作者感谢丹贝丝Sigma - Aldrich公司介绍视频。我们也感谢唐分公司，Evgenii Cherny，斯科特Chesla，伊丽莎白Hadac，阿曼达哈里森，达米安希思科特，安妮卡霍特，兰传青，苏珊娜麦金托什，Sarvani Komarraju，Korchagina埃琳娜，卡罗琳奥利弗，马丁奥尔森，斯蒂芬 - 帕克，伊戈尔Rodinov亚历山大Tuzikov，埃莉诺威廉姆斯其结果和FSL，结构设计，合成和确定生物活性的贡献。

以下协议描述插入FSL的构造（图1）生物膜的通用过程。简单的协议将仅是指细胞（kodecytes），在浓度为100％。然而，长期的细胞与病毒粒子（kodevirions）或生物体或细胞结构及其稀释剂的浓度互换并不重要，只要它总是在测试，控制和实验一致。与红血细胞，红细胞压积通常是80％，但与病毒粒子，胚胎和其他细胞，它通常小于1％。该方法是非常强大的，任何变化时，会产生改性膜，但将需要细化，优化和规范，修改为特定应用程序所需的程度。 1。 FSL的制备构造<ol><li>要准备FSL​​，构建原液第一次重组稀释液1.0mL（或在产品说明书中指定）除了产品小瓶干FSL的产品（见表1）。简言之超声（30秒）。这将准备一个1mg/ml的解决方案，它可以分装成100μL无菌容器和储存在2-8℃，最长不超过一周，或长达3个月冻结。 </li><li>准备工作FSL，构建插入的解决方案，在使用前简要超声（30秒）原液均质任何胶束。需要在一定范围内，或如果需要的浓度稀释FSL，在缓冲构造（最好不要含有血脂或高疏水性材料）。 </li></ol>注: <ol><li> FSL的结构通常会插入细胞脂质包含媒体，但通常会要求一个更高的50X FSL的工作浓度比在PBS或其他脂质自由媒体。 </li><li>工作范围内稀释，将取决于应用程序，检测方法灵敏度，FSL的构造类型和所需的修饰度。通常的稀释范围将介于10-500微克每毫升FSL的稀释剂碳水化合物FSL的结构和1-100微克/毫升，如荧光团和生物素等FSL，结构。 </li><li>如果准备插入稀释液中含有多个FSL的构造，只需添加构造在他们正常的工作浓度相同的稀释剂。如果一个构建在浓度远高于另一个是，一些集中的调整（增加）可能需要较小的构造。或者可能被添加构造顺序，最好用浓度最高的构造，由较小。 </li><li> FSL，构建超过1000微克/毫升的浓度可能会引入大量的脂质细胞膜，并可能改变细胞的形状，或使其更容易溶解。 </li><li> FSL的构建工作方案应储存在2-8℃，并在几天之内使用。 </li><li> FSL的结构可以在水稀释，但会减少的稳定，必须在几个小时内使用。如果包插入指定的重组稀释剂的水在瓶的产品也将包含盐（包中插入指定）。 </li></ol> 2。插入膜FSL的构造（S） <ol><li>先洗未绑定血脂FSL修改免费的细胞离心，洗涤液PBS或脂细胞介质。 </li><li>包中的细胞或暂停在100μL稀释液。 </li><li>同时准备控制理想情况下应该是双方未修改细胞孵育，用PBS，而不是FSL的解决方案和/或修改与FSL，构建一个良性的，但相关细胞。 </li><li>加入100μLFSL的解决方案（包含1个或多个FSL的构造）适当稀释细胞1-2个小时，在37 ° C。 6小时孵化获得了类似的结果可以通过在25 ° C或过夜（18小时）在4 ° C - 混合建议每隔几个小时，如果使用沉重的细胞悬浮液。 </li><li>清洗（可选）用PBS或媒体的两倍，以消除任何自由FSL的构造和准备适当的悬挂。 </li></ol>注: <ol><li>可以暂停细胞的稀释剂细胞培养基，PBS，细胞的存储解决方案等，但最好不脂类（如胎儿小牛血清）或清洁剂（如吐温）。 </li><li>可以使用不同的卷（超过100μL）的比值（大于1:1细胞/ FSL的解决方案）或孵育时间和温度条件，提供相同的比例，浓度和体积，用于获得可重复性的结果。 </li></ol> 3。处理和可视化<ol><li>一旦FSL，修改过程中已经完成了kodecytes可以存放在4-8 ° C脂质自由媒体或使用。 </li><li> Kodecytes和kodevirions一般行为未修改细胞/病毒颗粒相同，可handl教育署和在正常测试系统（显微镜，血清学，流式细胞仪等）来看。