这项工作是由美国国立卫生研究院R01 HG002995支持“翻耕斑马鱼基因组：一个反向遗传学方法”煤层气。

A部分：精子冷冻解决方案 1。金斯伯格鱼林格（做出新的，每3天;金斯伯格，1963年） 注：添加顺序重要的是要防止沉淀 在450毫升无菌ddH2O溶解： <ul><li>氯化钠3.25克</li><li>氯化钾0.125克</li><li>氯化钙•2水0.175克 然后添加： </li><li>碳酸氢钠每股面值0.10克</li></ul>使终体积为500毫升无菌ddH2O和储存在4 ° C 2。冻存液（请每日新鲜） <ol style="list-style-type:lower-roman"><li>无甲醇<ul><li>金斯伯格鱼林格10毫升（室温）。 </li><li>脱脂奶粉1.5 GM </li></ul></li><li>用甲醇 注：添加顺序重要的是要防止沉淀 <ul><li>金斯伯格鱼林格9毫升（室温） </li><li>甲醇1毫升</li><li>脱脂奶粉1.5克</li></ul> 组装后冻结的媒体，拌匀20分钟。轨道摇床或摇杆。在使用之前，分装成1毫升的Eppendorf管中，避免表面气泡。 </li></ol> B部分：精子冷冻所需的材料精子冷冻所需的材料<ol><li>一次性移液器10μL（Fisherbrand CAT＃22-358697） </li><li>鱼水250毫升的烧杯中包含MESAB / Tricaine </li><li>钟表眼镜（耐热猫＃9985-75） </li><li> P20的pipetman（或同等学历）和提示</li><li>海绵鱼持有人（持有男性，而挤压） </li><li>以上舞台灯光与解剖显微镜</li><li>扁钳（如微孔型） </li><li> 2.0毫升低温瓶（康宁猫＃430488） </li><li> 10X10 cryoboxes（猫NALGENE＃03 - 337 - 7AA） </li><li>发泡胶容器充满〜15厘米，细粉碎的干ICE1 </li><li> 15 ml锥形管（猎鹰＃352099） </li><li>含有液氮的杜瓦瓶</li><li>长钳（如CMS费舍尔保健猫＃10 - 316B） </li><li> Cryogloves </li><li>男性的回收罐</li><li>一个Eppendorf管中充满冻结无甲醇媒体</li><li>一个Eppendorf管中充满冻结媒体用甲醇</li><li> Cryofreezer（如泰勒 - 沃顿K系列） </li></ol> 1一个简单的方法是制作精细粉碎的干冰包裹在毛巾和粉碎锤。更有效的方法是使用干冰磨床，如克劳森模型RE - 2。 C部分：精子冷冻法<ol><li> 标记毛细管：开始前，马克与实验室笔的水平为3.33μL加入10μl毛细管（即16.7毫米，从底部，见图1.1） 。 </li><li> 男：麻醉包含MESAB / tricaine鱼水稀释（配方“的斑马鱼书”）进行烧杯中的男性（S ） </li><li> 干鱼：麻醉后，解除鱼的麻醉剂使用塑料勺轻轻吸干鱼纸巾上晾干，要特别注意干燥的腹侧方。水激活精子，因此重要的是彻底干燥泄殖腔周围。位置鱼海绵持有人，腹的一面向上（图1.2）。如果臀鳍周围地区仍是湿润，轻轻地用kimwipe干。小心，不要挤精子过早！ </li><li>放置标记在橡胶口吸管适配器，包括毛细管毛细管。适配器是橡胶软管，有一端的喉舌和毛细血管上的其他持有人。另外，毛细管，可以连接到一个汉密尔顿50μL注射器通过适当直径的聚乙烯管的一小段。 </li><li>男下的范围和揭露仔细传播腹鳍，除了使用毛细管年底泌尿生殖系统的开放。 </li><li> 收集精子：驱逐鱼的两侧轻轻地抚摸着顺利钳（如Millipore公司）的精子，或你的食指和拇指之间的鱼的两侧轻轻挤压。在毛细管收集精子，因为它是被驱逐用温和的吸力（图1.2）。避免可能与精子排出的粪便。 </li><li> 正常化精子量为3.3μL：如果精子到达量，或超过毛细管（即16.7毫米或更大）的笔标记，然后继续执行步骤8 。如果精子量没有达到目标量，然后正常化音量笔商标使用无甲醇（图1.3）冻结中等。精子的最低金额是可以接受的，与精子质量的变化。良好的精子是白色的，不透明的。可怜的精子看起来水汪汪的。在一般情况下，我们接受的最小值：1μL良好的精子（或1 / 3的目标）和2μl差的精子（或2 / 3的目标）。卷已归至3.3μL。 </li><li> 地址冷冻保护剂对精子：大吃冻结与M中等乙醇橙色标志（约总体积为20μL;图1.4）。驱逐到了手表的玻璃洁净区精子冷冻保护剂的混合物，要特别注意不要引入气泡。轻轻吹打向上和向下〜4倍（避免引入气泡）混合。 </li><li> 放到各2离心管加入10μl精子：吸取10μL的精子冷冻保护剂的解决方案：到已与相关信息（图1.5）标记的两个单独的离心管底部。快速移动，盖瓶和拖放到室温15毫升猎鹰管一离心管/管底部。第猎鹰管和一双猎鹰含离心管，管插入粉碎的干冰。 </li><li> 冻结精子20分钟。干冰：干冰应在细粉末状，没有颗粒。管插入深度不够，只是帽子秀（图1.6）的干冰。为了保持在干冰管的轨道，给每个一双猎鹰管数量从1-20（上限书面），并记录时间，他们的干冰。 </li><li> 地点，液氮冷冻管后的精子已经在20分钟的干冰冷冻，他们转移到含液氮杜瓦真空瓶。直到放入液态氮冷冻，精子是存储在这里。当放置在冷冻箱，放置在液氮，使样品不升温洗澡冷冻箱。使用长的金属钳，恢复杜瓦真空瓶小瓶和处理cryogloves。此外，离心管，可直接传送到冷冻箱，冷冻箱保持沉浸在泡沫塑料容器液氮干冰。 </li><li> 商店冷冻管长期在低温液态氮冷冻 。为了保持精子活力，重要的是保存在液氮中浸泡小瓶。根据我们的经验，在气相中存储的小瓶随着时间的推移失去生存能力。 速度是很重要的！加入冷冻保护剂和精子小瓶放置在干冰（即步骤6-10）之间的时间应不超过30秒 。 </li></ol> D部分：使用冻存精子体外受精方法我们发现，这个最好两个人一起工作。一人挤压从女性的卵子，而另一部分人解冻的精子。 <ol><li>设置水浴至33 ° C。 </li><li>取出离心管从液氮冷冻和转移到液态氮中，直到准备解冻杜瓦真空瓶。 </li><li>从女性挤入35毫米塑料培养皿的鸡蛋。如果可能，尽量获得3个鸡蛋离合器和合并成一盘。如果你不能得到您的第一个离合器1分钟内的三个好离合器，然后进行到第4步（一个很好的离合器就足够了）。