作者想感谢龙沙Walkersville协助封闭的系统知识，包设计，袋封口机和焊工的贷款，以及创造袋HYPERStack电路图。

1。 HYPERStack容器 - 透氧材料技术背景<ol><li> HYPERStack容器是一个封闭的系统依赖通过76.2micron透气性聚合物薄膜对气体交换细胞代谢，细胞培养多层次的船只。 </li><li> HYPERStack船只不同于更传统的细胞培养容器中，它并没有上述容器内的细胞有一个"顶空"。而不是包含这个"顶空"内船只的气体交换，透氧产品空气空间（简称为"气管"空间），这是开放的气氛下每个文化室。 </li><li>这气管的空间，使细胞中每个细胞培养室的船只越来越多，具有同等的气体交换。 </li><li>透氧膜，使气体交换发生，同时保持无菌的环境。发生多层次的所有的流体操作通过一个单一的入口端口。 </li></ol> 2。 HYPERStack容器组件摘要<ol><li> Stackette是个别的细胞培养室，是顶板和透氧膜。这些细胞是培养在这个车厢。 </li><li>液体流形连接12 stackette层​​内HYPERStack模块。模块与油管连接，形成12层的倍数的船只。多方面的，允许用户在一个流体操纵整个船只。 </li><li> stackette层​​空气歧管连接在一起，但它用来替代容器内的空气流体增加发生时。它包含了在填充过程中使用的填充线。 </li><li>气管空间是每个stackette层​​之间的开放领空，允许通过各层的透氧膜的气体交换发生。 </li><li>液体处理管连接到液体流形，是用来做全封闭系统的流体操作。此组件可定制。 </li><li>排气管连接到空气歧管，包含一个空气过滤器，是用来释放多余的空气，同时保持无菌。 </li><li>追逐管有一个过滤器，夹具和连接到液体处理管和用于疏散的流体从液体处理管充填后的船只。 </li><li>康宁堆栈机械手或CSM是一个处理设备，以协助配售的船只在使用过程中的正确位置。 </li><li>灌装楔形是一个不锈钢协助使用时手动灌装HYPERStack船只。 </li></ol> 3。使用的HYPERStack船只通过透氧膜的气体扩散结果<ol><li>在传统的细胞增长系统，通风顶空，在媒体中的氧气耗尽平均50％的，超过3天的文化。 （图1） </li><li>在同一细胞的生长系统，在一个3mm的媒体高度氧气梯度是在媒体几乎50％的顶空交界处，比它在细胞层。 （图2） </li><li>通过76.2micron透氧性聚苯乙烯薄膜的氧气的扩散是平等通过媒体2.6毫米的传播。透氧膜在HYPERStack船只的使用，使气体交换发生在细胞层。 </li><li> HYPERStack船只的气管空间中的氧气％保持不变，细胞生长至汇合。 （图3） </li></ol> 4。媒体编制袋<ol><li> HYPERStack - 12层容器媒体1.3L，HYPERStack - 36层容器媒体3.9L。 </li><li>您的袋装媒体接种​​之前，如果血清是用于，管焊接血清袋媒体包调匀。 </li><li>在使用袋大袋夹，钳断约300毫升媒体。这将确保所有接种细胞在填充过程中，并没有在媒体包的剩余使用。当地媒体袋袋上的立场。 </li></ol> 5。接种媒体<ol><li>与连接管细胞悬液填充注射器。管焊接3 / 16"管含有细胞悬液媒体包注射器。 </li><li>媒体袋注入细胞悬液，拌匀。 </li></ol> 6。填写程序<ol><li>管焊接接种的媒体袋HYPERStack船只的3 / 8"液体处理管（使用无菌连接或多用途连接器（MPC），你也可以附加媒体包船只）。 </li><li>关闭液体处理和上HYPERStack追逐管夹具。 </li><li> HYPERStack 36层容器放入负载位置的CSM;钩到夹具上的CSM发泄过滤管。拧紧盖子，填充的位置和移动的CSM。把正确的填写位置的船只;空气过滤器是在最高的位置，以防止在填充操作的润湿。 10 °角的许可证equilibr在灌装液体层ATION。 HYPERStack - 12层的船只在其一侧放置在灌装楔形是在正确的位置填补。 </li><li>清除空气填充线，定位在同一水平HYPERStack船（不高于）袋。保持追逐管钳封闭;打开液体处理管钳和媒体包钳，让液体进入船只。 </li><li>使用袋立场，提高媒体包，以帮助入血管的细胞悬液流。 </li><li>填写船只，直到所有的接种媒体进入血管; 300毫升的媒体仍然在媒体包的顶端部分。从媒体包中取出钳继续填写船只。由于液体接近上层的空气歧管，减慢填充率降低媒体包，以防止过度充盈。慢慢提起液位填充线和液体处理管钳。 </li></ol> 7。分离过程中，锁定到图层的液体<ol><li>两套歧管带来的隔离位置的CSM HYPERStack船只，所以在最高的位置，和媒体包以下的船只的高度降低。 HYPERStack - 12层容器，可以解除隔离位置放在一侧的船只上的一只手和使用其他解除填充楔形。 </li><li>在直立位置举行的追逐过滤器，打开追逐管钳。这将空或追在液体处理管媒体到媒体包。