<p class="jove_content"&gt;作者的工作得到了转炉研究资助ID-2006年的安特卫普大学（PPO和AVDL授予），研究资助G.0136.11和G.0130.11（授予AVDL，ZB和PPO）和1.5.021.09。 N.00（授予PPO）的科学研究，法兰德斯（FWO Vlaanderen，比利时），服务营办商研究资助内陆水运-60838：BRAINSTIM佛兰德科技研究所（ZB和AVDL授予），在基金1玛土撒拉的研究经费从弗拉芒政府（授予ZB型），部分由EC-FP6的NOE迪米（LSHB-CT-2005-512146），EC-FP6的NOE EMIL（LSHC-CT-2004-503569的部分）间大学景点波兰人IUAP-NIMI-P6/38（授予AVDL）。纳塔莉德Vocht FWO-Vlaanderen持有博士学位的奖学金。彼得Ponsaerts是的FWO-Vlaanderen的博士后研究员。</p

1。细胞的制备<ol><li>应开始实验使用体外培养干细胞基因工程表达的荧光素酶和绿色荧光蛋白记者蛋白质的人群。在这里，我们使用荧光素酶/ EGFP表达小鼠骨髓来源的基质细胞（BMSC-Luc/eGFP）以前Bergwerf 等。2，5。 </li><li>细胞标记，在8×10 5％的T75培养瓶中的细胞密度在15毫升完整的扩展介质（CEM），辅以1微克/毫升Puromycine板BMSC-Luc/eGFP细胞前两天。 </li><li>让细胞成长为48小时37°C和5％的CO 2。 </li><li>新增5 6冰川蓝微米大小的氧化铁粒子（GB MPIO）的每毫升培养基中日益增长的细胞培养（总数：75×10×10 6粒子中的T75培养瓶中，每15毫升的培养基）。 </li><li>孵育16小时，让粒子通过内吞作用摄取。 </li><li>洗去剩余的颗粒让细胞成长为一个额外的24小时，以获取细胞的汇合和均匀分布的GB的MPIO。 </li><li>视使用荧光显微镜验证粒子吸收EGFP + GB的MPIO +细胞计数的EGFP +细胞总量的比例。 </li><li>洗GB的MPIO标记BMSC-Luc/eGFP细胞10毫升PBS和收获细胞，胰蛋白酶EDTA治疗，每5分钟的T75培养瓶中（5毫升）的两倍。 </li><li>在终浓度为133×10 6细胞/毫升的PBS洗收获和重悬细胞。 </li><li>保持冰，直到移植的细胞。 </li></ol> 2。在小鼠中枢神经系统的细胞移植<ol><li>麻醉小鼠腹腔注射氯胺酮（80毫克/千克）+甲苯噻嗪（16毫克/千克）混合。 </li><li>等待5分钟，让老鼠入睡。 </li><li>刮胡子的无菌操作，让鼠标头。 </li><li>鼠标放置在一个立体框架。 </li><li> Desinfect“皮肤和湿老鼠的眼睛，以防止脱水。 </li><li>使头皮正中切口，暴露颅骨，并在使用在给定的坐标牙科钻毛刺的头骨钻洞后2毫米和2毫米，横向到囟门。 </li><li>涡简要的细胞悬液，在注射器吸出细胞悬液。 </li><li>放置在深度为2.5毫米的针头硬膜下，允许1分钟的压力平衡。 </li><li>注入硬膜下2毫米的深度和0.70μL/ min的速度在3μl的细胞悬液（4×10 5细胞）使用自动微量注射泵。 </li><li>等待一个额外的5分钟，注入细胞悬液，以防止回流。 </li><li>慢慢地缩回针头和消毒皮肤缝合皮肤前的边界。 </li><li>为了防止脱水，注入300μL0.9％NaCl溶液皮下注射，并将其置于鼠标下加热灯，从麻醉中恢复。管理带够按照制度标准的的OST手术镇痛。 </li></ol> 3。 在活体动物 <ol><li>麻醉小鼠的3％异氟醚和氧气的混合物。 </li><li>放置在光子成像的小鼠和麻醉水平降低至1.5％，异氟醚和氧气。 </li><li>注射每公斤体重150毫克的D-荧光素静脉滴注。 </li><li>收购照片Vision软件使用5分钟的形象。 </li><li>执行图像处理使用M3vision软件。量化观测到的信号，使用固定利率地区。 </li></ol> 4。 在体内磁共振成像<ol><li>与O 2的混合物在3％异氟醚麻醉小鼠的N 2 O（3:7）。 </li><li>限位水平的9.4T磁共振系统放置在老鼠和减少麻醉水平的1％O 2的混合物中的异氟醚的N 2 O（3:7）。 </li><li>润润的老鼠的眼睛，以防止脱水TION，重视直肠探头监测体温和鼠标肚子底下放置一个传感器监测呼吸频率。 </li><li>保持在110±10每分钟呼吸呼吸率和保持体温恒定在37±0.5℃的范围狭窄</li><li>将鼠标头和鼠标在磁铁的中间位置上的表面射频线圈。 </li><li>获得一套10冠状T2加权自旋回波（SE）的影像，以获得特定的解剖信息和T2 *加权梯度回波（GE）的图像，为了研究干细胞移植与平面的分辨率为70微米2。设置序列参数如下：重复时间（TR）：500毫秒，回波时间（TE）：8毫秒（GE序列）和重复时间（TR）：4200毫秒，回波时间（TE）：12.16毫秒（SE序列）;领域的视角（FOV）：18×18毫米2，矩阵：256×256，1毫米层厚1毫米的片分离（Paravision 5.1软件）。 </li><li>执行数据处理使用阿米拉4.0软件。 </li></ol> 5。后验切片<ol><li> ，isofluorane（4％），氧气（0.5升/分钟）和氮（1升/分钟）2分钟的混合物吸入麻醉小鼠很深。牺牲小鼠颈椎脱位。 </li><li>从头骨中取出的小鼠大脑和注视的脑组织在4％多聚甲醛的PBS 2小时。 </li><li>脑组织脱水，被放置在不同的蔗糖梯度大脑随后在5％蔗糖PBS，2小时2小时，在10％蔗糖PBS中，20％蔗糖的PBS过夜。 </li><li>使用液氮冻结的脑组织，并储存于-80°C至切片组织。 </li><li>第10使用恒温器微米厚的部分脑组织。 </li><li>屏幕不染的GB MPIO颗粒和EGFP表达细胞用荧光显微镜的绿色荧光的蓝色荧光的冰冻切片。 </li><li>屏幕不染冰冻切片为绿色/红色炎性细胞的背景荧光。 </li><li>执行进一步的免疫组化和免疫荧光染色，以确定内源性细胞群（如胶质细胞，星形胶质细胞，T细胞，...）与细胞移植相互作用。 </li></ol> 6。代表结果我们在这里直观地提出了一个优化多式联运成像成功的萤光素酶/ EGFP-气骨（干）在小鼠中枢神经系统的细胞群的事件序列。起初，描述GB的MPIO的标签高效的BMSC-Luc/eGFP细胞的标签，可以很容易地验证程序的结果用荧光显微镜（ 图2A）。下一步，根据GB的MPIO的嫁接标记BMSC-Luc/eGFP在小鼠中枢神经系统，移植细胞的生存可以被监测，由劳保局在体内荧光素酶的活性（ 图2B）的基础上。此外，移植细胞的精确定位， 在体内可以监控</em&gt;的基础上，铁含量的GB MPIO（ 图2C）MRI检查。最后，病理分析，使BLI和MRI所取得的成果进行验证。稳定的生存证实EGFP表达稳定在时间（ 图2D）。对于这一点，共存EGFP表达细胞与蓝色荧光的MPIO允许精确测定本地化和移植细胞的生存。此外，这种双重荧光标记的干细胞减少的可能性，以观察5炎性细胞产生的背景荧光的假阳性结果。 <img alt="图1。" src="/files/ftp_upload/3906/3906fig1.jpg" /> 处理顺序概述图1。 <ol class="lroman"><li>细胞标记：BMSC-Luc/eGFP 5×10 6 GB的MPIO的每毫升培养液中的颗粒标记。孵化后16小时和24小时以下的休息时间，细胞收获脑内植入。 </li><li>细胞植入的MPIO GB的标记BMSC-Luc/eGFP嫁接在老鼠大脑在以下坐标：前囟门-2毫米，从中线和硬膜下的2毫米2毫米的权利。 </li><li>产生的光在体内劳保局：下面的静脉给药150毫克D-luciferin/kg体重，收购期间使用的光子成像的5分钟。 </li><li> 体内 MRI：一套10冠状T2加权自旋回波（SE）的图像和T2 *加权梯度回波（GE）的图像与平面的分辨率为70微米2，被收购。序列的参数如下：重复时间（TR）：500毫秒，回波时间（TE）：8毫秒（GE序列）和重复时间（TR）：4200毫秒，回波时间（TE）：12.16毫秒（SE序列）;领域的视角（FOV）：18×18毫米2，矩阵：256×256，1毫米层厚1毫米的片分离。 </li><li>验尸报告组织学：10微米厚的冰冻切片，筛选表达EGFP和蓝色荧光标记的骨髓基质细胞ü唱荧光显微镜。 </li></ol><img alt="图2。" src="/files/ftp_upload/3906/3906fig2.jpg" /> 图2。多式联运成像干细胞移植<ol class="ualpha"><li>冰蓝色（GB）的MPIO直接免疫荧光显微镜标记BMSC-Luc/eGFP。 </li><li> GB 在体内注射4X10 5小鼠劳保局MPIO的标记在有BMSC-Luc/eGFP 1周和2周后植入细胞。劳保局信号的检测，在第1周和2周后植入的统计分析。从5分钟的时间，平均每平方厘米每秒每球面度的光子数（PH / S / SR /厘米2）数据表示为+ / -小鼠呈现出明显的劳保局信号从一个固定的区域利益计算标准错误在注射部位。 </li><li>日冕二维MRI GB的MPIO的标记BMSC-Luc/eGFP（左侧面板：（一）自旋回波T2加权图像和（二）T2 *加权梯度回波图像）和未标记的骨髓基质细胞 - LUC / EGFP（右图：（三）T2加权自旋回波图像和（六）T2 *加权梯度回波图像）在鼠脑移植。 </li><li> GB的MPIO的组织学分析标记BMSC-Luc/eGFP种植体植入后2周。 （一）直接免疫荧光分析EGFP表达和GB的MPIO的的标记BMSC-Luc/eGFP移植本地化。 （二）高倍率的细节（我）。 （三）免疫荧光染色，GFAP的抗原，表明在GB的MPIO的周围的星形细胞瘢痕组织发展BMSC-Luc/eGFP标记。 （六）为伊巴-1抗原的免疫荧光染色，表明入侵和周围GB的MPIO的标记的小胶质细胞BMSC-Luc/eGFP。 </li></ol>

