Name: regioselective biolistic targeting in organotypic brain slices using a modified gene gun
Uploaded: 2014-10-24T13:00:00.000+00:00
Duration: 6 min 40 s
Description: Watch this Scientific Journal Video about Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun at JoVE.com

笔者想感谢的MRC-LMB车间生产的增强型基因枪枪管。我们还要感谢大卫·R·汉普森博士在多伦多大学使用的设备和资源。

利用动物和动物组织中应严格遵守当地下的规则和法规的伦理委员会的批准。本研究中坚持的MRC-LMB的动物实验指南中获得的所有组织。 1，准备材料，文化传媒<ol><li>准备用微孔水所有的解决方案;仅使用分析纯试剂。准备并储存所有试剂在室温下（除非另有说明）。 </li><li>使500毫升HEPES缓冲的盐水中（10mM HEPES，120mM氯化钠pH 7.2）中的。通过0.2微米的无菌过滤装置进行过滤。保存在4℃。 </li><li>制成500毫升磷酸盐缓冲盐水（PBS;的10mM的Na 2 HPO 4，2mM的KH 2 PO 4，137 mM氯化钠，2.7 mM的氯化钾，pH 7.4）中。通过高压灭菌消毒。 </li><li> 100 N2ス:通过补充的DMEM（Dulbecco改良的Eagle培养基），用25mM HEPES，10％胎牛血清，30mM的葡萄糖，1制备的神经介质pplement，青霉素，链霉素1100单位/毫升; pH值为7.2。用0.2微米的无菌过滤装置进行过滤。保存在4℃。 </li><li>使聚乙烯吡咯烷酮（PVP）储备溶液用20毫克的PVP在1ml 100％乙醇。分装在1ml的量，冷冻并储存在-20℃的这一点。工作溶液是0.05毫克/毫升，因此加10微升的PVP原液每4毫升的100％乙醇。 </li><li>准备一个0.05M的亚精胺原液在微孔水;将pH调节至7.2。 </li><li>使在DMEM 2％的琼脂糖溶液（2克电泳级琼脂糖在100毫升的DMEM中的无菌螺旋盖瓶）。微波炉，直到琼脂糖完全溶解。储存在4℃冰箱数周，如果有必要的。 </li></ol> 2，准备器官切片的<ol><li>安乐死C57黑色6小鼠所需的年龄，用CO 2窒息后斩首。采用横向切割头骨开始在枕骨大孔，并在结束消除大脑嗅叶。轻轻提起颅骨从背面露出的脑。 </li><li>嗅叶和额叶皮层，并且与小脑延髓之间切断。轻轻提起大脑延髓部分，切断视神经。最后，取出大脑，并放置在培养皿中填充有在冰上冰冷的HEPES缓冲盐水。 </li><li>使用解剖显微镜下，用细镊子剥去软;小心地取出周围脑血管和脑膜。 </li><li>将新鲜的大脑进入一个小的收件人模具和覆盖它与琼脂糖溶液冷却到略高于室温。然后迅速地通过将其放置在冰上冷却模具至4℃。 注:处理片尽可能快地避免生存能力的损失。 </li><li>当琼脂糖设定（5 - 10分钟），除去从模具的琼脂糖包埋的脑（修剪如果必要的话），然后将其粘到vibroslicer平台。把这个变成含无菌冰冷的PBS vibroslicer室。迪被用70％乙醇，以尽量减少后续污染前sinfect的vibroslicer室。 </li><li>剪下使用定制vibroslicer或商业等同大脑。该模型的概念是设计一个组织切片，可以产生大振幅和高频率的运动水平，以在叶片的边缘，同时最小化垂直振动。 </li><li>使用90赫兹的振荡频率，以1.5的幅度 - 3.2毫米，并且所述切割刀片定位在15°到水平平面的角度;设置在安装块保持的琼脂糖包埋脑移动至1.7毫米/朝向叶片分钟。收集部（150微米）到腔室含有冰冷的培养基（DMEM青霉素/链霉素+10％FCS）。组织切片看切割和操纵作为阿瑟诺和奥布莱恩13补充视频。 </li><li>轻轻将切片放入细胞培养插件（0.4微米，直径30毫米）的6多井托盘与文化MEDIA上的插入件的外部，并孵育在湿润的培养箱中在37℃下用5％的CO 2。 注:为确保正常组织活力，是非常温和的，而传输和处理的切片。另外，过多的流体将防止器官切片附着在过滤器插入件的膜。如果发生这种情况，去除多余的液体，或再次板上。 </li><li>虽然器官切片可以是几个星期培养，电镀最佳转染率后继续转不晚于4天。 注意:为了避免在长期的培养条件下，用阿糖胞苷或无 ​​血清培养基2胶质增生。 </li></ol> 3，制备DNA包被的微型和纳米弹丸<ol><li>准备采用40纳米直径的金颗粒的纳米弹。简言之，加入50微升的0.05M的亚精胺和在1毫克/毫升（pEYFP-N1）至10mg的金颗粒的10微升的DNA。 <ol><li>添加钙和亚精胺的溶液混合在DNA的金微TURE颗粒制剂，以便于DNA分子的结合，以帮助到金纳米粒子。 注:每金​​载波毫克（DLR）使用的DNA的量，范围应的金每1〜5微克DNA的1毫克。此外，金载体​​颗粒，将每盒（MLQ）下拍摄的量，应本身的范围从0.1到0.5毫克的金。 DLR的和MLQ应尝试和根据所研究的特定的系统，以及使用的颗粒和基因枪的种类上的每个用户进行了优化。 </li></ol></li><li>混合使用涡旋，同时缓慢加入50微升的1M的CaCl 2中的10级分- 15微升。以下5分钟，偶尔涡旋，离心分离机以1000×g离心30秒，然后除去上清液。悬浮金颗粒沉淀物3.5毫升，0.075M的PVP。 </li><li>得出这样的悬挂成管（2.36毫米的直径）使用注射器。放置在管道准备站的管道。允许金颗粒沉淀并通过抽吸用注射器除去上清液。旋转管均匀分布的金颗粒，其随后在恒定的氮气流量在5升每分钟进行干燥。 </li><li>要创建DNA的子弹，用切管器1厘米的长度切断油管。或者立即插入到基​​因枪盒或保持干燥的（4℃）直到需要。 </li></ol> 4，基因枪转染对器官切片<ol><li>执行的无菌层流净化罩中进行如下操作。 </li><li>插入一个9伏电池和一个空墨盒架到赫利俄斯基因枪。 </li><li>附加基因枪氦罐氦气软管。消防2 - 3空白（空槽）在50psi加压氦气软管和喷枪的水库。 </li><li>加载包含所述DNA包被的金墨盒插入墨盒座和支架加载到基因枪。 </li><li>拧下基因枪间隔和高度经修饰的基因枪管的基因枪。 </li><li>用无菌镊子，将包含器官切片放入无菌塑料培养皿过滤器插件。 </li><li>从器官切片取出介质。 </li><li>设置在50psi的气压。 注:眼睛保护是必要的，保护耳朵为宜。 </li><li>通过使用测量到10mm的安装放置在适当的距离上的基因枪。仔细瞄准镜筒的中心以上的基因递送所期望的区域。 </li><li>烧成后，用新鲜培养基，5％CO 2培养箱更换刀片放回盘，并将它们返回到正常生长条件下， 即 37℃。 </li><li>一旦完成，关闭氦气罐，释放基因枪的压力和氦气罐分离基因枪。 </li><li>取出墨盒座和抛弃盒和清洁基因枪。 </li><li>检查组织的形态和成功渗透visualizing使用倒置显微镜切片。如果使用的微粒，均匀地分散在黄金;应该看到该切片上的金颗粒没有密集的区域。由于它们的小尺寸纳米颗粒不能被看作是单个单元通过常规的显微技术。 </li><li>在适当的时间间隔后（通常1 - 2天），固定于4％多聚甲醛（PFA）进行显微镜观察切片。 注:固定在PFA并不总是需要的。活细胞成像避免这一步。 </li></ol> 5，固定和脑片的可视化<ol><li>基因枪转染后，在PBS中2分钟，两次冲洗切片。 </li><li>孵化在新鲜出炉的，冰冷的4％PFA的PBS 20分钟修复片。 </li><li>为2分钟，在PBS中洗两次切片。 </li><li>轻轻切圆膜支持使用手术刀，不影响安装在他们的片。使用镊子放在载玻片每个膜支撑/片。休息organotyp集成电路片上载玻片上的膜的顶部上。 </li><li>加安装媒体的一滴在片的顶部，并直接与器官切片接触轻轻加盖玻片，没有任何力。 </li><li>固定盖玻片有一层薄薄的指甲油了。 </li><li>查看该切片上的适当的显微镜。 </li></ol> 6，激光共聚焦应用<ol><li>通过使用一个堂堂正正的激光扫描共聚焦显微镜用60X / 1.4数值孔径（NA）油浸物镜可视化的切片。 注:共焦显微镜的最成问题的特点是针孔大小（设置为1 AU），它能够隔离和收集焦点平面，从而消除了失焦"阴霾"常看到的荧光样本。细细节往往被这个霾遮蔽，不能在一个非共显微镜检测。 </li><li>氩激光器设置为488纳米的激发波长和优化用于收集青霉的itted光在500至520纳米波段。通常情况下，收集的图像，在1024×1024像素，并与堆叠的z轴节在0.5微米的间隔为包括分析每一个神经细胞的整个宽度。 <ol><li>以观察细胞核的形态，计数器染色这些细胞用DAPI（激发波长设定在420之间收集405 nm，发射光 - 4 40纳米）。最后，用于检查红色荧光，设置激光在561纳米激发和565 - 620纳米的发射。 </li></ol></li><li>通常情况下，获得通过使用4倍变焦扫描，涵盖57.6 平方微米的面积图像;图像尺寸均在1,024×1,024像素的区域。的z轴节在0.5微米的间隔记录栈和5的突起 - 8帧的总和。 </li></ol>

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Part A : the Journal of the International Society for Analytical Cytology. 77, 693-704 (2010).</li><li>Sasaki, T., Matsuki, N., Ikegaya, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+axon-attached+recording+with+fluorescent+patch-clamp+pipettes+in+brain+slices.">Targeted axon-attached recording with fluorescent patch-clamp pipettes in brain slices.</a> Nature Protocols. 7, 1228-1234 (2012).</li><li>Gan, W. B., Grutzendler, J., Wong, W. T., Wong, R. O., Lichtman, J. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transduction+of+the+central+nervous+system+after+intracerebroventricular+injection+of+adeno-associated+viral+vectors+in+neonatal+and+juvenile+mice.">Transduction of the central nervous system after intracerebroventricular injection of adeno-associated viral vectors in neonatal and juvenile mice.</a> Human Gene Therapy Methods. 24, 205-213 (2013).</li><li>Arsenault, 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=Botulinum+protease-cleaved+SNARE+fragments+induce+cytotoxicity+in+neuroblastoma+cells.">Botulinum protease-cleaved SNARE fragments induce cytotoxicity in neuroblastoma cells.</a> Journal of Neurochemistry. 129, 781-791 (2014).</li><li>Arsenault, 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=Stapling+of+the+botulinum+type+A+protease+to+growth+factors+and+neuropeptides+allows+selective+targeting+of+neuroendocrine+cells.">Stapling of the botulinum type A protease to growth factors and neuropeptides allows selective targeting of neuroendocrine cells.</a> Journal of Neurochemistry. 126, 223-233 (2013).</li></ol>

Dr. John A. O'Brien holds patents for the improved gene gun barrel. US patent number 10/380,452 and European patent Number 01974517.3.

本协议描述的方法通过使用增强基因枪提供染料和遗传物质进入成人器官切片。本质上，染料的种类和各种可能的基因使该方法多方面和适合于回答广泛的生物学问题。用，在这种情况下，以特定的大脑区域中的区域选择性递送方法，打开用于实验作为局部区域的异源基因的调制的新途径的生物学相关架构内尚未容易可行之前。我们先前已经确定的，该方法能对成人器官切片，其中成熟的突触被完全成形的，因为它显示出改善的细胞存活和异源基因表达的许多周13被使用。用的新的和改进的培养条件为哺乳动物大脑以及其它组织的出现，使用区域选择性遗传调制的，将成为各种各样bioche的日益有用MICAL分析。 正如我们将要在这里和前面突出显示由他人24所提到的，许多不同的因素可容易地影响基因枪递送的准确性和可靠性。首先，与染料或DNA的金颗粒的制备方法，必须有足够的平衡，以防止聚集，并促进暗盒货物驱逐。钙和亚精胺中的DNA的金微粒溶液可以帮助DNA分子结合于金纳米颗粒。穿透细胞减压能力的纳米颗粒被预先决定的17。其次，基因枪必须安装尽可能平稳和正确的角度和距离。一个可靠的压力表也是必要的重复性，以维持组织的完整性，并妥善加快金颗粒其预定的目标。事实上，距离，方向，和设备的稳定性也是一个重要的因素为吨argeting和精确的区域选择性交付。作为这些大脑结构是非常小的，在触发角的微小变​​化可以很容易地使整个过程更不可靠。最后，编写和维护的可行器官切片已经误人子弟用了几十年的困难对于不同的动物，大脑区域，年龄和共培养物还丰富，可靠的协议是目前可用的2,4,25,26。这些程序应该是用户提交的优化组织，以基因枪过程之前。这将会更容易地允许用户无偏确定该生物射弹粒子的影响，所述异源基因的上自己的文化的影响。为此，利用荧光基因如EYFP和EGFP的既可以使一个新的用户，以测试靶向精度和也遵循通过异源基因表达的细胞的存活率随着时间13持续的时期。许多可靠的关键步骤且可重复使用该协议在以下三段突出显示。 对于高效的转染，所述DNA浓度必须适当。浓度过低会妨碍转屈服而浓度是太高会导致纳米颗粒的附聚。黄金的聚集体可以显着降低转染效率，导致不均匀的分布，并且可以导致增加的细胞毒性和氧化胁迫。与涂覆效率的问题很可能是由于亚精胺溶液期满（应改为每2〜3个月）。对于适当的基因枪传播，标签必须是偶数。金纳米颗粒/ DNA悬浮液的非均相标记可能是由于PVP的溶液。它也应更换，每2至3个月。黄金纳米粒子的涂层，可以看到在琼脂糖凝胶电泳更高MW波段27。 每个生物SYST根据调查青霉以及使用可能需要在气体压力的微小变化，达到转染和基因枪传播的优化水平各个不同的仪器。关键参数是剥离从塑料盒中的微米或纳米载体和推动它们进入组织所需的氦脉冲的压力。高气体压力会影响细胞存活，因为它会造成整个目标冲击波，破坏或从基质17中分离的组织。针对基因枪管和具有完全垂直于组织的装置是用于精确基因枪递送重要。所选择的区域应当放置在枪管的直接中心在从外开口恰恰10mm以下。这将导致一个直径3毫米的震中周围的金颗粒递送。基因枪枪管应在每次使用后70％的乙醇进行清洗。为了防止大颗粒聚集体组织，桶中还含有尼龙网的SHOULD定期更换以保持最佳性能。为了更换筛网拧开盖子然后将盖和O形环之间的新的尼龙网。 2天震中周围染后 - 荧光标记的细胞应该至少1是可见的。没有或标签错位可导致制剂基因枪安装的不足和不稳定。使用共聚焦显微镜细胞的观察结果取决于许多因素:在激发/发射光的波长，针孔大小，物镜的NA，组件中的光路的折射率，所述组织，并且对齐的深度仪器。这些因素应为每个系统在调查进行优化。 在基因枪法的最新进展进行了利用这种方法的成功。在本研究中使用的基因枪管，以便减少所需的压力以及漏斗生物射弹已被修改散射图案融入了更多的限制和具体的区域17,19。其他桶改装试图限制基因枪传播，减少流出压力28但这种定制设计的基因枪枪管由奥布莱恩博士设计是安装在标准的Helios基因枪单个组件。接着，亚微米的金颗粒被用来减少我们先前已经确定的微米颗粒的浸润引起的细胞损伤，并最终持续异源基因表达的丧失。这些变化的生物射弹方法改进了方法13的整体可行性和可重复性。片上制备其他诸如深入的故障排除在切片过程中，使用的DMEM琼脂糖基质以保护大脑，和许多其他有用的进步，器官型脑片的制备此前报道13使我们能够探索利用成人片了已经开发成熟突触网络。 在我们的研究中，我们主要使用的染料和荧光异源基因以可视化细胞的形态作为转染效率的读出，并有能力将图像的小鼠脑组织的神经元的形态学特征。该信号所定义的范围内随机配送的信噪比使我们的母语环境中完全分离单个细胞的树突状海港。无数的努力被用来研究这些形态特征，绘制"connectomics"或标记单个细胞的各种分析，这种方法可以很容易地与现有的协议，如电生理和神经突起的测量29,30组合。显然，荧光异源基因的组合可以使一个更强大的划分单细胞荧光蛋白的随机分布，他们的组合将确定各个单元31,32的颜色。使用细胞特异性启动子，如突触蛋白和胶质原纤维酸性蛋白的异源基因的外显子的上游，也可以限制为表达对亚群33。事实上，无休止各种遗传物质，也可以在一个区域选择性地通过使用相同的过程金纳米颗粒递送。的总转染区域因此可以用传统的方法，如解剖并提交该区域的免疫印迹分析，异源基因的局部效果进行分析。 在器官切片程序的改进也将允许更广泛地使用脑组织的长期试验，以及允许使用其它组织和共培养的，并能促进转染程序。脂和聚合物的转染之前已经报道了影响膜稳态，内化，蛋白渗透率34和经常表现出非常低的效率，转染德rminally分化的神经元。小区特定货物的定位也适合仅向细胞中表达所需的内在化的细胞表面受体，并且虽然这种方法可以是高度选择性的，它可以缺乏靶向区域选择性35。病毒载体否则往往困难和昂贵的生产，需要严格的法规，并且对之际显示的安全性顾虑。基因枪递送因而具有无与伦比的能力，可行的组织中，以区域选择性转染神经元和其他类型的细胞。作为金是非在用基因枪法有毒，进一步进展将铺路生物医学用途，例如经皮药物递送，非侵入性的体内基因递送，以及局部的非病毒基因疗法。

最初的生物射弹技术中，点灯-的短语"生物弹道学"，建立了粒子介导的基因转移到植物细胞1。细胞转化的这种物理方法加速微 - 或纳米颗粒以高速来克服不渗透细胞膜的物理屏障，以提供货物，如DNA或染料。因为它不依赖于特定的配体 - 受体和/或在细胞表面的膜的生化性质，粒子介导的基因转移可以容易地适用于各种生物系统中，如器官型脑切片。 利用器官切片比其他优势， 在体外平台，因为他们认为很多解剖和生化性质是相关的体内生物学2-4。切片大多保存从那里起源和preser当地的建筑特色已经神经化学活性和突触连接。使用脑切片用于基础研究的，并在药剂的努力，已伴随增加，可能的生物技术操作来测量和监测大脑在体内的神经生物学行为，如上下文3,5-7数。使用器官切片为基础的检测系统的主要优点是它提供了容易的实验控制，并且允许细胞外环境的精确操作。 富有成效，器官切片培养系统已经建立了从各种脑区域，例如，但不限于，皮质，脊髓和小脑8-10。此外，一些共同培养已经证实，这允许在远端的脑区，以及神经元和神经病理细胞11,12间的连接通讯的评估。许多协议已经建立送uccessfully文化的器官切片，并能保持长期的活力和最近的许多研究，现在利用膜界面的方法和各种修改它13。这个原理通过将片上的多孔膜过滤器保持器官切片在培养基和培养器的加湿空气之间的界面。因此，媒体可以提供足够的营养物质通过毛细血管运动喂器官切片。通常切片已准备从出生后早期的动物（3 - 9天之久; P3 - 9）。然而，从这些切片的脑组织显示细胞可塑性的高水平和有机械应力的固有电阻，这是有益的，以获得可行的培养物，但成熟的突触和神经解剖学电路没有完全开发在体内直到2至3周龄14。例如，以前的观察结果表明，从P0得到的海马切片 - 1新生儿，虽然高度VIAB乐以下准备，逐渐失去了一些形态特征。本质上，它们被证明不适合于长期培养表明未成熟细胞更可能去分化相比从老年动物15,16器官型培养。由于这个原因，我们的方法进行了优化，成人器官大脑切片在其成熟和建筑发展已达到其终端阶段13,17-21。然而，这种方法也适用于新生儿和少年器官切片。 一旦可行器官切片已经生产含有切片的整个板可以被带到该基因枪安装并提交给区域选择性输送和转染。正确安装的基因枪（如在图1中描述的）的，面向90°的距离为10毫米的正上方的片（从开口至所述组织），允许快速基因枪递送的40纳米的去LD颗粒涂层货物。这些货物如染料和荧光DNA载体，以及任何感兴趣的基因，可容易地输送到活切片自如。这种方法从而说明了此协议需要交付，在区域选择性的方式，货物涂层的金纳米粒子转化为可行的器官大脑切片。基因枪枪管和最佳的安装可以看出，在图1中 ，有关改进枪管和纳米弹道的详细信息，请参阅奥布莱恩等17。 随后精炼还适用于用户优化的条件，如金每个目标区域传递适量的，其定义为微载体装载量（MLQ），并以确定的DNA的每金毫克装载量，定义为DNA加载比率（DLR）。此前沉淀的DNA在金颗粒和其装入Tefzel管，它包括实际基因枪"墨盒9;，它是必要的，以计算DNA和金的需要为每个转染，可以组织和条件之间略有不同的量（比率应保持在1毫克黄金和1微克DNA到1:5）。至关重要的是，制备MLQ的适当比例，以DLR另有DNA包被的金颗粒会粘附在一起，形成比预期的结块，这可能会降低基因枪传播的整体均匀性，以及增加组织损伤和细胞毒性更大。 这种方法显示了基因枪递送的最新改进，这已经过测试，确定为适合新生儿，青少年和成人的器官大脑切片。此外，通过利用基因枪枪管的降低基因枪传播，用户现在能够选择性地转染准时区域中的脑。下列适当的温育时间，所述荧光蛋白的表达，从而达到其24和48之间的最大速率小时，可通过荧光和共聚焦显微镜观察。这些荧光团允许范围内的生物相关结构的形态学分析和单个细胞的定位。然而，器官切片由其它异源基因的区域选择性调制，也可以通过适合于这些制剂中的任何其他测试来监测。可能的工作策略的局部基因递送示于图2。

<table><tbody><tr><td>Helios gene gun system</td><td>Bio Rad</td><td>165-2431</td><td>Gene gun and tube prep station</td></tr><tr><td>HEPES&amp;nbsp;</td><td>Sigma</td><td>83264-100ML-F</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S7653-250G</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333-500G</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S7907-100G</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P9791-100G</td><td></td></tr><tr><td>0.2 &amp;micro;m sterile filter unit</td><td>Millipore</td><td>SCGPU05RE</td><td>to filter media</td></tr><tr><td>DMEM</td><td>Gibco</td><td>21885-025</td><td></td></tr><tr><td>Fetal calf serum</td><td>Gibco</td><td>10270-098</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma</td><td>G8270-100G</td><td></td></tr><tr><td>N2</td><td>Life Technologies</td><td>17502-048</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Sigma</td><td>p4333</td><td></td></tr><tr><td>Polyvinylpyrrolidone</td><td>Sigma</td><td>856568-100G</td><td></td></tr><tr><td>Ethanol&amp;nbsp;</td><td>Sigma</td><td>277649-100ML</td><td></td></tr><tr><td>Spermidine</td><td>Sigma</td><td>S2626</td><td></td></tr><tr><td>Agarose</td><td>Bio Rad</td><td>161-3100EDU</td><td></td></tr><tr><td>Vibroslicer</td><td>Laica</td><td>VT1200</td><td>We used a custom design</td></tr><tr><td>Gene gun mount</td><td></td><td></td><td>Built in house at U of T</td></tr><tr><td>Humidified cell culture incubator</td><td></td><td></td><td></td></tr><tr><td>Gold nanoparticles</td><td>cytodiagnostics</td><td>G-40-20</td><td></td></tr><tr><td>pEYFP-N1</td><td>Clontech</td><td></td><td></td></tr><tr><td>Tubing cutter</td><td>Bio Rad</td><td>165-2422</td><td></td></tr><tr><td>Tefzel tubing</td><td>Bio Rad</td><td>165-2441</td><td></td></tr><tr><td>Barrel</td><td></td><td></td><td>Custom made by the LMB</td></tr><tr><td>Cell culture insert</td><td>Millipore</td><td>PICM0RG50</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>76240 Fluka</td><td></td></tr><tr><td>Dissecting microscope</td><td></td><td></td><td>for convenience</td></tr><tr><td>Confocal microscope</td><td></td><td></td><td>user defined for usage</td></tr><tr><td>Glass slide</td><td>VWR</td><td>2588-CA48323-185</td><td></td></tr><tr><td>Microcover glass</td><td>VWR</td><td>2448-CA48366-067-1</td><td></td></tr><tr><td>Vectashield mounting media</td><td>Vector laboratories</td><td>H-1400&amp;nbsp;&amp;nbsp;</td><td></td></tr></tbody></table>

regioselective biolistic targeting in organotypic brain slices using a modified gene gun

DNA转染是非常宝贵的生物科学和最新进展，以器官型脑片的准备，各种异源基因的影响因此可以很容易地调查，同时保持体内生物学的许多方面。已经有越来越多的关注，转染终末分化的神经元的传统转染方法一直困难重重，如低收益率和存活率显著损失。基因枪转染可以规避许多的这些困难，但只有最近，这种技术被修改，使得它适合于在哺乳动物组织中。 新修改的加速器腔增强了基因枪的射击精度，并增加了它的深度渗透，同时还允许使用较低的气体压力（50磅），而转染效率的损失，以及允许PA的聚焦区域选择性扩散rticles在3毫米。此外，这种技术是直线前进和速度比乏味显微注射来执行。瞬态和稳定表达是可能的纳米粒子轰击，其中附加型表达可以在24小时内被检测到，并在细胞存活被证明是优于或至少等于，常规的方法。该技术具有但是1关键优点:它允许转染到在单个约束半径之内本地化从而使用户能够将解剖分离的异源基因的影响。在这里，我们提出了一个深入的协议，准备可行的成人器官切片，并提交他们使用了一种改进的基因枪区域选择性的转染。

器官切片生存能力可以乳酸脱氢酶活性，碘化丙啶标记和染色的dUTP进行监测正如以前报道的13,22。显然，切片的可行性和完整性是针对遗传交付货物的长期持续表达很重要。下列基因枪递送到器官切片，在荧光的异源基因的表达通过共聚焦显微镜监测。基因枪传播的使用改性桶的气密性可以看出，在图3A图3B显示了转染的海马区，使用的DNA包被的金粒子的代表性图像， 图4示出了转染的成体小鼠器官切片的代表性图像。如在图4A中 ，浦肯野神经元具有显着的树突状海港的浦肯野层的界面和减量的分子层中可以看出ebellum示出以下瞬时基因枪转染用质粒pEGFP-N1的明显标记。 图4B中示出了这些树突其中棘可观察到较高的放大率。 图4C示出了以下基因枪递送pDSredFP涂布的金纳米粒子在海马的CA1区发现了一个锥体细胞。 染料代替荧光编码的DNA的货物，也可以用类似的方式来可视化的器官切片的形态特征。 DIO，该标签质膜，可以更迅速地划定各个细胞的形态，也可以配合使用递送至相同的区域的DNA涂覆的生物射弹以确认regioselection。如在图5A中可以看到，在海马沾上DIO神经元也显示出长枝晶与众多棘。的箭头表示由突触终扣的存在下确定相比于dentrit轴突集成电路刺的其它突起。 图5B示出了CA1区，突出用DAPI复染剂众多海马突起标记细胞核。 图5C示出了这些类型的并行树突与众多棘的高倍放大的另一个例子。 很显然，这些照片反映了下列细胞标记的形态特征。没有排除被施加以提供任何数量的涂覆在金纳米颗粒的外源基因相同的方法。以允许成功转染的视觉确认，单个质粒可以被构造成共同表达感兴趣两个基因，并在同一载体上的荧光报告蛋白。 <img alt="图1" src="/files/ftp_upload/52148/52148fig1highres.jpg" /> 图1基因枪枪管和安装。（一）提高<html>由奥布莱恩博士在MRC-LMB，有一个限制基因枪传播，通过降低所需的压力，粒子加速减少组织损伤的开发ð桶。（二）优化基因枪的侧视图安装了亨德森博士开发和安德拉斯·纳吉博士在多伦多大学。安装保持基因枪在精确90˚垂直于器官切片，同时允许用户精确地控制基因枪递送的距离。为了获得最佳的基因枪条件枪管孔应直接放置在所感兴趣的区域在从器官切片10毫米的距离。基因枪安装的（C）的后视图。感兴趣的被转染的区域需要被直接放置在枪管孔的下方。针对可与染料（diolistic）或荧光蛋白转染（基因枪）来确认。ANK"&gt;请点击这里查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/52148/52148fig2highres.jpg" /> 图2。器官切片制备和区域选择性靶向。（A）的DMEM嵌入式成年小鼠的大脑准备好切片（B）模具被粘到vibroslicer与刀片准备切割片安装（C）区域选择性异源基因表达策略基因枪递送到冠状器官大脑切片。从艾伦脑图谱解剖参考图谱23获得图片制作图片。左图显示了有针对性的，例如，两个不同的大脑区域。到限制的基因表达（1）向左侧半球的皮层区。到限制的异源基因的表达（2）至脑的下丘脑区域。中间的面板显示THA吨的两种异源基因的表达（图3和4），可以分离和两个半球的海马中的相同器官切片进行比较，基本上消除了来自使用不同的部分和/或给予的可能性，以解决来自的效果远端串扰任何偏压这些基因。右图显示转基因递送的可能重叠表达的第一个基因（5）精确的皮层区域，同时又表达基因（6）在一个完全不同但重叠的皮质区。这允许单独分析，也各外源基因在同一个片内组合的遗传调节的效果。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52148/52148fig2large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52148/52148fig3highres.jpg" /> 图3枪法散射图形上看到FILT呃纸和低倍率的图像（A）中的改进的桶被解雇在滤纸上，确定金颗粒的扩散。左侧示出了改进的筒的分配，而右侧表示正常桶。黑棒:1厘米。从奥布莱恩图像拍摄等人 17（B）低放大倍数图像表示内生物射弹蔓延6周龄小鼠的海马区中的3毫米随机转染图案。白条:100微米<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52148/52148fig3large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52148/52148fig4highres.jpg" /> 图4枪法交付荧光蛋白编码的DNA导入6周龄的小鼠器官切片的（A）无脊椎动物ð共聚焦图像的的pEGFP-N1涂金颗粒转浦肯野细胞的小脑固定器官切片的。图片摄于40倍的放大倍率。黑棒:30微米（b）为 60倍的另一个浦肯野细胞的树突状海港突出的刺。黑棒:5微米（c）所示 ，从海马区采取60倍的放大倍率4个固定片DSredFP转锥体细胞的共聚焦图像。黑棒:10微米<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52148/52148fig4large.jpg" target="_blank">，请点击这里查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52148/52148fig5highres.jpg" /> 图5 Diolistic递送膜标记荧光染料到器官切片（A）示出了海马神经元升的实时成像在放大20倍abeled与DIO。的树突棘可清楚地观察到，以及所确定的不存在树突棘和由白色箭头指示突触终扣的存在轴突。白条:标有DIO和counterstained用DAPI的海马区的30微米（二）发音的染色。白条:海马CA1区发现了树突棘的30微米（C）的放大倍数（60倍）。白条:5微米<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52148/52148fig5large.jpg" target="_blank">请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun at JoVE.com

Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

Recent improvements in organotypic brain slice preparations have permitted their exploitation for biotechnological applications. Organotypic slices maintain local structural characteristics of in vivo biology, including functional synaptic connections. Here we present a regioselective biolistic delivery method to label and genetically manipulate these slices.

区域选择性基因枪瞄准的器官脑片用改进的基因枪

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various ...

regioselective-biolistic-targeting-organotypic-brain-slices-using

Nanyang Technological University

Research

JoVE Journal

Neuroscience

16.3K 次观看.  University of Toronto. Recent improvements in organotypic brain slice preparations have permitted their exploitation for biotechnological applications. Organotypic slices maintain local structural characteristics of in vivo biology, including functional synaptic connections. