我们的实验室研究是由美国国立卫生研究院院内研究计划，国家酒精滥用和酒精中毒研究所的支持。

<html>一，制备注射本节介绍前注射到神经元的细胞核基因需要采取的准备步骤。神经元的分离过程是超出了本文的范围，但重要的是有分离成单个细胞的神经元和坚持以及35毫米的细胞培养皿的底部表面（见讨论有用的技巧来实现这一）。虽然可能被用于各种不同的神经元，如颈上神经节（SCG）或背根神经节的外围神经节解剖的首选，因为它们方便地访问，并产生大量的神经元。使用SCG神经元的附加 ​​优点是，他们是一个人口相对同质的细胞和良好的特点是 1,2,3,4 。 A.质粒制备的cDNA注射<ol><li>为了防止DNA的污染或分解，必须戴上手套在准备。 </li><li>克隆到一个合适的哺乳动物表达载体，如PCI（Promega公司，麦迪逊，威斯康星州）的高度和组成性激活的巨细胞病毒（CMV）启动子的调控下，DNA编码的蛋白质的利益。如果蛋白质没有标签，记者质粒编码的EGFP的pEGFP - N1，BD Biosciences公司Clontech公司，美国加州Palo Alto），例如（是合作​​注入确认成功的注入和表达。 DNA是使用高品质的分离柱（如QIAfilter的Midi - PREP试剂盒，QIAGEN，Chatsworth的，CA），并进一步纯化与PVDF过滤器（0.1微米，Millipore公司，贝德福德，马萨诸塞）使用离心式过滤装置去除微粒分离质粒的准备，它可以阻碍在注射过程中注射吸管。质粒是存储在一个约1微克/微升的浓度，在适当的DNA存储缓冲区（如TE缓冲液：10毫米，1毫米EDTA，pH值8.0的Tris）在-20 ° C。 </li><li>紧接显微注射，质粒的cDNA稀释和混合，以TE缓冲液中或H 2 O所需终浓度通常情况下，10纳克/μL记者基因是足够的标签注射细胞，由于DNA在高浓度的粘性，可以堵塞注射吸管的基因注入的总浓度不得超过200毫微克/μL。准备一个基因注射体积小（5-10μL）混合溶液的滴的封口膜（纸背底侧）的一小块干净的表面上。混合溶液滴pipeting向上和向下一个pipetor几次，混合过程中，避免引入气泡。 </li><li>使用microloader吸管尖（Eppendorf公司，布林克曼仪器，韦斯特伯里，纽约州），转移到专门准备的血细胞比容管（尔科技，宾夕法尼亚州匹兹堡）的cDNA解决方案。准备（材料表），血细胞比容管的说明材料。含有基因注射液1.5毫升的离心管，将血细胞比容管。 </li><li> 15-30分钟10 000克，在配备有一个固定角度或水平转头（Eppendorf公司）微量离心管，在室温（20-24 ° C）泥沙颗粒的cDNA的解决方案，可能会阻止显微注射吸管。的基因注射液可在室温下保存在注射会议。 </li></ol> B.注射液吸管的制备<ol><li>一次性玻璃微量注射吸管商业（如Femtotips，Eppendorf公司），这是方便，但价格昂贵（10元显微注射吸管）。可以生产注射用吸管在实验室用一个可编程的P - 97火焰山布朗吸管拉马（萨特仪器公司，诺瓦托，CA）和filamented，薄壁高硼硅玻璃毛细管（世界精密仪器，萨拉索塔，佛罗里达州）。此选项是更具成本效益和允许显微注射吸管的形状和大小的控制。 </li><li>一个两阶段的计划是用来拉窄显微注射吸管提示。由于开放显微注射吸管是过于狭窄，光镜下检查，质量注射吸管直接注入少量细胞程序设置完成之前，必须加以评估。以下是热，拉，速度和时间设置的近似设置：（拉1）600，115，15，250;（拉2）640，130，65，200。