我们要感谢加里·诺兰博士从斯坦福大学提供的凤凰GP包装细胞系用于生产逆转录病毒颗粒。我们感谢罗杰钱永健博士在加利福尼亚大学圣地亚哥分校提供TD番茄。我们还要感谢圣迭戈州立大学流式细胞仪核心设施为他们的服务和帮助。

1.准备哺乳动物细胞，病毒生产和转导遗传条形码的<ol><li>板2.5×10 6的贴壁细胞在10cm的板（或约50-60％汇合）的前一天，以转染在Dulbecco改进的Eagle培养基（DMEM）和10％胎小牛血清（FCS）。逆转录病毒生产使用的包装细胞系的选择，如凤凰-GP（从加里·诺兰，斯坦福大学是一种礼物）。 注：该包装细胞系稳定表达的Gag和Pol蛋白对逆转录病毒颗粒形成中反式 。确保细胞是健康的，并附着于所述板中的单层，在转染之前。 </li><li> 24小时后，准备DNA转染混合物。 <ol><li>到125微升无血清的DMEM中加3微克水泡性口炎病毒包膜糖蛋白载体（PCI-VSVG）和3微克携带感兴趣的荧光蛋白的基因转移载体的构建。混合，并添加15微升聚乙（2毫克/毫升）。 </li><li>孵育混合物在室温下搅拌15分钟，并加入到细胞中逐滴。 注：聚乙加入到均匀的DNA混合物作为转染试剂为多聚物的形成。为了获得病毒颗粒携带不同的荧光蛋白标记物，以供选择的标记独立地进行每次转染。请记住，逆转录病毒转移载体是大约7 kb的长度，并可以不被打包如果超过10 kb的。 </li></ol></li><li>孵育板在37℃。为了增加病毒的生产场所板在30℃或32℃，而不是。 注：24小时后的转染介质可以用7ml的新鲜培养基代替，虽然这可能会改善细胞的健康也可能在上清液降低病毒载量。 </li><li> 48小时转染后收集含上清液的病毒颗粒和过滤器以0.45微米的聚四氟乙烯过滤器。使用新鲜收集病毒上清转导。 OTHerwise保持上清液以等分试样在-80℃下，并避免冻融。请记住，病毒滴度将减少可能高达50％，与每个冻融循环。 </li><li>优先选择24小时的板块细胞系传导，如果贴壁，或转导如果不贴前一刻。种子2.0×10 5〜3.0×10 5个贴壁细胞（这里咦7.5.1和HEK 293T）或5.0×10 5〜 1×10 6个非粘附细胞（此处SupT1）每孔在6孔板中。 注意：请确认校准单元的数目之前应当转导接种，特别对于贴壁细胞，所以细胞达到约60％的一汇合在转导的时间。 </li><li>添加5微克/ ml的凝聚胺（hexadimethrene溴化物）以接种细胞，并再加入之前收集的病毒上清以获得左右20-40％感染（这里是2毫升）中。 注：MOI或感染的比例大多是任意的，因为排序将允许放大任何subpopulat离子电池。显然，更高的速率感染的简化和加快了扩增的过程。 </li><li>在1500×g离心120分钟转导的细胞通过离心在悬挂桶转子在32℃。悬浮非粘附细胞用新鲜培养基之前孵化。对于贴壁细胞，更换新鲜培养基后24小时转导。 注：建议密封离心分离用封口膜的前板，以避免溢出。