一旦管子是空的，密切的媒体袋管钳。 </li><li>追逐管过滤器在直立位置，打开HYPERStack钳允许任何剩余的液体进入并在平衡的船只上的液体处理管。等待1-2分钟，出现这种情况。 </li><li>在CSM使歧管在左边打开HYPERStack船只。降低负载位置上的CSM船只。稍微向左摇动HYPEStack - 12层容器将允许拆除填充楔形。关闭追逐和液体处理管夹具。 </li><li>媒体包现在可以从容器中删除，或它可能会保持在收获过程中使用附后。要存储媒体的附加袋，空袋囊中任何剩余的媒体，并放置卷下HYPERStack船只的存储托盘固定乐队。 </li><li>移动HYPERStack船只的孵化器。运载船只护理时，应采取保持进风口过滤器的液体。这是通过引爆的HYPERStack多方面的结束稍向上。 </li></ol> 8。收获协议<ol><li>要开始收获的过程，焊缝管的细胞分解的解决方案和淬火袋共同形式的采伐袋大会。确保关闭所有管夹。 （卡通证明） </li><li> HYPERStack船只从孵化器中取出，释放的保留频段下的媒体包，挂在袋的立场。放置在负载位置的CSM的船只，并拧紧盖子，以确保船只。钩到夹具的通风管和填充位置移动的CSM。 </li><li>确保包装袋上的立场的媒体包挂低于该HYPERStack;开放媒体包油管钳和船只的液体处理油管允许媒体的附袋流入。 </li><li>一旦船只约¾空，改变CSM设置到最后的空位置。当HYPERStack船只和液体处理管是空的，解除管追过去媒体上所花的媒体袋管钳，然后关闭液体处理管钳和上花的媒体袋管钳。 </li><li>管焊接的收获袋大会更换花的媒体袋，并提高细胞分解袋以上的HYPERStack使用袋立场的高度装配的高度。 </li><li>将在平衡位置的CSM。打开细胞解离液管钳，并转入HYPERStack解决方案。当传输完成后，关闭液体处理管和细胞分解解决方案油管上的夹子。 </li><li>将CSM的负载位置。控股横向定位杆打开，轻轻摇动的船侧方使用的轮，分布在细胞层的解决方案。船只可能偶尔会返回到平衡位置，在摇晃的解决方案保持平等分配。它可能不是直观明显，完全包覆层，但同样的解离解决方案将整个细胞表面分布。一旦解决方案已被充分分布，对CSM发布的杠杆锁定在负载位置HYPERStack。留在这个位置上的HYPERStack在解离所需的时间为您的细胞。 </li><li>一旦细胞分离;使用浊度为指导，CSM的平衡位置移动。应注意不要过度消化细胞。打开淬火袋钳和HYPERStack液体处理管钳，允许淬火介质进入船只。一旦传输完成关闭HYPERStack液体处理管钳。 </li><li>在CSM返回到负载的位置，并保持横向定位杆，岩石的船只一边到另一边。 </li><li>使用袋立场，降低收获袋大会HYPERStack水平以下的位置。调整CSM的空位置，打开船只的液体处理管钳和淬火袋转移到细胞溶液。 CSM的负载位置和管封口机使用的船只，从断开的收获袋大会。电池解决方案是准备进行处理。 </li></ol> 9。代表性的成果<ul><li>收获时间，需要优化时，从一个标准的聚苯乙烯表面转换的透氧性聚苯乙烯表面。细胞倾向于对HYPERStack产品释放得更快。 </li><li>要查看在显微镜下观察细胞的宣泄和液体处理管需要被夹住了。倒挂，与歧管朝下显微镜舞台上，将HYPERStack船只。 </li><li>预防可以采取措施，以避免在使用过程中的润湿过滤器，这将导致他们停止运转。不超过填补HYPERStack船只和高架发泄和大通管夹打开时，没有湿润应该发生。 </li><li>管封闭夹具的压力，使其保持关闭后夹打开。捏在相反的方向管会打开它。 </li><li>媒体流通过重力饲料HYPERStack船只进入和退出的时间是基于媒体的高度差之间的媒介在HYPERStack船的顶层和顶级的媒体容器中的介质。更大的距离，流动的速度就越快。最大流量是有限的过滤器，以1.5L/min的评级。 </li><li>在HYPERStack船保持合适的灌装量，将防止空气泡沫细胞生长室形成。气泡可能是由于取样或孵化unhumidified环境因蒸发失水。采样数量和频率，以及孵化条件，将决定应当由最终用户增加额外的流体。 </li></ul><img src="/files/ftp_upload/2499/2499fig1.jpg" alt="图1" /> 图1:媒体在一个标准的细胞培养容器的氧气耗尽。该图显示了氧的毫克/大号超过3天的细胞水平下降。 <img src="/files/ftp_upload/2499/2499fig2.jpg" alt="图2" /> 图2:在一个标准的细胞培养容器的媒体氧梯度。该图显示了氧的毫克/ L是最大的媒体不是在细胞层顶空交叉点。 <img src="/files/ftp_upload/2499/2499fig3.jpg" alt="图3" /> 图3。氧气在细胞生长的HYPERStack气管空间的百分比。的数字显示，超过96小时，各层之间的比例的氧气，提供每个透氧膜的底部，保持不变。这表明细胞的能力，在生长过程中的氧气，以汇合访问。