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O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+protein+binding+of+luciferase+substrates+influences+sensitivity+and+accuracy+of+bioluminescence+imaging.">Plasma protein binding of luciferase substrates influences sensitivity and accuracy of bioluminescence imaging.</a> Mol. Imaging. Biol. 13, 59-66 (2011).</li><li>Keyaerts, M., Verschueren, J., Bos, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+bioluminescence+imaging+for+quantitative+tumour+burden+assessment+using+IV+or+IP+administration+of+D:+-luciferin:+effect+on+intensity,+time+kinetics+and+repeatability+of+photon+emission.">Dynamic bioluminescence imaging for quantitative tumour burden assessment using IV or IP administration of D: -luciferin: effect on intensity, time kinetics and repeatability of photon emission.</a> Eur. J. Nucl. Med. Mol. Imaging. 35, 999-1007 (2008).</li><li>Zhang, Y., Bressler, J. P., Neal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ABCG2/BCRP+expression+modulates+D-Luciferin+based+bioluminescence+imaging.">ABCG2/BCRP expression modulates D-Luciferin based bioluminescence imaging.</a> Cancer Res. 67, 9389-9397 (2007).</li><li>Brightwell, G., Poirier, V., Cole, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-dependent+and+cell+cycle-dependent+expression+from+a+cytomegalovirus-based+mammalian+expression+vector.">Serum-dependent and cell cycle-dependent expression from a cytomegalovirus-based mammalian expression vector.</a> Gene. 194, 115-123 (1997).</li><li>Grassi, G., Maccaroni, P., Meyer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+DNA+methylation+and+histone+deacetylation+activate+cytomegalovirus+promoter-controlled+reporter+gene+expression+in+human+glioblastoma+cell+line+U87.">Inhibitors of DNA methylation and histone deacetylation activate cytomegalovirus promoter-controlled reporter gene expression in human glioblastoma cell line U87.</a> Carcinogenesis. 24, 1625-1635 (2003).</li><li>Krishnan, M., Park, J. M., Cao, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+epigenetic+modulation+on+reporter+gene+expression:+implications+for+stem+cell+imaging.">Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging.</a> FASEB J. 20, 106-108 (2006).</li><li>Svensson, R. U., Barnes, J. M., Rokhlin, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapeutic+agents+up-regulate+the+cytomegalovirus+promoter:+implications+for+bioluminescence+imaging+of+tumor+response+to+therapy.">Chemotherapeutic agents up-regulate the cytomegalovirus promoter: implications for bioluminescence imaging of tumor response to therapy.</a> Cancer Res. 67, 10445-10454 (2007).</li></ol>

作者宣称没有利益冲突。

在这份报告中，我们描述了一个在免疫小鼠中枢神经系统的细胞植入的详细描述。（BLI，MRI和组织学）三个互补的成像方式的组合优化的协议。记者细胞的基因标记，记者萤火虫荧光素酶和绿色荧光蛋白基因，并直接与GB的MPIO细胞标记基因改造的基础上，组合会导致体内的干细胞移植在准确的评估。 粒子标记的骨髓间充质干细胞，GB的的MPIO粒径组合与阴离子表?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> IMDM培养</td><td>龙沙</td><td> BE12-722F </td><td>细胞的生长介质的CEM组件</td></tr><tr><td>胎牛血清</td><td> Gibco公司</td><td> 10270-106 </td><td>细胞的生长介质的CEM组件</td></tr><tr><td>马血清</td><td> Gibco公司</td><td> 1605-122 </td><td>细胞的生长介质的CEM组件</td></tr><tr><td>青霉素链霉素</td><td> Gibco公司</td><td> 15140 </td><td>细胞的生长介质的CEM组件</td></tr><tr><td> fungizone </td><td> Gibco公司</td><td> 15290-018 </td><td>细胞的生长介质的CEM组件</td></tr><tr><td> PBS </td><td> Gibco公司</td><td> 14190 </td><td></td></tr><tr><td> puromycine </td><td> invivogen </td><td> ANT-PR-1 </td><td></td></tr><tr><td>胰蛋白酶</tD&gt; <td> Gibco公司</td><td> 25300 </td><td></td></tr><tr><td> GB的MPIO的</td><td>刘海实验室</td><td> ME04F/7833 </td><td></td></tr><tr><td>的D-荧光素</td><td> Promega公司</td><td> E1601 </td><td></td></tr><tr><td>氯胺酮（Ketalar） </td><td>辉瑞公司</td><td></td><td></td></tr><tr><td>甲苯噻嗪（Rompun） </td><td>拜耳医疗保健</td><td></td><td></td></tr><tr><td>异氟醚</td><td> isoflo </td><td> 05260-05 </td><td></td></tr><tr><td> 0.9％NaCl溶液</td><td>巴克斯特</td><td></td><td></td></tr><tr><td>多聚甲醛</td><td>默克公司</td><td> 1.04005.1000 </td><td></td></tr><tr><td>蔗糖</td><td> applichem </td><td> A1125 </td><td></td></tr><tr><td>微量注射泵</td><td>川崎病科学</td><td> KDS100 </td><td></td></tr><tr><td>光子成像</td><td> biospace实验室</td><td></td><td></td></tr><tr&gt; <td> 9.4T议员扫描器</td><td>布鲁克BioSpin </td><td> biospec 94/20的USR </td><td></td></tr><tr><td> BX51显微镜</td><td>奥林巴斯</td><td> BX51 </td><td></td></tr><tr><td> mycrom航模低温恒温器</td><td> prosan </td><td> HM525 </td><td></td></tr><tr><td>注射器</td><td>汉密尔顿</td><td> 7635-01 </td><td></td></tr><tr><td> 30号针头</td><td>汉密尔顿</td><td> 7762-03 </td><td></td></tr><tr><td>照片Vision软件</td><td> biospace实验室</td><td></td><td></td></tr><tr><td> m3vision软件</td><td> biospace实验室</td><td></td><td></td></tr><tr><td> paravision 5.1软件</td><td>布鲁克BioSpin </td><td></td><td></td></tr><tr><td>阿米拉4.0软件</td><td> Visage的影像</td><td></td><td></td></tr></tbody></table>

