Name: viral-mediated labeling and transplantation of medial ganglionic eminence (mge) cells for in vivo studies
Uploaded: 2015-04-23T14:00:00.000+00:00
Duration: 9 min 21 s
Description: Watch this Scientific Journal Video about Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence MGE Cells for In Vivo Studies at JoVE.com

这项工作是支持的补助，以JLRR:自闭症，尼​​娜爱尔兰，韦斯顿避风港基金会，NIMH R01 MH081880和NIMH R37 MH049428。 PRW是由来自台湾的国家科学委员会的奖学金支持。 SFS是由F32（MH103003）的支持。

伦理学声明:以下程序已通过我们的机构和动物的协议。确保获得批准，涉及生存手术开始实验前的所有程序，并确认所有的协议都是最新的。 1.慢病毒准备（可选步骤） <ol><li>拆分HEK293T细胞的3个10 CM平板的每个慢病毒被制成，以10ml的DMEM / 10％FBS的存在下，并生长至〜60-70％汇合。生长的细胞在培养箱中在37℃，5％的CO 2。 </li><li>转染以下DNA质粒（10微克总）转换成使用任何转染试剂各板:6.4微克CAG-FLEX-GFP的慢病毒载体（DNA载体序列在表1中提供），1.2微克pMD2.G（编码VSV-G），1.2微克PRSV-REV，和1.2微克pMDLg / pRRE。三个慢病毒包装载体是可商购的（表 注意:这种特殊的方法利用第三代lentivir人的载体，但第二代媒介和相关质粒也将正常工作。 </li><li>准备包含BSL2认证罩内10％的漂白粉溶液灭活的联系，或包含任何慢病毒解决方案或设备废料容器。接下来，转染到漂白粉溶液后，彻底去除媒体4-6小时，更换新媒体的同一卷，并允许细胞生长。 </li><li>后4天，收集培养基入50ml锥形管中并离心以1000×g离心15分钟以沉淀任何细胞碎片。接着，过滤所述上清液通过0.45μm的膜。任何低蛋白结合过滤器，如PVDF，效果很好。注意:将固体和液体废弃物病毒进入漂白粉溶液。 </li><li>负载30毫升过滤的上清液到超速离心管并进入超速离心机。离心机以100,000×g离心2.5小时，在4℃。打开离心管中BSL2罩和上清到10％的漂白粉。添加〜100微升的PBS到PEllet，这将产生1×10 ^ 7-8传染性单位/ ml的滴度。分装在次日并且储存在-80ºC。慢病毒等份是稳定的，在-80℃下至少6个月。 </li><li>重要的考虑因素:许多机构现在有病毒的核心。获得浓缩的病毒用1×10 7感染单位/ ml的滴度最低，以获得最佳的结果。 </li></ol> 2.供体小鼠的MGE解剖<ol><li>首先，建立了利益的Cre表达线，无论是野生型（WT）鼠标或鼠标，将表达Cre重组酶介导的重组后的记者之间的定时交配。 注:很多的Cre-驱动线转基因和维护，并孕育在半合状态。因此，只有一半的胚胎将包含酶Cre转基因。 DNA可从胚胎迅速准备一个短的PCR来检测酶Cre等位基因。所述酶Cre +胚胎然后可收获，使得所期望的基因型的仅MGE组织被使用。 Alternatively，一个依赖于Cre的报道，可以使用，以选择胚胎MGE解剖和移植。 </li><li>计算这一天将对应于E13.5。如果动物配对前的晚上，插头的一天是0.5。为了定时孕鼠或WT女性，P1幼崽，以时间为出生后（P）1小鼠当天的供体组织将E13.5。另外，在房子配对供体动物的前一星期设置这些交配。 注意:产生两个E13.5和P1胚胎的前策略/幼仔为同一天往往很难做到可靠。 </li></ol> 3.准备的媒体，工具​​和设备。 <ol><li>制备的试剂和工具的MGE细胞制剂（ 见表2）。 <ol><li>制备一个清洁的表面，并把清洁的镊子，剪刀以及一个显微刀或细镊子（用于精确组织解剖）的表面上。 </li><li>制备Hank氏平衡盐溶液（HBSS）中，f或剥离，并Dulbeco改进的Eagle培养基（DMEM）中，用10％胎牛surem（FBS），用于转导。 </li><li>预温育20-30毫升的DMEM / 10％FBS在37℃组织培养箱进行约1小时。该盖应松开，以允许介质气体交换和pH平衡。 注:媒体可以预孵育刚开始解剖前准备。 </li></ol></li></ol> 4. MGE细胞的制备<ol><li>根据批准的动物协议牺牲孕小鼠并取出胚胎成含有冰冷的HBSS 10cm的培养皿。 </li><li>从使用解剖范围每个胚胎取出脑（做一次一个，留下的胚胎的其余部分在冰冷的HBSS）中，然后进行切出的MGE组织。 注意:下面详细，并在图1中 ，也展示了如何删除MGE关键步骤。另外， 图2是示出电影的整个解剖流程。 <ol><li>定位大脑腹侧朝上（ 图1A，A"）。注意:它也还可以有背侧朝上。切开大脑中的一半沿矢状平面上以生成两片。一个例子切成对半的脑示（ 图1B，B&#39;）中 ，请注意，背侧（上覆皮质）和脑盖腹侧方面和包围该神经节隆起（GE）的组织。 </li><li>接着，用优势手，保持或者非常细的镊子或stabknife（ 表2），而固定带由非优势手保持镊子的半球。 （半球的内侧方面应朝上）。展开背，腹组织钳和刺一刀，露出底层的GE组织（ 图1C和图解图1C"）。 注意:删除从解剖培养皿一些媒体可以更容易稳定，而PE的组织rforming的MGE清扫。 </li><li>使直线切割与stabknife或细镊子分离专家咨询小组，LGE和室间隔组织（见例如削减记为红色虚线， 图1D和D&#39;）。这些切口可以以任何顺序进行。 注:想象一下，MGE的&#39;盒子&#39;被切出的周围组织。这样做有助于重复做类似的削减每个解剖，从而减少可变性的组织解剖。 </li><li>最后，打开MGE在其一侧并修剪掉所述底部（什么是脑的外侧面）。这消除了MGE的地幔区（丢弃在图1E和1E&#39;）从MGE的其余部分。 MGE其余方面主要包含脑室区和MGE，其中包含几乎所有的MGE祖细胞的脑室下区部分。当您的组织。 </li></ol></li><li>重要的考虑因素:添加硅氧烷凝胶至培养皿CA的底部ñ作为一个很好的剥离面，因为它可以保护镊子的细腻技巧，并提供了一​​个柔软的表面钉扎组织用钳子。 </li></ol> 4.3其他策略MGE解剖。 <ol><li>削减使用镊子似剪刀，迅速取出脑胚胎的皮肤。作为胚胎规定在其一侧，保持所述组织在头部与用于锚定组织的钳子的基极。首先，切割前腹侧到大脑和后部腹侧到大脑，然后连接所述两个切口沿着胚胎的一侧。然后抓住皮肤的护翼并在大脑的顶部拉出。大脑然后可以迅速解剖远离剩余的组织。 </li><li>可替代地，锚定皮质背后的全脑的后部的方面。同时，掌握每个半球的皮层的最背侧尾方面和撕裂皮层远离锚定组织中的前方向。以这种方式，使用Cortices可以被删除，双侧神经节隆起都显露出来，仍然附着于组织保持内侧 - 外侧解剖学的其余部分。 </li><li>切换到刺一刀，使周围的MGE精确的削减对每个半球。使用一个单独的干净的一套镊子收集MGE或一大笔小费吸管（ 即 ，P1000）。 注意:保持组织收集远离所有其他剖分材料，作为当MGE收集一些媒体不可避免地转移。 </li><li>收集MGE和把组织成含有DMEM / 10％FBS（500微升的体积是足以收集MGEs）1.5ml的收集管。重复其他半球的地方在同一个收集管。保持样品在冰上，直到所有组织收集。 </li></ol> 5.慢病毒标签和移植<ol><li>移动MGE组织的管成BSL2认证的发动机罩和取出介质。 注意:MGE组织是大到足以被肉眼可见的，并会沉降到管允许成为冷介质容易且轻轻除去的底部。 </li><li>添加〜500微升的DMEM / 10％FBS的培养基中，该预孵育在37℃组织培养孵化器。接下来，添加凝聚胺以促进传导到每个管中以8微克/毫升的最终浓度。磨碎的MGE组织以建立一个单一的细胞悬浮液，使用的是P1000的枪头。最后，添加〜15-20微升浓缩慢病毒向每个管中。 </li><li>重要的考虑因素:10-12倍甲研磨足以从单个胚胎分手MGE组织，但也可需要更triturations如果多个MGEs汇集在一起​​。尝试不同的研磨条件来决定什么效果最好。 </li><li>盖紧每个试管，颠倒混合，比地方所有的管子在37℃的孵化器。孵育细胞中的慢病毒为至少30分钟和最多1小时。更长的时间导致德折痕细胞活力。反转管每隔10分钟，直到时​​间到了。 </li><li>孵育慢病毒后，从孵化器和离心机在〜700×g离心试管3分钟以沉淀细胞。在BSL2罩，除去上清液并丢弃到10％的漂白剂。接着，加入1 ml的DMEM / 10％FBS和磨碎沉淀2-3次洗涤，然后再离心以相同的速度，以沉淀细胞。重复此洗涤步骤2-3多次，以除去过量的病毒。 注意:执行离心步骤在4℃下，然而，降低的生存力没有被观察到，当短的自旋都在室温进行。 </li><li>最后一次洗涤后，除去尽可能多的介质尽可能并把各管在冰上。由于在管的侧面残留介质，〜2-3微升介质将最终由移植过程的开始时间覆盖细胞沉淀。 </li></ol> 6.