这些设置随不同批次的玻璃加热丝在拉拔条件，并应根据需要进行调整。有关详细信息，请参阅5 。 </li><li>拉几个（2-5）注入每盘的神经元被注入注射会议期间损坏或问题的情况下，吸管。商店在有盖容器内注入吸管，以防止灰尘进入显微注射吸管尖。 </li></ol>二。核内微量注射帐篷“&gt;本节详细说明注射到神经元的细胞核基因的过程。一个基本的显微注射的设置包括一个倒置相差显微镜（如尼康Diaphot战区导弹防御系统，尼康，梅尔维尔，纽约州）可视化的过程中，显微（如5171 Eppendorf公司）控制运动注入吸管和一个高压注射器（如Eppendorf公司FemtoJet快递）开除吸管的cDNA的解决方案。，连接高压注射器的显微操纵器允许在注射后两个过程同步。可选，但强烈建议，组成部分包括一个安装视频监控系统（CCD摄像头，Cohu公司，加利福尼亚州圣迭戈;黑白视频监视器，索尼公司，东京，日本），本系统不注射的绝对要求;然而，黑色和白色的显示器，提供高对比度的提高可视化的细胞核，并提供一个更舒适的视觉体验，在长期注射会。此外，一台笔记本电脑上运行的软件操作手持式控制器可利用半注射过程自动化（见细节的讨论 ）。 注入SCG神经元的理想时间是3-6小时后离解。在这一阶段，细胞是球形的，紧紧的贴在底部的菜，是清晰可见的细胞核，相差显微镜下，作为一个中央的圆形深色膜细胞器，含有一个或多个核仁（图1A）。 <ol><li>游离神经元菜放在显微镜阶段的中心。调整的重点和优化相衬光学显微镜，使神经元的细胞核都清晰可见。 </li><li>吸取2μL编制成的显微注射用一个microloader吸管尖吸管的cDNA溶液。避免的血细胞比容管的底部附近的绘图解决方案，因为这可能会挑起颗粒在离心落户。灯丝在显微注射吸管应援解决方案给小费，但如果坚持小气泡，轻轻一抖一侧的玻璃，赶走他们。 </li><li>插入高压注射器毛细管持有人的显微注射吸管，然后安全持有人的显微。支架固定在45度角。 </li><li>下入菜的显微注射吸管。吸管进入培养液，半月板的形成，影响光线的折射，降低了神经元的图像质量。为了缓解这一问题，可略有偏移的阶段环炮塔，直到神经元的细胞核都清晰可见。 </li><li>一些高压喷射系统有一个“干净”的功能，适用于很短的时间内微量注射吸管尖的最高气压。使用此功能吸管开始之前和期间注射，如果提示出现堵塞的杂物清除。如果消除流体不显示在屏幕上同时使用此功能，请更换一个新的注射吸管。 </li><li>中心在观看监视器的显微注射吸管和对齐旁边的一个神经细胞，并在同一焦平面核仁提示。设置此位置较低的Z轴的显微操纵器上的限制。这一立场是显微注射吸管将提前在注射的深度。您现在的位置提示这一点大约30微米以上，因此，显微注射吸管清除神经元的顶部。 </li><li> “注入”的Eppendorf 5171显微操作模式可以定义以下设置执行核内注射。 注入功能设置上，而刺穿功能不放过。对角线注射路径（沿X轴和Z轴）产生量最少的细胞损伤，因此应注射使用的轴向模式。一个300微米/秒的注射速度是足够的，同步的压力上升时，显微注射吸管达到设定Z轴的限制。 </li><li>高压注射器控制注射到细胞核的基因解决方案的数量。在“自动”注射模式，提供的DNA是时间控制和连接显微发起的。 注射压力 （PI）应设置100至200百帕（HPA; 1百帕〜0.015 PSI）之间;较高的注射压力不提高成功率或注射质量，并可能会指出与其他显微注射吸管针尖大小或DNA的纯度问题。 注射持续时间（TI）0.3秒和30百帕，它提供了一个恒定的正压，以避免输入的咬合到吸管尖端的培养液中的颗粒（PC）的补偿压力，应该使用。 </li><li>显微注射使用显微镜的XY轴阶段控制的吸管尖下注射神经元的位置。吸管对准重点之间来回吨以上的核中心的一角他提示的核仁。 </li><li>按注入按钮（显微）注入细胞核。对于注射步骤，显微注射吸管和释放的cDNA溶液的运动控制的显微操纵器。这是用肉眼难以确认成功的核内注射，但相对较低的粘度cDNA的解决方案注射液（粘性的核环境相比），往往会出现白色羽下相衬光学和可用于注射的指标进入细胞核。有时显微注射的吸管不穿透核膜，只需轻推细胞核。在同一位置第二次注射的尝试可能会导致成功进入细胞核的DNA注射。肿胀的细胞通常是无意识的细胞质注射指示的。此外，重要核肿胀是没有很好的耐受性细胞死亡的神经元和平时成绩后不久，因此，注射压力和持续时间不应超过建议值。 </li><li>继续注资，通过重新定位，在显微镜阶段下显微注射吸管对齐下一个神经元的神经元。系统导航的菜约50-100细胞核注入过夜培养的神经元的菜，然后返回到孵化器。成功注入神经细胞可以识别翌日记者基因的表达。 </li></ol>三。代表性的成果<img src="/files/ftp_upload/1614/1614fig1.jpg" alt="图1" /> 图1：颈上神经节（SCG）神经元 。 SCG神经元的相位对比度的图像3小时后离解（一）。在这个阶段中，细胞核清晰可见，艾滋病的显微注射吸管尖对齐。原子核出现在中心的神经元（左）单一或多个（右）暗核仁。 SCG神经元以下16个小时的EGFP基因进入细胞核（二）注射。左，SCG相衬照明下观看的神经元。右键，啶形象的成功注入，以及由此产生的一个神经EGFP的表达相同的神经细胞。 白比例尺是20微米。 <img src="/files/ftp_upload/1614/1614figure2tmb.jpg" alt="图2" border="0" /> 图2：全细胞电流的录音通过异源表达的G蛋白偶联内向整流钾离子（GIRK）通道SCG神经元。 GIRK电流（我GIRK）诱发200 ms的斜坡电压从-60 mV的电位从-140至+40 mV的去甲肾上腺素（A的插图）以下激活内源性 α2受体（东北，10 μM） 。叠加的痕迹是由相同的神经元的录音，并指出前（黑色）和应用程序的任东北单独（蓝色）或东北加10毫米BA 2 +（红色）后。虚线表示零电流水平。 对于录音，外部溶液中（毫米）130氯化钠，5.4氯化钾，10 HEPES，10 2氯化镁， 氯化钙 ，0.8的15葡萄糖，15蔗糖，和0.0003 TTX，调整pH值用NaOH 7.4我GIRK。内部的录音解决方案中（毫米）135氯化钾，11 EGTA，1 2 2氯化钙，氯化镁2，10 HEPES，4 MgATP 和 0.3 NA 2的GTP，用KOH调节pH至7.2。 目前缺乏uninjected SCG神经元对NE引起的电压斜坡（一）演示，SCG神经元不表达内源性GIRK通道。成功的注射质粒cDNA编码功能GIRK通道11的电压斜坡上升期间，大电流时，接触到东北（B，左）。电流有电流通过GIRK通道的典型特征：活化的G蛋白偶联受体激动剂（NE），内向整流，和公认的GIRK通道阻滞剂，BA 2 +（B，正确的红色曲线）的电流块。开放和充满圆圈代表我GIRK分别控股和峰值电流。请<a href="https://www.jove.com/files/ftp_upload/1614/1614figure2.jpg">点击这里</a>看大图图2。

<ol>
	<li>Eccles, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+action+potential+of+the+superior+cervical+ganglion.">The action potential of the superior cervical ganglion.</a> J Physiol. 85, 179-206 (1935).</li><li>Ikeda, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-dependent+modulation+of+N-type+calcium+channels+by+G-protein+&#x03B2;&#x03B3;+subunits.">Voltage-dependent modulation of N-type calcium channels by G-protein &#x03B2;&#x03B3; subunits.</a> Nature. 380, 255-258 (1996).</li><li>Schofield, G. G., Ikeda, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+currents+of+acutely+isolated+adult+rat+superior+cervical+ganglion+neurons.">Potassium currents of acutely isolated adult rat superior cervical ganglion neurons.</a> Brain Res. 485, 205-214 (1989).</li><li>Schofield, G. G., Ikeda, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+and+calcium+currents+of+acutely+isolated+adult+rat+superior+cervical+ganglion+neurons.">Sodium and calcium currents of acutely isolated adult rat superior cervical ganglion neurons.</a> Pflugers Arch. 411, 481-490 (1988).</li><li>Dean, D. A., Goldman, R. D., Spector, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+4.">Chapter 4.</a> Live Cell Imaging: a Laboratory Manual. 1, 51-66 (2005).</li><li>Ikeda, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterologous+expression+of+receptors+and+signaling+proteins+in+adult+mammalian+sympathetic+neurons+by+microinjection.">Heterologous expression of receptors and signaling proteins in adult mammalian sympathetic neurons by microinjection.</a> Methods Mol Biol. 83, 191-202 (1997).</li><li>Ikeda, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+G-protein+signaling+components+in+adult+mammalian+neurons+by+microinjection.">Expression of G-protein signaling components in adult mammalian neurons by microinjection.</a> Methods Mol Biol. 259, 167-181 (2004).</li><li>Ikeda, S. R., Jeong, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+RGS-insensitive+G&#x03B1;+subunits+to+study+endogenous+RGS+protein+action+on+G-protein+modulation+of+N-type+calcium+channels+in+sympathetic+neurons..">Use of RGS-insensitive G&#x03B1; subunits to study endogenous RGS protein action on G-protein modulation of N-type calcium channels in sympathetic neurons..</a> Methods Enzymol. 389, 170-189 (2004).</li><li>Washbourne, P., McAllister, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+gene+transfer+into+neurons.">Techniques for gene transfer into neurons.</a> Curr Opin Neurobiol. 12, 566-573 (2002).</li><li>Zhang, Y., Yu, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+microinjection+technology+in+cell+biology.">Single-cell microinjection technology in cell biology.</a> Bioessays. 30, 606-610 (2008).</li><li>Vivaudou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+G-protein+regulation+of+GIRK1+and+GIRK4,+the+two+subunits+of+the+KACh+channel,+using+functional+homomeric+mutants.">Probing the G-protein regulation of GIRK1 and GIRK4, the two subunits of the KACh channel, using functional homomeric mutants.</a> J Biol Chem. 272, 31553-31560 (1997).</li></ol>

遇到的常见问题和建议： 如前所述，成功的核注射依赖于组织培养板的细胞粘附。推迟注射开始后，细胞分离程序的几个小时，使神经元坚持的菜肴底部。此外，0.1毫克/毫升的超高分子量聚- L -赖氨酸（Sigma公司，圣路易斯，密苏里州）艾滋病神经元的菜肴底部表面的粘附涂层的菜肴。 显微注射吸管堵塞，尤其是当试图注入高浓度的DNA，可以是一个经常出现的问题。使用高品质分离柱分离和纯化质粒DNA，并提到（旋转过滤器，血细胞比容管纺丝）执行额外的净化步骤，可以帮助到注射前去除颗粒。在注射剂，“干净”的压力，喷油器的功能可以使用（0.2秒就足够了），但如果不解决这个问题，一个新的注射吸管，应使用。如果大多数显微注射吸管成为堵塞注射会议期间，考虑改变玻璃吸管拉马的设置，以生产具有较大的开放显微注射吸管提示。 在核注射产生的另一个问题是组织培养皿的底部表面的不平衡。这种差异直接影响定位精度注射吸管的小窍门到细胞核内，在注射吸管穿越以来的深度是固定的。因此，Z轴的限制，在注射在同一盘过程中必须调整。这将是明显的调整是必要的：不会有核注的迹象（没有细胞核或细胞是完全错过的白色羽），或注射吸管尖接近底部的菜（压缩单元甚至轰然到吸管尖盘底部）。在神经元的电镀玻璃克隆气瓶（外径10毫米，猫号2090-01010，Bellco玻璃，葡萄​​园，新泽西州），可用于限制在一个较小，多局限于区域，因此，最大限度地减少需要移动的距离之间的注射剂注射吸管。这些克隆气瓶被删除之前执行microinjections。 技巧的缺点： 核内microinjections技术要求高，这需要耐心和手巧由运营商的高度。这种技术的熟练程度，与任何其他技能一样，是与实践。因此，建议尝试实际的实验之前，记者基因的核注射单独练习。为了进一步协助注射过程中，它可能会巩固到计算机程序中的显微操纵器和高压注射器的步骤。伊戈尔临（WaveMetrics，奥斯威戈湖，或），如程序可以利用存储显微注射的设置和程序注射过程半自动化多功能控制器（ShuttlePro，外形设计，温德姆，NH）（见6,7,8细节）。 核内显微注射技术的另一个缺点是成功率很低。企图核注射约为10-20％，导致蛋白的表达。虽然这种效率可能是足够的应用程序，不需要大量表达的细胞，例如电，各种生化分析，这可能不足以的。 应用技术： 尽管它的缺点，细胞核内的DNA显微注射法是一个极为有用的方法，为异源表达蛋白在神经元。 蛋白的表达水平高，是实现核microinjections因为大量的过程中释放到细胞核内注射，一个高度活跃的CMV启动子调节基因的转录，并在细胞核中的质粒的长期稳定的质粒。蛋白质的过度表达显性负的外源蛋白的高层次需要压倒的内源性蛋白的实验，尤其是在有用的。核内注射的另一个优点是能够提供一致的基因结构比例注入到细胞核内，当多个构造的。与其他转染方法，是很难再现介绍每个DNA相同的比例，构建成不同的细胞在同一盘之间的转染。细胞核的cDNA溶液直接注入消除这种变异的来源，并允许有效滴度表达的蛋白质的比例。 传统的转染方法在有丝分裂后细胞的成效有限，因为DNA 的细胞核9转REAG化学毒性低纳入经济需求测试10。核microinjections克服这些问题，通过引入到细胞核内的遗传物质机械。虽然更多地参与，这种技术是研究蛋白质功能的生物系统的影响的能力，完成与内源性信号通路，多费力这项技术的性质进行补偿。 所有实验动物的程序进行，按照与美国国立健康的动物护理和使用指南研究院。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Ultrafree-MC Filter Unit</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>UFC30VV00</td>
<td>Durapore PVDF filter; 0.1 &mu;m pore-size</td>
</tr>
<tr>
<td>TE buffer</td>
<td>&nbsp;</td>
<td>Quality Biological, Inc</td>
<td>351-010-131</td>
<td>10 mM Tris, 1 mM EDTA, pH 8.0</td>
</tr>
<tr>
<td>Micro-hematocrit capillary tubes</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>22-362-574</td>
<td>Unheparinized Preparation: 
Soak in 100% ethanol overnight. Wash thoroughly with deionized water three times and dry bake glass in an oven (95&deg;C). Cut the cleaned hematocrit tubes into 3-4 cm length pieces using a diamond-tipped scriber, and seal one end closed with a flame and forceps to hold the glass. Store in a clean glass beaker and cover to prevent dust build-up. </td>
</tr>
<tr>
<td>Microcentrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5417 R</td>
<td>With a fixed angle or swinging-bucket rotor</td>
</tr>
<tr>
<td>Microloader 20 &mu;l pipet tips</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5242 956.003</td>
<td>&nbsp;</td>
<tr>
<td>Microinjection capillary glass</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>TW120F-4</td>
<td>Thin-walled with filament, 1.2 mm outer-diameter, 0.9 mm inner-diameter, 100 mm length</td>
</tr>
<tr>
<td>Flaming/Brown Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument Co.</td>
<td>P-97</td>
<td>With platinum-iridium box filament (part # FB330B)</td>
</tr>
<tr>
<td>Nikon Diaphot TMD Inverted Microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>20x and 40x phase-contrast objective lenses, mounted on a vibration isolation table</td>
</tr>
<tr>
<td>Charge-coupled device (CCD) camera </td>
<td>&nbsp;</td>
<td>Cohu Inc.</td>
<td>6410</td>
<td>&nbsp;</td>
<tr>
<td>Video monitor</td>
<td>&nbsp;</td>
<td>Sony Corporation</td>
<td>PVM-122</td>
<td>Black and White, 9&#x201D; screen </td>
</tr>
<tr>
<td>Micromanipulator system</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5171</td>
<td>&nbsp;</td>
<tr>
<td>Microinjection Unit</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>FemtoJet Express</td>
<td>&nbsp;</td>
<tr>
<td>Microinjection controller</td>
<td>&nbsp;</td>
<td>Contour Design</td>
<td>S-PROV2</td>
<td>Programmable multimedia controller</td>
</tr>
<tr>
<td>Igor Pro</td>
<td>&nbsp;</td>
<td>WaveMetrics</td>
<td>Version 4.05A</td>
<td>Command program to control the microinjection controller written by S. Ikeda</td>
</tr>		</tbody>
		</table>
		</div>

intranuclear microinjection of dna into dissociated adult mammalian neurons

主神经元细胞培养，都是宝贵的工具来研究蛋白质的功能，因为它们代表了永生细胞株的生物学相关的系统相比。然而，初级神经元的有丝分裂后的性质，防止有效的外源蛋白的表达，使用常用的程序，如电或化学介导的转染。因此，必须采用其他技术，以有效地在这些非分裂细胞中表达的蛋白质。 在这篇文章中，我们描述了执行分离成人交感神经元核内注射基因结构所需的步骤。这种技术已经应用到不同类型的神经元，可以成功地诱导外源蛋白的表达。为显微注射过程中所必需的设备包括一个可视化的细胞，注射玻璃吸管充满cDNA的解决方案， 连接到一个N 2（G）的压力传递系统，一个显微的倒置显微镜。显微坐标与弹出吸管尖端的cDNA解决方案的一个简短的脉冲加压N 2注射微量注射吸管的议案。 这种技术没有相关的毒性与许多其他的转染方法，使多个DNA结构是一致的比例来表示。注射细胞的数量很少，使得非常适合，如单细胞电生理记录和光学成像研究的显微注射过程，但未必需要大量的细胞和转染效率较高的生化分析的理想选择。虽然细胞核内microinjections需要时间和设备的投资，有关生理环境的能力，实现外源蛋白的表达水平高，使得这种技术非常有用的工具来研究蛋白质的功能。

Watch this Scientific Journal Video about Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons at JoVE.com

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

核内基因直接注射是有效的有丝分裂后细胞转染技术。此方法提供从单个或多个基因结构的外源蛋白的表达水平高，使蛋白质的功能与生理有关环境的各种单细胞分析研究。

游离成年哺乳动物元DNA的细胞核内内微量注射

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

intranuclear-microinjection-dna-into-dissociated-adult-mammalian

Research

JoVE Journal

Biology

16.8K 次观看.  National Institutes of Health (NIH). 核内基因直接注射是有效的有丝分裂后细胞转染技术。此方法提供从单个或多个基因结构的外源蛋白的表达水平高，使蛋白质的功能与生理有关环境的各种单细胞分析研究。

视频: Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

游离的小鼠皮层神经元文化的制备

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

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 ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

在哺乳动物细胞中的DNA双链断裂（DSB）的维修分析

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

腹腔注射到成年斑马鱼

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

在哺乳动物细胞通过显微注射mRNA的核出口动力学分析

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

为极化上皮细胞注射质粒的小GTP酶的功能分析

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

直接进入成年斑马鱼的肾细胞移植

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

在体内电穿孔Morpholinos到成年斑马鱼视网膜

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

高效的小鼠胚胎干细胞分化成运动神经元

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.