他们通常有没有特殊的要求，除了避免溶剂/清洁剂/血脂可能洗脱从细胞膜的脂质结构。 </li></ol>注: <ol><li>结构仍将在不活跃的细胞，如红细胞，膜，如果储存在细胞的寿命脂自由媒体。积极膜细胞内的构造和代谢的速度依赖于细胞膜活动。 </li><li>死细胞通常会包含FSL，结构的高层次，这可以用来从活细胞中分离出来的自生能力的细胞。 </li><li>如果kodecytes存储（ 或体内 ）的脂质包含媒体/血清/血浆的构造，如环境，会慢慢从膜上洗脱过了几个小时或几天内，取决于温度和血脂浓度/成分。 </li></ol> 4。代表结果: <ol><li>以下代表的结果取决于构造（见表1），其插入的浓度和检测系统（S）的相对灵敏度和特异性。一般情况下，所有FSL的结构将插入细胞后1小时，但每一种情况的最佳条件，将需要建立。 </li><li> FSL的结构可以用来标记一个活细胞的各种 3-9,11,14外。在如图所示的例子中，被用来在不同阶段的胚胎（因为他们是一个脆弱的细胞），但其他细胞（图3）或笼罩病毒颗粒（图4）也可以被标记。 FSL荧光素可以直接表面的标签（图2-4），而FSL抗生物素蛋白和其他FSL的结构可能需要一所中学的检测系统（通常是抗生物素蛋白/抗体/凝集素）的使用，以显示他们的存在（图5） 。 </li><li>血型的人类或动物的特异性标记，可以附着到细胞，包括红细胞 4-9（图6） 。由于FSL的金额上kodecyte兴建的可控性和重现性好4-6，抗体定量kodecytes可（见表2和图6）。细胞的实际来源并不重要，他们可以是任何物种，甚至同一产地的源抗体，从而确保唯一不相容的抗原是由FSL 7,8（图6）介绍。 </li></ol><img src="/files/ftp_upload/3289/3289table2.jpg" alt="表2" /> 表2。测试代表了三种不同的FSL - A（三）红细胞kodecytes的血清学反应的单克隆抗- A稀释。与同等体积的O型红细胞孵育FSL - A（三）50微米的解决方案产生了强烈的抗原阳性细胞，这可能是由单克隆抗- A 1:512稀释检测。 5 - 10倍低FSL - A（三）解决方案的生产与少血型A抗原的表达kodecytes。 <img src="/files/ftp_upload/3289/3289fig1.jpg" alt="FSL的构造概述" /> 图1。FSL构造概述。像花一样FSL的结构由三个主要部分组成组成。功能头（F）中的固定利差贷款结构的情况下可以是各种不同的生物功能群体，远离膜诱导的F间距，提高水分散性和血清非活性间隔（S），而酰基脂质允许建构自发地纳入表面。 （一）FSL - GB3与血型GB（或P K）三糖抗原表位Galα4Galβ4Glcβ。 （二）FSL - A（三）与血型一个三糖抗原表位GalNAcα3（Fucα2）Galβ。 （三）FSL荧光素。 （四）FSL抗生物素蛋白与一个单一的生物素F基团。 I - III结构功能组结合dioleoylphosphatidylethanolamine激活己二酸衍生物（DOPE），而第四结构的间隔是基于carboxymethylglycine -己二酸。 <img src="/files/ftp_upload/3289/3289fig2.jpg" alt="FSL荧光素标记的小鼠胚胎" /> 图2。FSL荧光素标记的小鼠胚胎。所有图像都被打成当他们透明带（ZP）是完整的（即FSL的构造通过ZP标签内的胚胎通过）的活胚胎。图片我的四酸tyrodes FSL荧光素后标签从他们ZP发布的ZP - 胚胎。相同的染色发生时，不管是否胚胎FSL，与ZP完好或ZP免费修改。胚胎直接与FSL荧光素标记，2小时37℃，在无血清细胞培养基的方法，洗净，然后在荧光显微镜下观察。 （一）ZP自由两个细胞的小鼠胚胎，也呈现出经典的激烈极体染色。 （II - III）ZP免费四单元和8个细胞的小鼠胚胎细胞奏阴影，既显示染色IDE显微镜平面。 （四）ZP免费16细胞的小鼠胚胎。 （五）ZP完整的小鼠囊胚的胚胎（D4 - D5）胚胎和卵被标记。 <img src="/files/ftp_upload/3289/3289fig3.jpg" alt="FSL荧光素和斑马鱼" /> 图3。FSL荧光素和斑马鱼14。 （一）Microangiography 52 HPF（小时受精后）斑马鱼幼虫直接注入流通与FSL荧光素。