移植和验证<ol><li>掌握主机P1的幼仔，并准备程序区域通过喷洒下来，用70％的乙醇。以在操作过程中保持无菌，建议在一个专用清洁过程室执行这些过程。另外，使用高压灭菌手术工具消毒表面，该幼仔将在接触与使用70％的乙醇。执行移植和有代表性的程序区所需代表性的设备的一个例子示于图3。 </li></ol> 注意:虽然此步骤是注射小体积，并且不会在外科手术过程，将需要一个切口，开放性伤口或缝合线，它仍然是建议一个新微量用于每个鼠被注入。的微量是热拉和斜面上被喷上70％的乙醇被存储在密封容器中的表面时进行灭菌。 <ol><li>产生微量具有直径在前端40-80微米之间。以下这些方面将产生最佳结果。热灭菌微量时拉出并斜面上的表面和存储微量​​在密封容器中之前喷以70％的乙醇。 注意:可替代地，如果获得所需要创建这些针状物（ 表2）的装置是限制性的，可以使用任何其他注射装置。但是，这些可能不那么适合作为由于不同直径的微量。 </li><li>制定矿物油成1ml注射器，并用一个30 1/2 G针，完全填满玻璃吸管与矿物油。接下来，安装活塞到立体定位装置，然后装上玻璃吸管的立体装置和负载到活塞。使用液压驱动器，移动大约一半柱塞进入玻璃吸管，除去矿物油泄漏出来，用干净干净的纸巾。 <ol><li>可替代地使用注射器，淹没在矿物油中的移液管的后端和填充通过毛细管作用。为了防止气泡在pipettE，维持注射器正压，直到完全脱离玻璃吸管。 注:其他的设备可以用来成功地移植MGE细胞14。任何设备，可提供一个容积超过或接近100 NL作品以及在此过程中少。 </li></ol></li><li>接下来，使用消毒纸巾，或任何吸收材料，具有扭曲成一个细点的角落，微妙去除上面的MGE细胞沉淀多余的介质。这一步浓缩的细胞密度，以便较小的注射体积就可以实现。接下来，使用低容积（P2优先）吸管慢慢地拟定细胞沉淀和移动细胞悬液到疏水表面之前磨碎1-2次。量应略低于1微升。移动针进入细胞悬浮液的尖端，并使用液压驱动，绘制出细胞悬浮到吸管。 </li><li>麻醉通过低温一个P1小狗（包装在一个乳胶手套，并雪藏了〜2-4分钟）。检查麻醉与有害刺激效果。捏脚趾之间的皮肤用细镊子:如果捏了一把小狗应该是反应迟钝。接着，将小狗到一个模具，它是注射装置之下，然后使由拉回皮肤上的头部，并与标准实验室胶带固定拉紧皮肤。 注:虽然低温是对新生小鼠，并导致低死亡率非常有效，程序必须要快，因为幼崽将开始在10分钟中恢复。此外，在冰上4分钟是什么是可以容忍的上限。试图只诱导低温一次，如果需要用于注射的时间较长。但是，如果小狗的早期复苏，首先让小狗再次引起体温过低前完全恢复。此外，仅诱导低温一个更多的时间来执行注射。幼崽将在低温5-10分钟恢复。这可以通过将垃圾，在妈妈的幼崽，或者将小狗在一个温暖的面传热提供便利E要恢复。 </li><li>使用立体定位装置，定位玻璃吸管的尖端的表面上的小狗的头部，在垂直于头部的表面上。当吸移管是在与头部接触，认为这是"0"。接着，通过皮肤和颅骨插入皮层的表面推移液管，然后插入并停止之前缩回移液器〜2倍。为了获得最佳的细胞靶向到新皮质，注射时执行微量是在0.1毫米的深度。然而，这应该由用户进行优化（见下面的注释）。 注意:当该深度可靠地与上述设备的目标更深新皮层的层，几个深度应测试经验确定。此外，一系列的深度不同rostro-尾鳍和内 - 外位置应当进行测试，以确定是什么深度是最佳的在每个站点。另外，在初始穿刺应迅速，因为缓慢渗透到大脑皮层减弱的能力到cleanly穿透所述组织。尝试不同的速度时，首先启动该程序，找出最。此外，用于测试的坐标，没有真正的替代使用标记的细胞，如染料已经靠不住来表示的位置。 </li><li>一旦针在位置，旋转液压装置来推进所述柱塞进入玻璃吸管，推细胞悬浮液的设定容积进皮层。注入每个站点50-70 NLS。重复此过程在3-6不同站点在不同的延髓 - 尾的水平，如果目标是获得细胞的整个皮层一个宽的分布。 注意:100 NL或更高的卷可能导致损伤和导致注射细胞未能有效地迁移出注射部位的大的团块。因此，较小的进样体积（〜70 NL或更少）是最优的，在更多的网站，如果时间允许。 </li><li>当注射完成后，从舞台上拆下小狗并标记（趾裁剪是一种可靠的方式来识别PUPS更高版本）。一旦小狗已经恢复，并能够将其自己的（这种情况发生时从舞台中取出后几分钟内），把它带回了妈妈和垃圾。 注:由于这是一种微创手术，没有手术后的程序一旦小狗可自由移动和温暖。然而，幼仔应检查不仅程序，但也翌日，以确保后它们具有健康状况恶化的迹象。 </li><li>开始的下一个小狗的过程之前，喷向下面积，用70％的乙醇，以保持无菌的工作​​区。允许注入的幼仔开发然后评估所述组织在适当的发育阶段（多个）。 </li></ol>

