Manipulate injection needle</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Three-Axis Joystick Type Oil Hydraulic Fine Micromanipulator MMO-202ND</td>
			<td style="width:200px;">Narishige</td>
			<td style="width:211px;">9212</td>
			<td>Manipulate injection needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Transfer pipettes</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:211px;">13-711-9AM</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vertical needle puller </td>
			<td style="width:200px;">Narishige</td>
			<td style="width:211px;">PC-10</td>
			<td>Pull injection needles</td>
		</tr>
	</tbody>
</table>

high throughput microinjections of sea urchin zygotes

显微注射到细胞和胚胎是用来研究广泛的生物学过程的一种常用技术。在该方法中少量的处理溶液被装入一个用于物理地注入单个固定的细胞或胚​​胎显微注射针。尽管需要对初始训练才能执行此程序用于高通量输送，显微注射提供最高的效率和可重复性交付各种各样的治疗方案（包括样品的复杂混合物）进入细胞，卵子或胚胎。应用显微注射包括递送的DNA构建体，mRNA表达，重组蛋白，增益功能，以及功能的试剂的损失。荧光或比色染料被加入到注射溶液，以使有效地提供实时可视化，以及提供可靠的递送溶液的量的归一化的工具。所描述的方法，使产生100-400海胆Ž显微注射在10-15分钟ygotes。

GFP和的mCherry记者构造进行体外转录和显微注射到新的受精卵。胚培养在15℃下放置24小时（直到囊胚期），并使用蔡司观察Z1显微镜成像。注射记者构造并没有导致任何发育缺陷（ 图6）。用于荧光信号的量化，图像采集是在低放大倍数（100X）进行，以最大程度地捕获荧光像素（ 图6D-F）。荧光信号使用的AxioVision 4.8.2.0定量。供的荧光信号中的100-200囊胚人口的强度?...

Watch this Scientific Journal Video about High Throughput Microinjections of Sea Urchin Zygotes at JoVE.com

High Throughput Microinjections of Sea Urchin Zygotes

显微注射是用于递送DNA构建体，mRNA表达，吗啉代反义寡核苷酸或其它处理成卵，胚胎，以及不同物种的细胞中的常见技术。

海胆受精卵的高通量显微注射

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

high-throughput-microinjections-of-sea-urchin-zygotes

Research

JoVE Journal

Biology

14.4K 次观看.  University of Delaware. 显微注射是用于递送DNA构建体，mRNA表达，吗啉代反义寡核苷酸或其它处理成卵，胚胎，以及不同物种的细胞中的常见技术。 

视频: High Throughput Microinjections of Sea Urchin Zygotes

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography.  We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

斑马鱼幼虫的研究急性肾损伤的静脉Microinjections

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections ...

科研

生物学

High-throughput Yeast Plasmid Overexpression Screen

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

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

高通量酵母质粒过表达屏幕

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

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

木质纤维素材料的高通量检测糖化

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

流式细胞仪分离纯化<em&gt; S.酵母</em&gt;合子

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

萤光素酶表达的重组病毒的高通量滴定

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

A Method for Microinjection of Patiria minata Zygotes

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

A Method for Microinjection of Patiria minata Zygotes

Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks. The sea star Patiria miniata is an emerging model system for these studies. Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species.

用于显微注射的方法<em&gt; Patiria minata</em&gt;合子

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

恢复成年斑马鱼的心脏的高通量应用

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

激酶底物对鉴定使用高通量筛选


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...