斑马鱼的血管染色。 （二）FSL荧光素异构斑马鱼的肾组织细胞（ZK kodecytes）创建了体外，然后进入流通的52 HPF收件人斑马鱼微注射的ZK kodecytes体内观察注射后2小时的成像与时间推移显微镜下的荧光血管。显示的是一个大的滞销或不动的细胞（橙色箭头表示）和快速移动的细胞（由于运动模糊图像 - 绿色箭头指示）的单个视频帧。标签允许在体内的行为和生物分布的ZK kodecytes观察实时。 （三）FSL荧光素口服摄取实现沉浸在FSL -荧光斑马鱼的胚胎中含有长达5天的媒体。洗衣机是通过媒体没有固定利差贷款的结构（至少6个小时是必需的，但可以几天）的胚胎转移到。 （IIIA）FSL荧光素处理后的斑马鱼相应相邻的荧光图像（IIIB）明场显微镜。优先设在肠道中的荧光。在未经处理的控制胚胎（IVA和IVB）无染色观察。 <img src="/files/ftp_upload/3289/3289fig4.jpg" alt="FSL -荧光标记的病毒粒子" /> 图4。FSL - 10 VSV的病毒粒子和H1N1荧光标记。 （一）水泡性口炎病毒（VSV）是直接与10微克/毫升2小时FSL荧光素标记在37 ° C，4％多聚甲醛，然后流式细胞仪固定。没有净化VSV的kodevirion后FSL标签是必要的。 （二）kodevirions使用FSL荧光素标记流式细胞仪检测与人类/波多黎各Rico/8/1934（H1N1）感染的猪睾丸细胞。被视为未受感染的细胞的黑线，而融合的细胞在荧光细胞（红色线）与ST的H1N1 kodevirion。 <img src="/files/ftp_upload/3289/3289fig5.jpg" alt="FSL -生物素标记的细胞" /> 图5。FSL，生物素标记的细胞，并通过标记的抗生物素蛋白7,8随后的可视化。所有细胞与FSL -生物素标记在37 ° C为1小时，洗净，然后与标记的抗生物素蛋白的荧光团，水洗和湿装荧光显微镜反应。 （一）编制一个小鼠胚胎囊胚共聚焦图像。 （二）中央焦片的胚胎从前面的形象。 （三）现场能动的人类精子 - 模糊作为其运动的后果发生。 （四）固定（4％多聚甲醛后插入）人类精子。 （五）人红细胞。 （六）固定（4％多聚甲醛前插入）RL95人类子宫内膜癌。 （七）不固定的RL95子宫内膜人类癌细胞系。 （八）（VIIIA），光学显微镜下观看了现场后2小时kodecytes静脉滴注（VIIIb）是同一领域，在荧光看，确定两个kodecytes目前在该领域所采取的血液样本中观察到的生物素红细胞kodecytes的视图。 kodecytes未标记的细胞比例计算，可使用作为一个生存的指标。 （九）现场可视化人体子宫内膜的生物素kodecytes结合avidinylated珠。 <img src="/files/ftp_upload/3289/3289fig6.jpg" alt="FSL，碳水化合物添加血型标记的血清学分析" /> 如图6。FSL碳水化合物添加血清分析血型标志。 （一）人类红细胞Galili kodecytes创建一个FSL - Galili浓度（500微克/毫升）和对人体血清稀释测试。人类红细胞不自然地发生xenoantigen Galili抗原。这种kodecytes可用于血清中抗体定量水平。在这个例子中，捐助者，被确定为反Galili滴度1:32。 （二）通过建立与量的日益减少的固定利差贷款（"抗原抗体滴度"），最佳抗原水平kodecytes检测抗体可确定（在这个例子中乐一个 ）。细胞可以创建只给予了积极的结果，当抗体水平超过某个特定的滴度。给予了积极的结果所需的FSL的抗原水平取决于被检测抗体的质量和水平。对于碳水化合物抗原，为100微克/毫升的FSL的解决方案通常会导致在强阳性反应。 （三）人类O型红细胞进行了修改，有蚂蚁的特定水平IGEN（标准A kodecytes），并以此来准确和可重复的定量分析人血清中抗A。在这个例子中，反的一个在O型血清测试的滴度是1:32 kodecytes准备从捐赠者的红血球。 （四）血型A和B抗原同时插入到一个单一的O型红细胞样本被用来创建一个弱弱 kodecytes乙。这些kodecytes可以用于ABO血型质量控制的目的。这个结果显示了一个专门制订一个弱乙弱抗A和抗B试剂测试，并给出了预期弱反应kodecyte的分析。