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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interneurons+from+embryonic+development+to+cell-based+therapy.">Interneurons from embryonic development to cell-based therapy.</a> Science. 344 (6180), 1240622 (2014).</li><li>Hunt, R. F., Girskis, K. M., Rubenstein, J. L., Alvarez-Buylla, A., Baraban, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+progenitors+grafted+into+the+adult+epileptic+brain+control+seizures+and+abnormal.">GABA progenitors grafted into the adult epileptic brain control seizures and abnormal.</a> Nature Neuroscience. 16 (6), 692-697 (2013).</li><li>Braz, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forebrain+GABAergic+Neuron+Precursors+Integrate+into+Adult+Spinal+Cord+and+Reduce+Injury-Induced+Neuropathic+Pain.">Forebrain GABAergic Neuron Precursors Integrate into Adult Spinal Cord and Reduce Injury-Induced Neuropathic Pain.</a> Neuron. 74, 663-675 (2012).</li><li>Vogt, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lhx6+Directly+Regulates+Arx+and+CXCR7+to+Determine+Cortical+Interneuron+Fate+and+Laminar+Position.">Lhx6 Directly Regulates Arx and CXCR7 to Determine Cortical Interneuron Fate and Laminar Position.</a> Neuron. 82 (2), 350-364 (2014).</li><li>Alvarez-Dolado, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+inhibition+modified+by+embryonic+neural+precursors+grafted+into+the+postnatal+brain.">Cortical inhibition modified by embryonic neural precursors grafted into the postnatal brain.</a> J. Neurosci. 26 (4), 7380-7389 (2006).</li><li>Du, T., Xu, Q., Ocbina, P. J., Anderson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NKX2.1+specifies+cortical+interneuron+fate+by+activating+Lhx6.">NKX2.1 specifies cortical interneuron fate by activating Lhx6.</a> Development. 135 (8), 1559-1567 (2008).</li><li>Arguello, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dapper+Antagonist+of+Catenin-1+Cooperates+with+Dishevelled-1+during+Postsynaptic+Development+in+Mouse+Forebrain+GABAergic+Interneurons.">Dapper Antagonist of Catenin-1 Cooperates with Dishevelled-1 during Postsynaptic Development in Mouse Forebrain GABAergic Interneurons.</a> PLoS ONE. 8 (6), e67679 (2013).</li><li>Lee, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyramidal+neurons+in+prefrontal+cortex+receive+subtype-specific+forms+of+excitation+and+inhibition.">Pyramidal neurons in prefrontal cortex receive subtype-specific forms of excitation and inhibition.</a> Neuron. 81 (1), 61-68 (2014).</li><li>Chen, Y. J. J., et al. <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+&#8220;MGE+Enhancers&#8221;+for+Labeling+and+Selection+of+Embryonic+Stem+Cell-Derived+Medial+Ganglionic+Eminence+(MGE)+Progenitors+and+Neurons.">Use of &#8220;MGE Enhancers&#8221; for Labeling and Selection of Embryonic Stem Cell-Derived Medial Ganglionic Eminence (MGE) Progenitors and Neurons.</a> PLoS ONE. 8 (5), e61956 (2013).</li><li>Pattabiraman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+enhancers+active+in+protodomains+of+the+developing+cerebral+cortex.">Transcriptional regulation of enhancers active in protodomains of the developing cerebral cortex.</a> Neuron. 82 (5), 989-1003 (2014).</li></ol>