好离合器的定义：&gt; 150均匀漂亮的蛋（即偏黄，没有退化的白色碎片指示）。保持覆盖精子解冻时菜。 </li><li>从液氮中取出小瓶，并删除第。快速浸入小瓶〜1 / 2到33 ° C为8-10秒水浴方式。 </li><li>快速添加70μL房间温度汉克斯吹打向上和向下的小瓶和混合。立即加入鸡蛋和枪头轻轻混匀。 </li><li>毫不拖延地加入750μL鱼水，激活精子和卵子。涡流来混合。在室温下孵育5分钟。 </li><li> 5分钟后，填写鱼水和孵化盘菜在28 ° C。 2-3小时后，计数和肥沃的胚胎转移到（50/dish）百毫米菜。不孕鸡蛋计数，使施肥频率可确定。 </li><li>幼虫以照顾好！第5天，每天换水，并去除死胚胎。只放幼虫已进入第5天育苗鱼鳔。 </li></ol> E部分：代表结果我们用这种crypreservation方法，以弥补2001年和2003年之间的园艺8600冷冻精子样本库。我们已经解冻这些样品106个，并确定他们的生育能力。生育率是可行的胚胎在体外与冷冻精子受精的卵子的总数除以在施肥的比例。新鲜（非cryporeserved）精子通常&gt; 95％的生育能力;冻存精子的生育率低得多：平均29％（N = 106瓶）。生育率的小瓶，小瓶变异大，范围从不到1％到高达62％的高。这种变异是代表图的标准差较大的误差酒吧。 2。然而，在106个样本的精子解冻日期只有一次，我们有经验丰富的0％生育两个精子样本，未能收回相应的TILLING突变。 从我们在过去的几年中库解冻精子小瓶（2001-2008），我们发现，通过这种方法冷冻精子样本的平均生育率仍随着时间的推移，稳定（图2）。在2001年的冷冻精子解冻样品的平均生育率为23.5％（基于11个精子样本），并于2008年为25％（基于21个精子样本）。 其他一些调查，现在使用这个精子冷冻协议，在他们的实验室。有些报道与生育率和类似我们所观察到的样品，样本变异良好的成功。据报道，其他未能使本议定书的工作在自己手中。我们认为，可能的来源的问题： <ol><li>未使用的确切的离心管或锥形管，我们已经在协议的建议，导致一个不同的速率在干冰冷却; </li><li>不使用粉末状干冰，导致次优的冷却速度; </li><li>使用差生育雄性鱼：你应该从一个单一的男性至少有1μL精子。还可以测试男性生育能力与新鲜精子体外授精，但是这可能会造成误导，因为它需要很少的新鲜精子得到接近100％生育。 </li><li>使用不合标准的优质卵子在体外受精。使用任何东西，但完美的离合器总是导致生育率接近0％水平，即使是从精子样本，具有良好的生育能力（重复小瓶的生育率为基础）。 </li></ol><img src="/files/ftp_upload/1395/1395fig1.jpg" alt="图1" /> 图1。精子冷冻方法的示意图。 <img src="/files/ftp_upload/1395/1395fig2.jpg" alt="图2" /> 图2。冻存精子生育仍然随着时间的推移稳定。 ％的生育是可行的胚胎与冷冻精子受精的卵子的总数除以在体外授精产生。 “N”指的冻存精子的小瓶，其生育能力测试是在相应年份数量。误差线表示标准偏差。由于每个从不同的男性的精子小瓶，瓶，小瓶的变异观察。