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L., Brad Jones, R., Ramkumar, S., Sakac, D., Binnington, B., Henry, S., Lingwood, C. A., Branch, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synthetic+globotriaosylceramide+analogue+inhibits+HIV-1+infection+in+vitro+by+two+mechanisms.">A synthetic globotriaosylceramide analogue inhibits HIV-1 infection in vitro by two mechanisms.</a> Glycobiology. 27, 515-524 (2010).</li><li>Barr, K., Diegel, O., Parker, S., Bovin, N., Henry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function-Spacer-Lipid+(FSL)+constructs+enable+inkjet+printing+of+blood+group+antigens.">Function-Spacer-Lipid (FSL) constructs enable inkjet printing of blood group antigens.</a> FEBS J. 277, 235-235 (2010).</li><li>Lan, C. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Modeling inflammatory bowel disease [dissertation]. , (1988).</li></ol>

德博拉答:空白和斯蒂芬M亨利雇员和股东KODE生物技术有限公司KODE技术的专利所有者。丹贝丝是Sigma - Aldrich公司，生产这种视频文章，至今已资助的雇员。

修改细胞和病毒粒子与FSL，构造是一个非常简单和强大的技术 3-11 。描述和区分FSL，构造修饰细胞和从未修改细胞的病毒颗粒，它们被称为kodecytes和kodevirions分别为6-10，但只有当引入官能团可以证明其膜。 kodecytes FSL的构造不同浓度时，他们可以被称为FSL，构建用于创建它们，例如15μg/ mL的一个kodecytes，或如果使用一个以上的FSL是一个结合长期的解决方案的浓度，如通过A +生物素kodecytes 7,8。当不同的细胞进行比较，包括在描述的细胞类型是建议，如红细胞素kodecytes或子宫内膜素 kodecytes 7,8。 虽然插入的过程中是非常稳健，插入（尽管不同的利率）会发生在一个大的温度范围从4 - 37 ° C和几分钟到几小时内，获得重复性插入严格控制接触时间，温度，FSL的浓度（包括稀释剂配方），并在较小程度上的细胞浓度是必需的。至目前为止，所有FSL的结构，可以用来修改相同的技术 4-11细胞，但它预计一定的条件将更加有利的工作流程和/或细胞/要修改的病毒粒子的最佳处理要求。 FSL，确切的浓度，以达到预期的效果，需要确定用户 4 。结果FSL，构建了修改过的细胞/病毒体通常可以使用同样的方式在未修改的细胞/生物或分析系统的病毒粒子4-11。由于FSL结构修饰的表面会慢慢洗脱时，在与脂质的解决方案7,8接触，或消耗由11个活跃的细胞，这些问题必须考虑一段时间内获得的信号或活动水平。 Kodecytes可以是固定的（如戊二醛/福尔马林）插入后提供固定液不包含脂质洗脱溶剂和功能组是兼容固定液。 FSL，构造或者固定的细胞可以修改。 除了 ​​细胞和病毒表面的改性FSL的构造也可以直接注入到实验室动物造成体内循环细胞的修改7流通， 抑制病毒12，毒素，12 和抗体7。财务和后勤系统的结构也被用来修饰脂质体，并可以打印到纸张表面，在那里他们固定，然后就可以用于诊断分析。 能够很容易地修改与FSL的构造的范围日益扩大的活细胞被证明是一个有益的研究和发展为细胞表面的生物学研究工具。

<img src="/files/ftp_upload/3289/3289table1.jpg" alt="表1" /> 表1。市售FSL的构造本文描述。

fsl constructs: a simple method for modifying cell/virion surfaces with a range of biological markers without affecting their viability