The authors declare that they have no competing financial interests.

从胚胎神经节隆起（GES）为基础的细胞治疗使用皮质GABA能中间神经元前体正显示出的承诺了很多条件，12 -1 4。需要精确的分子技术进行跟踪，并表示在特定的中间神经元亚组感兴趣的基因。在这里，我们提供了一个详细的协议，用于在移植前用慢病毒标记胚胎MGE细胞并展示如何这种技术可以用来表达的体内分析中的具体的皮层中间神经元亚群的兴趣基因。 本?...

GABA能皮质的interneurons包括在哺乳动物大脑皮质的神经元〜20-30％，而其余的对应兴奋性，谷氨酸主要神经元。的interneurons在电生理特性，轴突和树突形态和突触目标1高度多样化，并在兴奋/抑制色调失衡假设是背后的神经/精神神经疾病，包括孤独症，精神分裂症和癫痫2部分的表型。本文所描述的协议的总体目标是提供对移植的体内分析之前有效地遗传修饰GABA能皮层中间神经元祖细胞的方法。 皮质GABA能中间是出生在内侧和尾神经节隆起（MGE和CGE，分别）3,4，以及视前区5。皮质中间神经元祖细胞经过长距离迁移切线后面通过放射状迁移，达到他们的最终目标。抵达目的地，这些皮质的interneurons必须正确集成到现有网络中的神经元，每个独特的中间神经元亚组会以特定的方式有助于皮质电路。四个主要子组，可以区分由分子标记:MGE衍生的促生长素抑制素（SST）+和小清蛋白（PV）+亚组，和CGE衍生血管活性肠肽（VIP）+和颤+; SST -亚组6。不同皮层中间神经元亚群都在MGE和CGE 7,8胚胎发育过程中所生了不同的时间。这些和其他皮质GABA能中间神经元标记物已被用于产生特异的Cre驱动线许多这些亚组9-11的。 MGE祖细胞的移植已成为一个潜在的基于细胞的疗法来治疗可通过不平衡引起的疾病励磁/抑制12 - 24。这些治疗益处可能部分是由于MGE祖细胞（分散，分化和整合入宿主脑），或可能是因为许多关节周围的体细胞抑制性的PV +细胞是从MGE衍生的独特能力。 MGE细胞也可以快速和有效地转导的移植15之前慢病毒，允许被遗传修饰在体外的细胞在体内进行研究。开发这种方法的原理是克服障碍在研究GABA能中间神经元皮质发育和成熟。特别是，移植的MGE允许研究人员研究突变的细胞在体内的发展，当突变小鼠将否则在早期时间点死亡。此外，通过引入移植前感兴趣的基因上的突变体的表型的特定基因的作用，可以在一个EFF评估icient方式。 在这里，我们提供了一个详细的协议转导的MGE细胞移植前的慢病毒。此外，我们显示如何这种技术可以适用于从皮层中间神经元前体的一组异质性表达在特定中间神经元亚群的目的基因，采用依赖于Cre的表达慢病毒和可用Cre的驱动鼠标线的组合。此外，该协议引入的技术和研究人员的一个平台，以基因修饰GABA能中间神经元的皮质前体的体内研究以独特的方式。这种技术比其他现有方法的一个优点是，移植的MGE细胞将分散远离注射部位。此外，与焦病毒注射后的MGE细胞分散它们的形态是更容易评估。这种方法可用于研究引入感兴趣基因引入野生型或突变体细胞中，引入的细胞类型特异性的效果记者评估形态，或潜在学习的疾病等位基因体内的效果。