根据美国专利号:7745209 HYPERStack容器透氧技术覆盖。作者康宁生命科学部，公司的生产在这篇文章中使用的仪器的员工。

HYPERStack船只已成功地成长工程，小学和干细胞。通过无菌袋连接液添加到船只已进行吊瓶，还从无菌媒体容器中抽水。容器提供了一个增长领域增加了2.5倍大于对叠板产品的相同容积的足迹。 HYPERStack - 12 6,000平方厘米的增长领域，HYPERStack - 36有18000平方厘米的增长领域，并HYPERStack - 120 60,000厘米的增长领域2。通过增加表面积，用户能够产生更多的细胞相同的细胞群或很多，并减少变异。他们还可以通过使用更少的船只来达到同样的电池的输出，或增加电池的输出，没有加入的人，洁净室套房，或孵化器，以节省空间和/或劳动。该系统是生产低颗粒的装配方法，不使用粘合剂。它是一个封闭的系统，这是预装管套和过滤器，三合一袋装和提供无菌。

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		<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>1.5L Bagged Media</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>TBD</td>
<td>1350ml media + 150ml serum (if required)</td>
</tr>
<tr>
<td>4.0L Bagged Media</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>TBD</td>
<td>3600ml media + 400ml serum (if required)</td>
</tr>
<tr>
<td>HYPERStack Vessel-12</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>10012</td>
<td>&nbsp;</td>
<tr>
<td>HYPERStack Vessel-36</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>10036</td>
<td>&nbsp;</td>
<tr>
<td>Filling Wedge</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>10040</td>
<td>&nbsp;</td>
<tr>
<td>Stack Manipulator</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>TBD</td>
<td>&nbsp;</td>
<tr>
<td>HyQtainer Sub-Assembly Sampling Device</td>
<td>&nbsp;</td>
<td>HyClone/LonzA</td>
<td>SH3B0324.01</td>
<td>&nbsp;</td>
<tr>
<td>600ml Bagged Media Quencher (10% FBS IMDM)</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>TBD</td>
<td>&nbsp;</td>
<tr>
<td>600ml Cell Dissociation Solution</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>TBD</td>
<td>&nbsp;</td>		</tbody>
		</table>
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closed system cell culture protocol using hyperstack vessels with gas permeable material technology

大容量的贴壁细胞培养目前叠板细胞生长产品的标准化，微载体珠时，是不是最佳的选择。 HYPERStack船只允许关闭系统的规模从目前的叠板产品，并提供在相同体积的足迹&gt; 2.5X更多的细胞。 HYPERStack船只通过透气性材料，使气体交换发生的功能，从而消除了需要在容器内部的顶空。顶空消除允许发生在细胞的生长，以尽量减少车厢，以减少空间，允许在相同的体积占用更多层细胞生长表面积。 对于许多应用中，如细胞疗法或疫苗生产，需要一个封闭的系统是细胞生长和收获。 HYPERStack船只允许细胞和试剂除了通过媒体袋或其他方法从油管和清除。 该协议将解释背后的透氧性材料，用于在HYPERStack船只，气体扩散的结果细胞，封闭的系统细胞生长的协议，以及各种采伐方式，以满足代谢的需要的技术。

Watch this Scientific Journal Video about Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology at JoVE.com

Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology

介绍到高产贴壁细胞培养使用康宁HYPERStack船只及配件的技术，协议和处理。该协议将展示如何使用目前的叠板产品的封闭系统中增加细胞收集船只。

封闭的系统使用透氧材料科技HYPERStack船只的细胞培养协议

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice.   ...