修改/可视化的生物表面，然后在体外的范围和体内环境研究修改后的细胞/病毒粒子的能力是必不可少的，以争取到的特定分子或整个实体的功能的进一步了解。生物表面改性的研究一般仅限于基因工程的生物或化学基团的细胞表面 1,2的共价结合。然而，这些传统技术细胞暴露在化学反应物，或他们需要显着操纵，以达到预期的结果，使他们繁琐，而且他们也可能在不经意间影响的可行性/修改后的细胞的功能。无害修改细胞表面的一种简单的方法是必需的。 最近的一项新技术，KODE科技已推出了一系列新颖的结构由三个部分组成:功能头组（F组），一个间隔（S）和脂质的尾巴（L）和功能间隔脂质或FSL， constructs3。选择间隔（S）是提供一个构造，它是在水中分散，但会自发地和稳定纳入膜。 FSL，构建功能基团（六）到目前为止，包括一系列的糖类，包括血型相关的决定因素，唾液酸，透明质酸多糖，荧光，生物素，radiolabels和肽3-12的范围。 FSL的结构已经修改的胚胎，精子，斑马鱼，上皮细胞/子宫内膜细胞，红细胞，病毒颗粒，建立质量控制系统和诊断面板，修改细胞粘附/互动/分离/固定，并在体外和活体成像细胞/病毒颗粒3-12。 修改细胞/病毒粒子的过程是通用的，极其简单。最常见的程序是解决FSL构造1-2个小时在37 ° C 4-10细胞（脂质自由媒体）的孵化。在孵化FSL，构造自发纳入膜，和过程是完整的。洗是可选的。被称为kodecytes 6-9 FSL的结构修饰细胞，而病毒粒子kodevirions 10。 FSL的构造直接注入和kodecytes / kodevirions已在实验动物模型7,8,10。所有kodecytes / kodevirions出现保留其正常的活力和功能的同时，获得新功能的F基团 7,8,10,11 。 生物相容性媒体，自发纳入到细胞膜，并明显的毒性低，分散组合，使得FSL构造的细胞和病毒粒子的研究有价值的研究工具。

Watch this Scientific Journal Video about FSL Constructs: A Simple Method for Modifying Cell/Virion Surfaces with a Range of Biological Markers Without Affecting their Viability at JoVE.com

FSL Constructs: A Simple Method for Modifying Cell/Virion Surfaces with a Range of Biological Markers Without Affecting their Viability

功能间隔脂质（FSL）的构造允许活细胞和病毒粒子的表面特性，没有丧失活力的情况下修改。该方法只需要简单的接触FSL构建的解决方案与一个细胞/病毒粒子和自发的和稳定的表面掺入发生。

FSL，构造一个简单方法修改细胞/病毒粒子表面的生物标志物的范围:在不影响他们的生存能力

The ability to modify/visualize biological surfaces, and then study the modified cell/virion in a range of in vitro and in vivo environments is essential ...

fsl-constructs-simple-method-for-modifying-cellvirion-surfaces-with

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

13.8K 次观看.  AUT University and KODE Biotech Ltd. 功能间隔脂质（FSL）的构造允许活细胞和病毒粒子的表面特性，没有丧失活力的情况下修改。该方法只需要简单的接触FSL构建的解决方案与一个细胞/病毒粒子和自发的和稳定的表面掺入发生。

视频: FSL Constructs: A Simple Method for Modifying Cell/Virion Surfaces with a Range of Biological Markers Without Affecting their Viability

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

焦磷酸测序：一个准确的基因分型的简单方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

科研

生物学

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

一个线虫多巴胺神经元退化含量的应用验证潜在的帕金森氏症基因

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

制作生肌工程的组织结构

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls&mdash;fucosylated and sialylated human lactoferrin glycopeptides&mdash;and negative controls&mdash;high mannose glycopeptides from Saccharomyces cerevisiae&mdash;that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

一个外源凝集素的高效液相色谱法从复杂生物样品，以丰富的选择性糖基化肽

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood or interstitial fluid); 2) the object is illuminated with light of appropriate wavelength in order to excite the probe into its triplet state; 3) the emitted phosphorescence is collected, and its time course is analyzed to yield the phosphorescence lifetime, which is converted into the oxygen concentration (or partial pressure, pO2). The probe must not interact with the biological environment and in some cases to be 4) excreted from the medium upon the measurement completion. Each of these steps imposes requirements on the molecular design of the phosphorescent probes, which constitute the only invasive component of the measurement protocol. Here we review the design of dendritic phosphorescent nanosensors for oxygen measurements in biological systems. The probes consist of Pt or Pd porphyrin-based polyarylglycine (AG) dendrimers, modified peripherally with polyethylene glycol (PEG's) residues. For effective two-photon excitation, termini of the dendrimers may be modified with two-photon antenna chromophores, which capture the excitation energy and channel it to the triplet cores of the probes via intramolecular FRET (F&ouml;rster Resonance Energy Transfer). We describe the key photophysical properties of the probes and present detailed calibration protocols.

We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.

氧成像在生物系统中的磷光纳米探针的合成和校准

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

天堂斑马鱼的细胞标记与激光Uncagable荧光葡聚糖

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

一个简单的协议提取血细胞从野生毛毛虫

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

重组工程同源重组构建<em&gt;果蝇</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

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 ...

FSL，构造一个简单方法修改细胞/病毒粒子表面的生物标志物的范围:在不影响他们的生存能力

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