<table><tbody><tr><td colspan="4">Reagents and equipment for MGE cell transplantation</td></tr><tr><td>1 ml syringe</td><td>REF 309602</td><td>BD, Becton Dickinson and company</td><td></td></tr><tr><td>30 1/2 G needle</td><td>305106</td><td>BD, Becton Dickinson and company</td><td></td></tr><tr><td>Precision bore to deliver 5 &amp;micro;l (comes with plunger)</td><td>5-000-1005</td><td>Drummond scientific company</td><td></td></tr><tr><td>Parafilm</td><td>PM-999</td><td>Polysciences, Inc.</td><td></td></tr><tr><td>Stereo microscope with boomstand **</td><td>MZ6</td><td>Leica</td><td></td></tr><tr><td>Digital just for mice stereotaxic instrument **</td><td>51725D</td><td>Stoelting company</td><td></td></tr><tr><td>Single-axis oil hydraulic fine micromanipulator **</td><td>MO-10</td><td>Narishige</td><td></td></tr><tr><td>Diamond coated rotary beveler</td><td></td><td>made in house</td><td></td></tr><tr><td>Needle pipette puller</td><td>730</td><td>Kopf instruments</td><td></td></tr><tr><td>Mineral oil</td><td></td><td>Any drug store or pharmacy</td><td></td></tr><tr><td>Any pipette and tips that can reliaby measure 1 &amp;micro;l of volume</td><td></td><td></td><td></td></tr><tr><td colspan="4">** These are the particular models we use, but many other setups should work</td></tr><tr><td colspan="4">Optional Reagents (for making Lentiviruses)</td></tr><tr><td>25 mm, 0.45 &amp;micro;m filter</td><td>09-719B</td><td>Fisher Scientific</td><td></td></tr><tr><td>Ultra-clear centrifuge tubes (25 x 89 mm)</td><td>344058</td><td>Beckman Coulter&amp;reg;</td><td></td></tr><tr><td>Lipofectamine &lt;sup&gt;2000 &lt;/sup&gt;(1.5 ml size)</td><td>11668019</td><td>Invitrogen</td><td></td></tr><tr><td colspan="4">Other commercially available supplies</td></tr><tr><td>pMDLg/pRRE plasmid encoding gag/pol</td><td>12251</td><td>Addgene</td><td></td></tr><tr><td>pRSV-Rev plasmid encoding Rev</td><td>12253</td><td>Addgene</td><td></td></tr><tr><td>pMD2.G plasmid encoding VSVG</td><td>12259</td><td>Addgene</td><td></td></tr><tr><td>pAAV-Flex-GFP</td><td>28304</td><td>Addgene</td><td></td></tr></tbody></table>

viral-mediated labeling and transplantation of medial ganglionic eminence (mge) cells for in vivo studies

GABA能皮质的interneurons，从胚胎内侧和尾神经节隆起（MGE和CGE）得出，在功能和形态多样。进展已在理解的不同的皮层中间神经元亚群的作用，但是，也有可能向不同类型的GABA能细胞的发育和成熟仍有许多机制来制定。此外，改变的GABA能信令可以有助于孤独症，精神分裂症和癫痫的表型。特异性表达Cre-驱动线已经开始包裹出独特的中间神经元亚群的功能。尽管在小鼠模型中的进步，它往往是难以有效地研究GABA能皮层中间神经元祖细胞与分子的方法在体内进行 。用于研究这些细胞的细胞中自主编程的一个重要的技术是移植的MGE细胞向宿主皮层。这些移植的细胞广泛迁移，分化，一第二功能集成。此外，MGE细胞可以有效地转导的慢病毒立即移植前，允许多种分子的方法。下面我们详细的协议移植体内分析之前，有效地转导MGE细胞，利用现有的Cre-驱动线和依赖于Cre的表达载体。这种做法是有利的，因为它结合了精确的遗传操作与这些细胞的分散移植后，允许在体内更大的细胞类型特异性的分辨率的能力。

因为MGE细胞具有迁移并在移植到宿主新皮层16整合的独特能力，它们在体内的研究之前提供用于遗传操作的优良模型系统。这里，我们显示如何能够从E13.5胚胎分离的MGE组织（ 图1和2），然后可以用慢病毒在体外或在移植体内研究前一个快速的方式转导。通过慢病毒标记MGE已经使用了增强驾驶GFP的GABA能神经元15日前已执行。但是，表达能力从异质群...