<ol>
	<li>Ginsburg, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm-egg+association+and+its+relationship+to+activation+of+the+egg+in+salmonid+fishes.">Sperm-egg association and its relationship to activation of the egg in salmonid fishes.</a> J. Embryol. Exp. Morphol. 11, 13-33 (1963).</li><li>Harvey, B., Kelley, R. N., Ashwood-Smith, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+zebra+fish+spermatozoa+using+methanol.">Cryopreservation of zebra fish spermatozoa using methanol.</a> Can. J. Zoology. 60, 1867-1870 (1982).</li></ol>

其他一些调查，现在使用这个精子冷冻协议，在他们的实验室。有些人报告了类似的生育率和我们所观察到的同一样品，样本变异。据报道，其他未能使本议定书的工作在自己手中。我们认为，可能的来源的问题： <ol><li>未使用的确切的离心管或锥形管，我们已经在协议的建议，导致一个不同的速率在干冰冷却; </li><li>不使用粉末状干冰，导致次优的冷却速度; </li><li>使用差生育雄性鱼：你应该从一个单一的男性至少有1μL精子。还可以测试男性生育能力与新鲜精子体外授精，但是这可能会造成误导，因为它需要很少的新鲜精子得到接近100％生育。更大，更有力彩色的男性通常会提供更多更高质量的精子。 </li><li>使用不合标准的优质卵子在体外受精。使用任何东西，但完美的离合器总是导致生育率接近0％水平，即使是从精子样本，具有良好的生育能力（重复小瓶的生育率为基础）。 </li></ol>

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>10 &mu;l disposable pipettes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-358697</td>
<td>&nbsp;</td>
<tr>
<td>Watch glasses</td>
<td>&nbsp;</td>
<td>Pyrex</td>
<td>9985-75</td>
<td>&nbsp;</td>
<tr>
<td>2 ml cryogenic vials</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430488</td>
<td>&nbsp;</td>
<tr>
<td>10 x 10 cryoboxes</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>03-337-7AA</td>
<td>&nbsp;</td>
<tr>
<td>15 ml conical tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352099</td>
<td>&nbsp;</td>
<tr>
<td>Tongs</td>
<td>&nbsp;</td>
<td>Fisher Health Care</td>
<td>10-316B</td>
<td>&nbsp;</td>
<tr>
<td>Cryofreezer</td>
<td>&nbsp;</td>
<td>Taylor-Warton</td>
<td>K-series</td>
<td>For example</td>
</tr>		</tbody>
		</table>
		</div>

a high-throughput method for zebrafish sperm cryopreservation and in vitro fertilization

这是一个斑马鱼的精子冷冻保存方法，是一种适应哈维方法（哈维等人，1982年）。我们引进了两个转变到原来的协议，既简化程序，提高样品的均匀性。首先，我们所有精子量，使用不包含的冷冻保护剂的冻结媒体正常化。二，精子冷冻保存，储存在离心管代替毛细管。精子的冷冻和解冻（δ° C /时间）的利率可能控制在此过程中最关键的两个变量。出于这个原因，不代替指定的那些不同的管子。 2，团队的工作是可能的，冻结100〜2小时，每队男性的精子。精子冷冻保存，使用此协议，有25％的平均生育率（除以总数的受精卵在体外授精产生可行的胚胎数量来衡量），多年来，这个百分比生育率稳定。

Watch this Scientific Journal Video about A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization at JoVE.com

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

这是一种高通量的精子冷冻保存对斑马鱼的协议。精子冷冻保存，使用此协议，有25％在随后的体外受精生育的平均多年来保持稳定。

对于斑马鱼精子超低温冷冻保存的一个高通量的方法和<em在体外施肥

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

a-high-throughput-method-for-zebrafish-sperm-cryopreservation-vitro

Research

JoVE Journal

Biology

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

30.8K 次观看.  University of California, Davis. 这是一种高通量的精子冷冻保存对斑马鱼的协议。精子冷冻保存，使用此协议，有25％在随后的体外受精生育的平均多年来保持稳定。

视频: A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

一种高通量的方法，在全球范围内研究在酿酒酵母中的细胞器形态

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

科研

生物学

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

一种快速高通量的方法映射核糖核蛋白（RNPs）人类的前体mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

冰帽：为发展方法拟南芥和番茄植株在96孔板高通量的基因分型

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

使用油脂全线Bicelle方法的膜蛋白的高通量结晶

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Mouse Sperm Cryopreservation and Recovery using the I&amp;middot;Cryo Kit

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

精子冷冻保存和恢复使用I ·冷冻包

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

高通量清醒小鼠肾小球滤过率的测定方法

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

生产单倍体斑马鱼的胚胎通过<em&gt;体外</em&gt;施肥

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

手册小分子屏幕接近高通量使用斑马鱼胚胎

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

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 ...

对于斑马鱼精子超低温冷冻保存的一个高通量的方法和施肥