closed-system-cell-culture-protocol-using-hyperstack-vessels-with-gas

Research

JoVE Journal

Biology

18.7K 次观看.  Corning Life Science. 介绍到高产贴壁细胞培养使用康宁HYPERStack船只及配件的技术，协议和处理。该协议将展示如何使用目前的叠板产品的封闭系统中增加细胞收集船只。

视频: Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology

Principles of Site-Specific Recombinase (SSR) Technology

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular biology. Site-specific recombinases are proteins that bind to distinct DNA target sequences. The Cre/lox system was first described in bacteriophages during the 1980's. Cre recombinase is a Type I topoisomerase that catalyzes site-specific recombination of DNA between two loxP (locus of X-over P1) sites. The Cre/lox system does not require any cofactors. LoxP sequences contain distinct binding sites for Cre recombinases that surround a directional core sequence where recombination and rearrangement takes place. When cells contain loxP sites and express the Cre recombinase, a recombination event occurs. Double-stranded DNA is cut at both loxP sites by the Cre recombinase, rearranged, and ligated ("scissors and glue"). Products of the recombination event depend on the relative orientation of the asymmetric sequences.
SSR technology is frequently used as a tool to explore gene function. Here the gene of interest is flanked with Cre target sites loxP ("floxed"). Animals are then crossed with animals expressing the Cre recombinase under the control of a tissue-specific promoter. In tissues that express the Cre recombinase it binds to target sequences and excises the floxed gene. Controlled gene deletion allows the investigation of gene function in specific tissues and at distinct time points. Analysis of gene function employing SSR technology --- conditional mutagenesis -- has significant advantages over traditional knock-outs where gene deletion is frequently lethal.

Principles of Site-Specific Recombinase SSR Technology

The advent of site-specific recombinase (SSR) technology and the Cre/lox system has led to numerous advances in molecular biology, and has proven itself as a valuable tool for assessing gene function in transgenic animals. This interview discusses the mechanism of site specific recombination by Cyclization recombinase (Cre) and how the use of this enzyme has led to the development of conditional mutagenesis, which has significant advantages over traditional knock out strategies.

特定地点的重组酶（SSR）技术的原则

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular ...

科研

生物学

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

工程细胞透水蛋白

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

鼠类全胚胎培养方法，使用的肩型瓶培养系统

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.
Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed1 who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate2.
To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry1. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments1.
The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection3 in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.

The Scepter Cell Counter is a handheld automated device that can be used to count cells, monitor cell diameter and volume, and be used to check the health and quality of cellular populations from one culture to the next.

确定在细胞培养的细胞数量，使用权杖细胞计数

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

对糖皮质激素信号A化学筛选程序与斑马鱼幼虫的荧光素酶报告系统

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous proteomes, including those from viruses, bacteria, and yeast.&#160; In comparison, analysis of the human proteome lags behind, partially due to the sheer number of proteins which must be studied, but also the complexity of networks and interactions these present. To specifically address the challenges of understanding the human proteome, we have developed HaloTag technology for protein isolation, particularly strong for isolation of multiprotein complexes and allowing more efficient capture of weak or transient interactions and/or proteins in low abundance. &#160;HaloTag is a genetically encoded protein fusion tag, designed for covalent, specific, and rapid immobilization or labelling of proteins with various ligands. Leveraging these properties, numerous applications for mammalian cells were developed to characterize protein function and here we present methodologies including: protein pull-downs used for discovery of novel interactions or functional assays, and cellular localization. We find significant advantages in the speed, specificity, and covalent capture of fusion proteins to surfaces for proteomic analysis as compared to other traditional non-covalent approaches. We demonstrate these and the broad utility of the technology using two important epigenetic proteins as examples, the human bromodomain protein BRD4, and histone deacetylase HDAC1.&#160; These examples demonstrate the power of this technology in enabling &#160;the discovery of novel interactions and characterizing cellular localization in eukaryotes, which will together further understanding of human functional proteomics. &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;

HaloTag technology is a multifunctional technology which has shown significant success in isolation of both small and large protein complexes from mammalian cells.&#160; Here we highlight the advantages of this technology compared to existing alternatives and demonstrate its utility to study numerous aspects of protein function inside eukaryotic cells.

发现蛋白质相互作用和检定蛋白质功能使用的HaloTag技术

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

一个<em&gt;体外</em&gt;企业文化系统来研究甲状腺发展

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

逆转录病毒感染的原发性肠化培养的视频协议

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

蛋白质直接送货到哺乳动物细胞利用细胞渗透半胱氨酸<sub&gt; 2</sub&gt; -His<sub&gt; 2</sub&gt;锌指域

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

用基团特异 rDNA 引物对水生无脊椎动物进行遗传肠道含量分析的实验室规程

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...