Watch this Scientific Journal Video about Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence MGE Cells for In Vivo Studies at JoVE.com

Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence MGE Cells for In Vivo Studies

GABA能中间神经元皮质祖分散，开发和突触融入移植后的主机皮层。这些细胞可以移植在体内研究转基因GABA能前体的前容易地转导。这里，我们表明病毒标记技术到目标使用现有的Cre线和依赖于Cre的记者特定中间神经元亚群。

病毒介导的标签和内侧神经节隆起移植（MGE）细胞<em&gt;在体内</em&gt;研究

GABAergic cortical interneurons, derived from the embryonic medial and caudal ganglionic eminences (MGE and CGE), are functionally and morphologically ...

viral-mediated-labeling-transplantation-medial-ganglionic-eminence

Research

JoVE Journal

Developmental Biology

Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence (MGE) Cells for In Vivo Studies

11.4K 次观看.  University of California San Francisco.  GABA能中间神经元皮质祖分散，开发和突触融入移植后的主机皮层。这些细胞可以移植在体内研究转基因GABA能前体的前容易地转导。这里，我们表明病毒标记技术到目标使用现有的Cre线和依赖于Cre的记者特定中间神经元亚群。 

视频: Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence MGE Cells for In Vivo Studies

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

颅内注射腺相关病毒载体

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

科研

神经科学

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

在成年小鼠V-SVZ细胞的神经分析的稳定，高效的基因修饰干细胞自主和非自主的影响

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

发育生物学

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

工程神经前体宫内移植,用于Vivo神经结构研究

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

遗传学

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

在中央小鼠中枢神经系统的干细胞移植的多模态成像

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

扩展胚胎及成体神经干细胞<em&gt;在子宫内</em电穿孔或病毒立体定向注射

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

腺相关病毒介导的脊髓基因神经元的转基因表达

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative cell replacement therapies while also creating tools to study cell fate in situ. To date, it has been possible to obtain neurons via in vivo reprogramming, but the precise phenotype of these neurons or how they mature has not been analyzed in detail. In this protocol, we describe a more efficient conversion and cell-specific identification of the in vivo reprogrammed neurons, using an AAV-based viral vector system. We also provide a protocol for functional assessment of the reprogrammed cells&#8217; neuronal maturation. By injecting flip-excision (FLEX) vectors, containing the reprogramming and synapsin-driven reporter genes&#160;to specific cell types in the brain that serve as the target for cell reprogramming. This technique allows for the easy identification of newly reprogrammed neurons. Results show that the obtained reprogrammed neurons functionally mature over time, receive synaptic contacts and show electrophysiological properties of different types of interneurons. Using the transcription factors Ascl1, Lmx1a and Nurr1, the majority of the reprogrammed cells have properties of fast-spiking, parvalbumin-containing interneurons.

This protocol aims at generating directly reprogrammed interneurons in vivo, using an AAV-based viral system in the brain and a FLEX synapsin-driven GFP reporter, which allows for cell identification and further analysis in vivo.

在Vivo直接重新编程居民胶质细胞到内神经通过病毒载体的脑内注射

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point measurements and can therefore only evaluate longitudinal changes of cell migration and survival to a limited extent. This paper provides a reliable, minimally invasive protocol to transplant and longitudinally track neural progenitor cells (NPCs) in the adult mouse brain. Before transplantation, cells are transduced with a lentiviral vector comprising a bioluminescent (firefly-luciferase) and fluorescent (green fluorescent protein [GFP]) reporter. The NPCs are transplanted into the right cortical hemisphere using stereotaxic injections in the sensorimotor cortex. Following transplantation, grafted cells were detected through the intact skull for up to five weeks (at days 0, 3, 14, 21, 35) with a resolution limit of 6,000 cells using in vivo bioluminescence imaging. Subsequently, the transplanted cells are identified in histological brain sections and further characterized with immunofluorescence. Thus, this protocol provides a valuable tool to transplant, track, quantify, and characterize cells in the mouse brain.

