作者没有确认。

1 Concatemerization <ol><li>引物悬浮FW和RV（见表材料的引物序列），以10微克/微升的分子生物学级别H 2 O的浓度然后，将5微升的FW和5微升旅行车底的1.5 ml离心管中。 </li><li>退火引物混合物通过加热到正确的退火温度（75℃，此处描述的引物）15分钟。离开板凳上冷静下来，在室温（RT）。通过溶解在DDH 2 O准备孵育期间，60％的PEG8000 </li><li>加入50μl的60％PEG8000（达到30％的终浓度），10微升的10倍多核苷酸激酶（PNK）缓冲液，27微升H 2 O和3微升PNK向引物混合物。在37℃下搅拌2小时。 </li><li>冷却室温，加入11微升DNA连接酶缓冲液中。再加入DNA连接酶3 U和孵化，在37°C过夜。 </li></ol> 2，DNA纯化与分析<ol><li>加入300微升H 2 O的以增加连接混合物的体积。注：氯仿​​蒸汽：在通风橱中，由于酚的毒性继续。 </li><li>加入1倍体积的苯酚（400微升）：氯仿并通过摇动剧烈混合。 </li><li>离心机中以最大速度在台式离心1分钟。水性和溶剂层将分离在最上面的水层和沉淀的蛋白的两个层之间的白色层。 </li><li>传送上层至新管通过小心移液（避免传输中，白层） </li><li>重复步骤2.2和2.3的至少两倍或直到没有沉淀的蛋白质离心后。 </li><li>加入1体积的氯仿（400微升），然后重复步骤2.3-2.4。 </li><li>添加100％预冷乙醇2卷（800微升），并培育在-20℃至少1小时或过夜。注：此步骤沉淀DNA的连环体。 </li><li>降速以最大的速度和吸苏培rnatant。加入1ml的70％乙醇洗一次，并允许在空气中干燥在通风橱中。 </li><li>重悬的DNA在50μl不含内毒素的DDH 2 O。注意：使用不含内毒素的试剂，是发生在细胞和体内的DNA特异性先天免疫反应的关键。 </li><li>取下concatemerized DNA的2微升等分到一个新的管加入1 ml的DNA凝胶上样缓冲液。 </li><li>溶解在TAE缓冲液的1％琼脂糖进行加热全功率在微波炉中2分钟（或时间，这是合适的烘箱和体积的功率）。 </li><li>倾入凝胶浇注装置，加入4微升荧光DNA螯合染料，插入梳子，并在室温下离开的凝胶来设置大约20分钟。 </li><li>一旦设定，取出梳子和地点的凝胶到含TAE缓冲液电泳槽。负载3微升的DNA样品或尺寸标记成井。 </li><li>跑电泳30-40分钟，在90 V与恒定电流。 Visuali泽下紫外光将DNA观察concatemerization（ 图1A）。 </li><li>测量在样品由UV吸收光谱的其余部分的DNA浓度。空白分光计具有相同的DDH 2 O的，是用来重悬DNA和从200-300纳米测量的吸收光谱。注：DNA浓度可以使用比尔 - 朗伯定律，A = E * C * L，其中A是吸光率来计算，e为消光系数，并且升的光路的距离。用于测量浓度DNA -1 -1所用的260nm处的吸光度应测定和0.027（微克/毫升）的消光系数。光路距离，升，将根据分光光度计变化。在测量浓度，注意到260/280吸光度比值作为DNA的纯度的指示;建议对上述2.00这个比例值。 </li></ol> 3，转双链DNA连环中的Vi卓<ol><li>种子细胞在组织培养皿或板为70％汇合，在刺激的时间。 </li><li>计算DNA的需要使用的1-10微克/毫升的最终浓度的细胞的刺激物质。 DNA的所需的质量将会注意到为X以下。例如，如果在5微克/毫升的DNA的浓度则质量为2×5 = 10微克的DNA在6孔板中以2毫升培养基的单井刺激细胞是必需的。 </li><li>添加50 * X微升无血清培养基中成适当大小的无菌管中。移液管小心地插入介质的中心位置，而不接触管，2 * X微升阳离子脂质转染试剂和离开5分钟的一侧。 </li><li>添加DNA的第X微克到管和涡简要介绍。发表于室温至少20分钟。 体外转染直接添加该混合物的细胞，刺激细胞质DNA的检测途径。 </li></ol> 4，转双链DNA连环I的Ñ​​体内<ol><li>如在使用3.1-3.5 1微克的DNA，2微升的脂质转染试剂和18微升血清每只小鼠耳培养基被刺激描述制备转染混合物。准备一个无菌的20微升Hamilton注射器，瓶盖用无菌地下30针，并加载用DNA转染混合物。 </li><li>麻醉来源于C57BL / 6小鼠用1-2％的异氟醚。通过移动和固定呼吸频率的损耗和捏反射测试，如果有必要确认麻醉。麻醉过程中监测动物的眼睛，用药膏的要求，以避免过度干燥。 </li><li>使用无菌技术，注入小心到耳廓10微升转染混合物，使得液体的单个“泡沫”是耳朵的真皮层之间形成的。注意：使用的橡胶套管上的拇指或食指，伸展耳过它，并注入成锐角，以确保针穿透耳廓皮肤只有第一层。 </li><li>收服troduce鼠标放回笼子，并定期监测后麻醉复苏。使用加热灯，以避免麻醉小鼠的过度冷却。不要让老鼠无人看管，直到他们重新获得足够的意识来维持胸骨斜卧。 </li><li>闪光通过浸入液氮中2分钟，冻结在微量离心管中，整个耳廓。 </li></ol> 5，处理的DNA刺激的样品和分析定量PCR技术（定量PCR） <ol><li>从细胞培养：中吸用吸管和丢弃垃圾。 </li><li>加入1 ml无菌PBS。重复5.1和5.2的两倍，并继续执行步骤5.3 </li><li>从耳组织：使用无菌陶瓷杵和研钵在液氮下研磨组织，用消毒压舌板地组织转移到离心管中，并继续执行步骤5.4 </li><li>添加350μL裂解液，并继续与商业RNA提取试剂盒的协议。洗脱RNA的提取纯化试剂盒在40微升DDH 2 O列注：商业RNA提取试剂盒（见材料/设备表）工作得很好;其他技术进行了测试与欠佳的结果。 </li><li>如对以上的DNA（第2.13）量化紫外光谱的RNA。注意：消光系数为RNA是0.025（微克/毫升）-1厘米-1。 </li><li>加入到无菌的PCR管中加入1μl的寡聚（dT）引物，1微升的10mM dNTP混合物，和250微克的RNA。补足体积至11微升用无菌DDH 2 O。 </li><li>孵育5分钟，在65℃下。再加入4微升第一链缓冲液，1μl的0.1M的二硫苏糖醇，0.25微升反转录酶和1.75微升DDH 2 O的到所述管。孵育在50℃下进行1小时，随后在72℃下15分钟。使用DDH 2 O 5：可选，稀释cDNA产物1 </li><li>对于定量PCR使用2微升的cDNA，2微升DDH 2 O 5微升的qPCR预混液和1μL每10 uM的正向和反向引物储备来扩增所选择的基因（次）。混合，在96孔板中，并使用定量PCR仪（ 图1B和图1C）进行分析。 </li></ol>

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The authors have nothing to disclose.

这里所述的协议被用于产生双链DNA，以刺激与在广泛的细胞类型并在体内可再现的结果胞质先天免疫应答。因为所用的主要底物的试剂是人工合成的寡核苷酸，存在于该系统中，使用替代性的DNA序列或化学修饰的灵活性。这是可能的，例如，以取代在本协议与一个包含荧光标记或生物素部分跟踪定位或评估与其它生物分子的相互作用所描述的正向链寡核苷酸。 之一的DN...

DNA是所有生物和细胞，真核细胞保持在特定车厢的重要组成部分。先天免疫系统的一个功能是确定当这种隔离发生故障或当异物经由违反物理，外部屏障引入到生物体中。在人体内有大量存在，以帮助降低少量的DNA，它可能逃过了正确的条块分割的蛋白质; DNA酶I，II和III分解的DNA在等离子体中，核内体，和胞质溶胶，分别。然而，已经证明，缺乏DNA酶II的小鼠免受过量产生干扰素1表明的严重贫血死了先天免疫系统响应外核DNA。这个反应发生时，即使在小鼠这是在Toll样受体信号转导的能力完全缺失。现在许多研究表明干扰素基因（STING）2和坦克结合激酶1（结核病的刺激K1）3顷涉及在细胞质中的DNA的检测，并且此信号转导途径激活干扰素调节因子（IRF）-3 4。细胞因子，趋化因子和干扰素基因的后续IRF3依赖性转录是要么病原体或危险相关分子模式（PAMP或者DAMP）5先天免疫应答的特性。 来研究细胞内的核酸检测通路的愿望已经导致要求开发可再生的方法，通过将DNA或RNA导入细胞中，而不激活其它先天免疫反应刺激细胞。一种方法，通过它的刺痛/ TBK1 / IRF3途径可刺激是通过使用阳离子脂质体转染试剂来递送核酸进入其中它可以通过DNA的传感器，如DNA-PK，IFI16，和C气体6-8被访问的细胞质中。 的DNA是目前的理解，让EFF的特点没有其他途径的刺激细胞质先天免疫应答的icient活化是其长度，质量，纯度和缺乏的CpG基序4,7,9。合成的寡核苷酸是高度适合于产生先天免疫应答，因为​​它们允许所述DNA序列进行优化，并制备成纯化学物种的目的。 A 45个碱基对的免疫刺激DNA（ISD）的序列进行鉴定的Stetson 等 4为是合适的，CpG的自由序列用于此目的。该双链DNA序列是随机的，否则，没有具体的功能超出了其缺乏CpG基序的。我们已经发现，通过concatemerizing的ISD寡成显著长链增加刺激7的大小。 concatemerized新闻处的产生，因此刺激细胞质先天免疫反应。这里，我们提出的协议用 ​​于制备该试剂，以及它如何被用来激活该DNA传感通路在体外和体内 。 注：所有动物研究是根据批准的机构的协议和动物保健指导进行。

<table border="0" cellpadding="0" cellspacing="0" width="1066">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Forward Primer</td>
			<td style="width:221px;">Integrated DNA technologies</td>
			<td style="width:144px;">n/a</td>
			<td style="width:407px;">TACAGATCTACTAGTGATCTATGACTGATCTGTACATGATCTACA</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:295px;">Reverse Primer</td>
			<td style="width:221px;">Integrated DNA technologies</td>
			<td style="width:144px;">n/a</td>
			<td>TGTAGATCATGTACAGATCAGTCATAGATCACTAGTAGATCTGTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">PEG8000</td>
			<td style="width:221px;">Sigma-Aldrich</td>
			<td style="width:144px;">P-4463</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Molecular biology grade water</td>
			<td style="width:221px;">Sigma-Aldrich</td>
			<td style="width:144px;">W4502-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">T4 Polynucleotide Kinase</td>
			<td style="width:221px;">Promega</td>
			<td style="width:144px;">M4101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">T4 DNA Ligase</td>
			<td style="width:221px;">Promega</td>
			<td style="width:144px;">M1801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Phenol:Chloroform</td>
			<td style="width:221px;">Fisher Chemical</td>
			<td style="width:144px;">BPE1752p-400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Chloroform</td>
			<td style="width:221px;">Fisher Chemical</td>
			<td style="width:144px;">C/4960/PB08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Ethanol</td>
			<td style="width:221px;">Sigma-Aldrich</td>
			<td style="width:144px;">32221-2.5L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">DNA gel loading buffer</td>
			<td style="width:221px;">New England biolabs</td>
			<td style="width:144px;">B7021S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Agarose</td>
			<td style="width:221px;">Bioline</td>
			<td style="width:144px;">BIO-41025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Optimem</td>
			<td style="width:221px;">Life Technologies</td>
			<td style="width:144px;">11058021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TransIT-LT1&#160;</td>
			<td style="width:221px;">Mirus Bioscience</td>
			<td style="width:144px;">MIR 2300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Hamilton Syringe</td>
			<td style="width:221px;">Hamilton</td>
			<td style="width:144px;">80901 Model 1705</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">30G needles&#160;</td>
			<td style="width:221px;">BD bioscience</td>
			<td style="width:144px;">304000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">SYBR Safe DNA gel stain</td>
			<td style="width:221px;">Life Technologies</td>
			<td style="width:144px;">S33102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Rneasy Plus Mini Kit</td>
			<td style="width:221px;">Qiagen</td>
			<td style="width:144px;">74134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Oligo(dT) primer</td>
			<td style="width:221px;">Thermo Scientific</td>
			<td style="width:144px;">SO131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">dNTP mix</td>
			<td style="width:221px;">Fermentas</td>
			<td style="width:144px;">R0192</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Super Script III reverse transcriptase</td>
			<td style="width:221px;">Life Technologies</td>
			<td align="right" style="width:144px;">56575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">First strand cDNA synthesis buffer</td>
			<td style="width:221px;">Life Technologies</td>
			<td style="width:144px;">y02321</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">SybrGreen qPCR Fast master mix</td>
			<td style="width:221px;">Applied Biosystems</td>
			<td align="right" style="width:144px;">4385612</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">cxcl10 murine forward primer&#160;</td>
			<td style="width:221px;">Integrated DNA technologies</td>
			<td style="width:144px;">n/a</td>
			<td>ACTGCATCCATATCGATGAC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">cxcl10 murine reverse primer</td>
			<td style="width:221px;">Integrated DNA technologies</td>
			<td style="width:144px;">n/a</td>
			<td>TTCATCGTGGCAATGATCTC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">il6 murine forward primer&#160;</td>
			<td style="width:221px;">Integrated DNA technologies</td>
			<td style="width:144px;">n/a</td>
			<td>GTAGCTATGGTACTCCAGAAGAC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">il6 murine forward primer&#160;</td>
			<td style="width:221px;">Integrated DNA technologies</td>
			<td style="width:144px;">n/a</td>
			<td>GTAGCTATGGTACTCCAGAAGAC</td>
		</tr>
	</tbody>
</table>

stimulation of cytoplasmic dna sensing pathways in vitro and in vivo

为了有效地刺激先天免疫应答的DNA必须具有足够的长度和纯度。我们提出了一个方法，其中双链DNA（dsDNA）的有必要的特性，刺激细胞质DNA的检测途径可以廉价且容易产生。由短的，合成的寡核苷酸（其缺乏CpG基序）的concatemerization，双链DNA可产生具有足够的长度，以激活细胞内的DNA检测途径。这个协议包括在聚乙二醇的存在下（PEG），它提供了有效的连接，以产生一个环境的寡核苷酸的平端连接。该双链DNA连环体，可以使用以下的纯化通过苯酚/氯仿萃取，以模拟由标准转染方案在体外的先天免疫应答。该DNA也可以用来刺激先天免疫在体内通过皮内注射到小鼠的耳廓，例如。由规范concatemerization过程和随后的刺激方案中，先天性免疫系统的可靠和可重复的活化可以生产。

<html>下面的结果表明，可以相对容易地产生concatemerized DNA和能够刺激在细胞和小鼠健壮的先天免疫应答。与使用PEG8000的，它可以清楚地看出，DNA的生成长度为显著较长，同样能够诱导CXCL10的转录。所看到的琼脂糖凝胶分析中，标准concatemerization具有高达800个碱基对（bp）的DNA的长度，而对于与PEG8000的样品，DNA的长度，包括在超过10,000 bp的（ 图1A）链。这些DNA插入小鼠胚胎成纤维细胞?...

Watch this Scientific Journal Video about Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo at JoVE.com

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

该协议的目的是要使用的最佳方法用于刺激细胞质DNA传感途径在细胞和体内 。这是通过平末端连接时改善长时间，双链DNA的生成来实现的。细胞或小鼠用脂质转染试剂转染。

细胞质DNA的检测途径的刺激<em&gt;在体外</em&gt;和<em&gt;体内</em

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA ...

stimulation-of-cytoplasmic-dna-sensing-pathways-in-vitro-and-in-vivo

Research

JoVE Journal

Biology

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

8.8K 次观看.  University of Cambridge. 该协议的目的是要使用的最佳方法用于刺激细胞质DNA传感途径在细胞和体内 。这是通过平末端连接时改善长时间，双链DNA的生成来实现的。细胞或小鼠用脂质转染试剂转染。 

视频: Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

在体外和体内生物发光记者人类胚胎干细胞的基因显像

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

科研

生物学

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

使用哺乳动物骨骼肌的DNA转染在体内电穿孔

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

In vivo and in vitro Studies of Adaptor-clathrin Interaction

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs are distinguished by a polyhedral lattice of clathrin that coats the vesicle membrane and serves as a mechanical scaffold. Clathrin coats are assembled during vesicle formation from individual clathrin triskelia , the soluble form of clathrin composed of three heavy and three light chain subunits7,8. Because the triskelion does not have the ability to bind to the membrane directly, clathrin-binding adaptors are critical to link the forming clathrin lattice to the membrane through association with lipids and/or membrane proteins9. Adaptors also package transmembrane protein cargo, such as receptors, and can interact with each other and with other components of the CCV formation machinery9.
Over twenty clathrin adaptors have been described, several are involved in clathrin mediated endocytosis and others localize to the trans Golgi network or endosomes9. With the exception of HIP1R (yeast Sla2p), all known clathrin adaptors bind to the N-terminal -propeller domain of the clathrin heavy chain9. Clathrin adaptors are modular proteins consisting of folded domains connected by unstructured flexible linkers. Within these linker regions, short binding motifs mediate interactions with the clathrin N-terminal domain or other components of the vesicle formation machinery9. Two distinct clathrin-binding motifs have been defined: the clathrin-box and the W-box9. The consensus clathrin-box sequence was originally defined as L[L/I][D/E/N][L/F][D/E]10 but variants have been subsequently discovered11. The W-box conforms to the sequence PWxxW (where x is any residue).
Sla1p (Synthetic Lethal with Actin binding protein-1) was originally identified as an actin associated protein and is necessary for normal actin cytoskeleton structure and dynamics at endocytic sites in yeast cells12. Sla1p also binds the NPFxD endocytic sorting signal and is critical for endocytosis of cargo bearing the NPFxD signal13,14. More recently, Sla1p was demonstrated to bind clathrin through a motif similar to the clathrin box, LLDLQ, termed a variant clathrin-box (vCB), and to function as an endocytic clathrin adaptor15. In addition, Sla1p has become a widely used marker for the endocytic coat in live cell fluorescence microscopy studies16. Here we use Sla1p as a model to describe approaches for adaptor-clathrin interaction studies. We focus on live cell fluorescence microscopy, GST-pull down, and co-immunoprecipitation methods.

In vivo and in vitro Studies of Adaptor-clathrin Interaction

Clathrin-mediated endocytosis depends on adaptor proteins that coordinate cargo selection and clathrin coat assembly. Here we describe procedures to study adaptor-clathrin physical interaction and live cell imaging approaches using as a model the yeast endocytic adaptor protein Sla1p.

适配器，网格蛋白相互作用在体内和体外研究

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs ...

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

MG53介导的细胞膜使用维修的可视化在体内在体外系统

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

激活细胞凋亡的细胞色素的细胞质显微注射 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a &#34;double decker&#34; microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm.

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

The giant ciliate Stentor coeruleus is a classical system for studying regeneration and wound healing in single cells.&#160;By imaging Stentor cells simultaneously at low and high magnification it is possible to measure cytoplasmic flows before, during, and after wounding.

可视化细胞质流动过程中单细胞伤口愈合<em&gt;蓝斑宏声</em

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

在c-fos蛋白免疫组织学检测：一个有用​​的工具，中央通路在特定的生理反应所涉及的标记<em&gt;在体内</em&gt;和<em&gt;离体</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤：一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...