multimodal imaging of stem cell implantation in the central nervous system of mice

在过去十年中，造血干细胞移植已越来越为原发性或继发多种疾病，在临床前和临床研究的治疗方法的兴趣。然而，迄今为止，有关功能的结果和/或组织再生以下干细胞移植的结果是多种多样的。一般来说，临床受益观察没有深刻理解底层机制（S）1。因此，多的努力已导致不同的分子成像的发展，以监察其最终目的的干细胞移植，以准确评估移植的干细胞和/或他们的微环境的生存，命运和生理。分子成像确定一个或多个参数的变化可能与观察到的临床效果。在此背景下，我们的研究侧重于生物发光成像的结合使用。（BLI），磁共振成像（MRI）和病理analysis到评价干细胞移植。 ，劳保局常用非侵入性细胞跟踪和监视细胞的生存时间，移植后2-7细胞表达荧光素酶报告基因的生化反应的基础上能够发出光以下的相互作用，其基板（例如D-荧光素），8，9。另一方面，MRI是一种非侵入性的技术，这是临床上适用10，可用于精确定位，具有很高的分辨率11-15细胞移植，但其灵敏度高，取决于细胞标记后生成的磁共振造影剂的对比。最后，验尸的病理分析的首选方法，以验证获得最高的分辨率和灵敏度的非侵入性技术的研究成果。此外终点的病理分析，让我们进行详细的表型分析移植的细胞和/或周围组织，BASED上使用荧光记者蛋白质和/或直接与特异性抗体细胞标记。 总之，我们在这里直观地展现劳保局，MRI和组织学的互补性，解开不同的干细胞和/或环境相关的特征后，干细胞移植在小鼠中枢神经系统。作为一个例子，骨髓基质细胞，基因工程表达增强型绿色荧光蛋白（EGFP）和萤火虫荧光素酶（fLuc），和蓝色荧光微米大小的氧化铁粒子（MPIOs）标记，将嫁接中枢神经系统免疫能力的小鼠和成果将监测劳保局，MRI和组织学（ 图1）。

Watch this Scientific Journal Video about Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice at JoVE.com

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

本文介绍了优化多式联运成像啮齿动物脑细胞的使用移植的事件序列：（i）在活体动物和磁共振成像，及（ii）验尸的病理分析。结合在一个单一的动物允许这些成像分辨率高，灵敏度和特异性细胞移植评估。

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, ...

multimodal-imaging-stem-cell-implantation-central-nervous-system

Research

JoVE Journal

Neuroscience

11.0K 次观看.  University of Antwerp. 本文介绍了优化多式联运成像啮齿动物脑细胞的使用移植的事件序列：（i）在活体动物和磁共振成像，及（ii）验尸的病理分析。结合在一个单一的动物允许这些成像分辨率高，灵敏度和特异性细胞移植评估。

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

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

视神经横断：一个成人在中枢神经系统的神经元凋亡模型

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

科研

神经科学

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

从中枢神经系统转导细胞的慢病毒载体生产

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

标记的单细胞在中枢神经系统<em&gt;果蝇</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

在中枢神经系统Translatome由多核糖体分馏体内拷问

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

生物神经毒素在茎的网络文化功能评估细胞衍生的中枢神经系统神经元

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

在肠神经系统的原位 钙成像

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

鼠标中枢神经系统的光透明使用被动CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

第三星果蝇中枢神经系统活动的电生理记录

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...