为了增加转基因条形码细胞多样性，转导的细胞与携带不同的荧光蛋白（来自步骤1.4）得到的病毒颗粒。当选择荧光标记物，要确保它们与仪器兼容，它们有区别的荧光参数。 </li></ol> 2.选择基因条码细胞和扩增<ol><li>通过流式细胞仪分析转导哺乳动物细胞至少72小时后传导到定量的百分比率转导。用可比较的非转导的细胞系作为阴性对照。 注意：作为排序将被用于纯化单个条形码的细胞群，高比例的速率初始转导，而不是必要的，应加快扩增的过程。 </li><li>利用细胞分选仪选择的，以获得表达目的蛋白质的细胞。 <ol><li>为了这个目的，确保栅极在右声道适当设定。 <ol><li>这样做使用相同的幼稚细胞系作为阴性对照，并设置参数/通道电压，使得负极人口低于10 3上的每个荧光轴上的值。确定选通荧光阳性如出现高于阴性对照为每个荧光通道的事件。 </li></ol></li><li>请注意，每个仪器可能会发生变化，并在正确的电压，用于确定选通应作相应的调整，使得必须在每一个记录阳性和阴性对照erimental设置。 <ol><li>荧光检测在本实验装置使用的FITC通道（由502长通滤波器进行三十零分之五百三十○带通滤波器），用于eGFP的，在PE通道（由556长通滤波器进行42分之585带通滤波器），用于TD番茄，和APC通道（二十〇分之六百六十〇带通滤波器），用于E2深红。 注：488 nm激光激发绿色荧光蛋白和TD番茄，而E2深红由633 nm激光激发。 </li></ol></li></ol></li><li>排序在15ml锥形管中，用1ml 100％的FCS。 注：排序荧光细胞群的过程不是强制作为一个可以直接进行单个克隆（见下文）的排序。排序根据所选荧光紧身人群，确保备份的人口为将来选择单个克隆。请记住，个体细胞是在时间难以生长，同时人口众多的细胞可以容易地冷冻，然后解冻，并放大在稍后阶段。 </li><li> SP在向下排序种群悬挂桶转子离心机以524克在32℃下5分钟以沉淀细胞。重新悬浮在1ml新鲜的培养基，并重新盘细胞，使细胞汇合是60％以下，以允许生长和分裂为至少两天。 <ol><li>例如，板的贴壁细胞在约3×10 5个细胞在2毫升每孔媒体在一个6孔板和2×10 6个细胞在2ml培养基中6厘米板。用DMEM培养贴壁细胞和RPMI对非贴壁细胞（或任何指明介质如果需要）。放置铺板细胞在培养箱中在37℃下进行数天，以允许扩增。 </li></ol></li></ol> 3.获取不同强度基因条码细胞克隆群<ol><li>从细胞中最初转导得到克隆群（步骤1.7）或从排序人口（步骤2.3）。 注意：这将确保在所有的细胞荧光蛋白的表达相同或相似的水平内的人口秒（一紧人口具有小的CV中的高达40％的平均荧光强度）。 <ol><li>为此目的，分类单个细胞到96孔板用自动细胞沉积单元（ACDU）。根据兴趣人群分类设置门。保证闸门都设置至少一个对数标度时，除了在不同的荧光强度选择细胞，以获得可区分种群。对于此处示出的过程中，使用相同的流式细胞仪实验设置为在步骤2.2中描述的。 </li></ol></li><li>扩增在培养箱单个克隆在37℃下为至少2周。通过扩增的过程中进行分析，在显微镜下的细胞，以确保不断增长的人口源自单个细胞而不是从多​​于一个。 注意：请记住，分拣机将置于每孔一个细胞的平均值，而一些孔可以不具有任何细胞可言，其他人可能有一个以上的。 <ol><li>为了确保婆pulation产生从一个小区只，是非常温和的与板和从未通过移液打扰的孔中。观察板块，深受很好，在显微镜下。 注意：虽然单个细胞有时可能难以确定，一旦他们开始除以他们将很容易辨认。 </li><li>要知道，往往细胞的丛&#39;沿边缘。抛弃那些井在多个无性系种群可以观察到，保留那些有一个严格的人口。 注意：最好是转移到那些较大的板块进行放大。 </li></ol></li><li>通过流式细胞仪分析这些筛选假定克隆群。比较它们的阴性对照使用相同流式细胞实验设置。 <ol><li>分析样品的唯一的百分比，并保持其余作为备份。选择的基础上平均强度和人口密封性最明显的不同人群。放大他们的需要或冻结股冷冻，用20％的甘油培养基在-​​80℃下的时间或液氮更长短时间。 注：请记住，“真正的”克隆群体应该有一个非常紧张的荧光图案。 </li></ol></li></ol> 4.确保复用功能的高通量筛选（HTS） <ol><li>根据需要，可以以多路复用格式进一步利用结合许多不同的克隆群。选择具有可区别的吸收/发射光谱和/或不同强度的克隆。如果一个96孔板格式的情况下，其适用于高温超导，种子50,000个细胞在200μl每孔。 注意：请注意，单元的数目只是一个估计，并且可以更改基于细胞类型以及它是否是附着或没有。 </li><li>使用流式细胞仪，以确保每个独立建立荧光细胞系可以相互区别开来的样品进行分析。在分析之前制备负和单一的颜色控制设置参数和补偿值。 注：设置的参数，以明确区分的混合人群，使他们进一步用在复用格式。 </li></ol> 5.适应基因条形码的细胞系选择的生物应用<ol><li>转导如斯托尔普等人，2013 59描述在这里建立遗传条形码的细胞系与包含所选择的测定元件的病毒颗粒。请记住，所选择的测定法应当占据额外的和可区别的荧光信道，因而它是被转移到相应的条形码的细胞系。 注：每个条码细胞系可承受不同的检测。 </li><li>排序基因萤光条形码细胞为那些含有测定的元素。 注：检测元件可改造耦合到标签或荧光蛋白（作为融合或通过内部核糖体进入位点）F或者简单的检测，通过免疫印迹，流式细胞仪和显微镜。在这个协议中使用的系统依赖于HA抗原表面表达单独或与另外的FLAG表位的表达。 <ol><li>含染色与医管局及APC-荧光抗体耦合的实验的克隆群。然后分析克隆群与抗FLAG和FITC缀合的抗体染色。 </li><li>为了跟踪背面的测定中，分析或“译码”在适当的信道的种群，其中所述不同的遗传条形码的细胞系能够被区分。这里，通过在对于TD番茄检测的体育频道分析解码所述衬底的性质。 </li></ol></li></ol>