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

We describe the intraparenchymal transplantation of human neural progenitor cells transduced with a dual reporter vector expressing luciferase-green fluorescent protein (GFP) in the mouse brain. After transplantation, the luciferase signal is repeatedly measured using in vivo bioluminescence and GFP-expressing grafted cells identified in brain sections using fluorescence microscopy.

小鼠大脑中人类神经祖细胞的脑内移植和 体内 生物发光跟踪

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point ...

大鼠三叉神经节与钙指示剂的体内可视化

In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their large signal-to-noise ratio makes GECIs a powerful tool for detecting stimulus-evoked activity in sensory neurons. GECIs facilitate population-level analysis of stimulus encoding with the number of neurons that can be studied simultaneously. This population encoding is most appropriately done in vivo. Dorsal root ganglia (DRG), which house the soma of sensory neurons innervating somatic and visceral structures below the neck, are used most extensively for in vivo imaging because these structures are accessed relatively easily. More recently, this technique was used in mice to study sensory neurons in the trigeminal ganglion (TG) that innervate oral and craniofacial structures. There are many reasons to study TG in addition to DRG, including the long list of pain syndromes specific to oral and craniofacial structures that appear to reflect changes in sensory neuron activity, such as trigeminal neuralgia. Mice are used most extensively in the study of DRG and TG neurons because of the availability of genetic tools. However, with differences in size, ease of handling, and potentially important species differences, there are reasons to study rat rather than mouse TG neurons. Thus, we developed an approach for imaging rat TG neurons in vivo. We injected neonatal pups (p2) intraperitoneally with an AAV encoding GCaMP6s, resulting in &gt;90% infection of both TG and DRG neurons. TG was visualized in the adult following craniotomy and decortication, and changes in GCaMP6s fluorescence were monitored in TG neurons following stimulation of mandibular and maxillary regions of the face. We confirmed that increases in fluorescence were stimulus-evoked with peripheral nerve block. While this approach has many potential uses, we are using it to characterize the subpopulation(s) of TG neurons changed following peripheral nerve injury.

作者聚焦：探索三叉神经损伤中神经性疼痛的外周机制 

Genetically encoded calcium indicators (GECI) enable a robust, population-level analysis of sensory neuron signaling. Here, we have developed a novel approach that allows for in vivo GECI visualization of rat trigeminal ganglia neuron activity.

体内 大鼠三叉神经节感觉神经元的钙成像

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their ...

病毒介导的标签和内侧神经节隆起移植（MGE）细胞

摘要

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此视频中的章节

颅内注射腺相关病毒载体

在成年小鼠V-SVZ细胞的神经分析的稳定，高效的基因修饰干细胞自主和非自主的影响

小鼠视网膜神经节细胞的转染在体内电穿孔

工程神经前体宫内移植,用于Vivo神经结构研究

在中央小鼠中枢神经系统的干细胞移植的多模态成像

扩展胚胎及成体神经干细胞

腺相关病毒介导的脊髓基因神经元的转基因表达

在Vivo直接重新编程居民胶质细胞到内神经通过病毒载体的脑内注射

小鼠大脑中人类神经祖细胞的脑内移植和体内生物发光跟踪

体内大鼠三叉神经节感觉神经元的钙成像

病毒介导的标签和内侧神经节隆起移植（MGE）细胞

摘要

探索更多视频

此视频中的章节

颅内注射腺相关病毒载体

在成年小鼠V-SVZ细胞的神经分析的稳定，高效的基因修饰干细胞自主和非自主的影响

小鼠视网膜神经节细胞的转染在体内电穿孔

工程神经前体宫内移植,用于Vivo神经结构研究

在中央小鼠中枢神经系统的干细胞移植的多模态成像

扩展胚胎及成体神经干细胞

腺相关病毒介导的脊髓基因神经元的转基因表达

在Vivo直接重新编程居民胶质细胞到内神经通过病毒载体的脑内注射

小鼠大脑中人类神经祖细胞的脑内移植和 体内 生物发光跟踪

体内 大鼠三叉神经节感觉神经元的钙成像

小鼠大脑中人类神经祖细胞的脑内移植和体内生物发光跟踪

体内大鼠三叉神经节感觉神经元的钙成像