作者什么都没有透露。

这里有两个既定的程序已合并;通过逆转录病毒技术和检测利用流式细胞荧光蛋白的基因工程。荧光蛋白的基于遗传条码用于生产独特的细胞系提供了一个强有力的和简单的方式用于多路复用应用。通过反转录病毒的技术产生的基因工程条码的细胞，最初是一个漫长的过程，但允许人们获得，一旦建立，电池材料的可靠和稳定的来源。此技术的性质一致地产生遗传改造的细胞系与成为自己真实区分...

技术，如荧光光谱，荧光显微镜，流式细胞仪，全部依靠荧光，属性广泛利用生物化学，生物医药，化工等应用。荧光，无论本征或经标记，已经开发用于蛋白质表达的模式和分布，细胞命运，蛋白质相互作用和生物功能1-9的分析，以及通过对生物分子的相互作用和构象变化的荧光检测/荧光共振能量转移10-13。由于Aequorea victoria的绿色荧光蛋白（GFP）14的分离，从其他腔肠动物，特别是珊瑚附加天然存在的荧光蛋白质的发现，已经在很大程度上增加了现有的荧光蛋白质与可区别的激发/发射光谱的数目。这些，在他们的基因15-19引入突变在一起，甲肝Ë进一步扩大的可能性，获得提供给科学家们利用显微镜，流式细胞仪等荧光技术用于他们的研究荧光蛋白的真实调色板。 与此同时，虽然独立地，逆转录病毒技术的发展已大大促进了异位遗传信息的稳定表达在哺乳动物细胞20-23。因此，这是不奇怪，这种技术已被用来传输的荧光蛋白的基因导入细胞类型和组织24-28的广泛号码或用于生产转基因动物的29-31。以下逆转录病毒的性质，异位荧光蛋白的遗传信息的小区32的基因组中导入和细胞就荧光`为ever&#39;。该属性允许内细胞群的单个细胞的细胞命运的跟踪，或。现在荧光细胞也由此阿奎红其自身biosignature并且可以被定义为条形码的。其独特的biosignature来自其他小区的识别它，并且重要的是，从细胞中遗传操作以表达不同的荧光蛋白与可区分的吸收/发射光谱区分它。生物应用，例如重编程因子的跟踪朝向多潜能33的亚核因素的分析为核仁定位34的阐明，荧光报告质粒的构建转录研究35或神经元的神经元网络体系结构36的研究中的遗传标记，是那些利用不同的荧光蛋白基因相同的实验设置的许多只是四个例子。 流式细胞术已被广泛用于生物过程在单细胞水平，如基因表达，细胞周期，细胞凋亡的分析，并通过磷的信令荧光蛋白基因在哺乳动物细胞ATION 37-43 .The稳定表达进一步增强了流式细胞仪的细胞分析38,44和配体-受体相互作用45的效用。增强的功能已经允许流式细胞成为高通量和高含量筛选46一种广泛使用的方法。尽管可以耦合板读数器系统，成像和流式细胞术荧光计和机器人技术现在扩大数量，似乎有一个缺少的实验设计，可利用和适合这些增强技术能力。 有大幅度需要复用的应用进一步提升的高吞吐能力快速，可靠，简单而强大的基于细胞的方法。这是在药物发现领域尤其如此，其中以多路复用格式的工程基于细胞的测定可增强高通量筛选39,47-50电源</sup&gt;。复用的，因为它允许同时分析一个样本，进一步增强了高吞吐量能力51-54。荧光遗传条码不仅允许优雅复用，而且，一旦改造，规避了耗时的协议的需要，降低了成本伴随抗体，珠子和污渍39,52,55，并且可以减少所需的高屏幕数吞吐量的应用。我们最近已描述的技术如何逆转录病毒可以增强通过荧光遗传条码多路复用生物应用，通过表达以前开发并监控HIV-1蛋白酶活性的56,57具有不同的临床流行变体58的测定法。该方法是在一个更具描述性的方式解释着眼于如何选择和基因扩增荧光条码细胞如何产生克隆群体的表达板不同的荧光蛋白和/或不同的荧光intensitIES。细胞群区分基于其荧光特性的面板增强多路复用能力，它可以被进一步利用结合基于细胞的试验是对付不同的生物的问题。该协议还描述了如何对工程师条形码细胞轴承先前在实验室开发的基于细胞的测定中的一个的板，如实施例59。这个协议是因此不意图以显示既定的逆转录病毒/慢病毒技术进行基因转移，荧光蛋白的价值或流式细胞仪60,48的应用程序，而是以显示结合三个用于多路复用应用的增强能力。

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			<td height="21" style="height:21px;width:345px;">Name of Material/ Equipment</td>
			<td style="width:160px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:515px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">10mL syringes</td>
			<td style="width:160px;">BD&#160;&#160;</td>
			<td>309604</td>
			<td>used for filtering the virus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.45&#181;m plytetrafluoroethylen filter</td>
			<td style="width:160px;">pall corporation</td>
			<td>4219</td>
			<td>used for filtering the virus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">DMEM (Dulbecco&#39;s Modified Eagle Medium)</td>
			<td style="width:160px;">Corning</td>
			<td style="width:119px;">45000-304</td>
			<td>cell growth media for HEK 293T cells</td>
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		<tr height="21">
			<td height="21" style="height:21px;">PEI (Polyethylenimine)</td>
			<td style="width:160px;">poly sciences</td>
			<td>23966-2</td>
			<td>&#160;2mg/mL concentration used</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Hanging bucket centrifuge (refrigerated)</td>
			<td style="width:160px;">Eppendorf&#160;</td>
			<td style="width:119px;">5805 000.017</td>
			<td>used for spin infection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">PBS (phosphate buffered saline)</td>
			<td style="width:160px;">Corning</td>
			<td>21-040-CV</td>
			<td>used for washing of cells</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Polybrene (hexadimethreen bromide)</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td>107689</td>
			<td>Used to increase viral infection efficiency.&#160; Used at a 5&#181;g/mL concentration.&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:345px;">FACSAria</td>
			<td style="width:160px;">BD Biosciences</td>
			<td style="width:119px;"></td>
			<td>instrument used for sorting cell populations</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">FACSCanto</td>
			<td style="width:160px;">BD Biosciences</td>
			<td style="width:119px;"></td>
			<td>instrument used for cell analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Phoenix-GP</td>
			<td style="width:160px;">Gift from Gary Nolan</td>
			<td style="width:119px;"></td>
			<td>cell line used to produced retroviral particles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Fetal calf serum</td>
			<td style="width:160px;">Mediatech</td>
			<td>MT35015CV&#160;</td>
			<td>used for cell growth and sorting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">&#160;SupT1 cells</td>
			<td style="width:160px;">ATCC</td>
			<td>CRL-1942</td>
			<td>Human T lymphoblasts</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">HEK 293T cells</td>
			<td style="width:160px;">ATCC</td>
			<td>CRL-11268</td>
			<td>Human Embryonic Kidney cells that also contain the SV40 large T-antigen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">RPMI 1640</td>
			<td style="width:160px;">Corning</td>
			<td>10-040-CV</td>
			<td>cell growth media for SupT1 cells</td>
		</tr>
	</tbody>
</table>

genetic barcoding with fluorescent proteins for multiplexed applications

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded &#180;indefinitely&#180;. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

复用荧光基因条形码的细胞的生物应用的目的，才能达到一次个别克隆群已经产生。当条形码的人群与最低频谱重叠清晰鲜明的特色荧光复用是最有效的。在图1与哺乳动物SupT1细胞的克隆群所示的例子中示出了条形码的细胞mCherry和氰基荧光蛋白（CFP）可以容易地同时进行分析，而不会失去它们各自的荧光特性。此矩阵从而体现荧光基因条形码的单元的面板是用于在又一个附加的可利?...

Watch this Scientific Journal Video about Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications at JoVE.com

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

遗传条形码与荧光蛋白的复用的应用程序

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

genetic-barcoding-with-fluorescent-proteins-for-multiplexed

Research

JoVE Journal

Biology

9.2K 次观看.  San Diego State University. Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications. 

视频: Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

与非侵入性检测荧光染料标记的干细胞与光学成像

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

科研

生物学

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

双整装原位杂交技术在早期鸡胚

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

对活细胞成像的SNAP - Tag融合蛋白的荧光标记的COS - 7

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

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

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice. HYPERStack vessels allow closed system scale up from the current stacked plate products and delivers >2.5X more cells in the same volumetric footprint. The HYPERStack vessels function via gas permeable material which allows gas exchange to occur, therefore eliminating the need for internal headspace within a vessel. The elimination of headspace allows the compartment where cell growth occurs to be minimized to reduce space, allowing more layers of cell growth surface area within the same volumetric footprint.
For many applications such as cell therapy or vaccine production, a closed system is required for cell growth and harvesting. The HYPERStack vessel allows cell and reagent addition and removal via tubing from media bags or other methods.
This protocol will explain the technology behind the gas permeable material used in the HYPERStack vessels, gas diffusion results to meet the metabolic needs of cells, closed system cell growth protocols, and various harvesting methods.

An Introduction into the technology, protocol and handling of the Corning HYPERStack Vessels and accessories used for high yield adherent cell culture. The protocol will show how to use the closed system vessels for increasing cell harvesting over current stacked plate products.

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.   ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

粉末样品的绝对量子产量测量

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

DNA电穿孔，肌纤维的分离和成像

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.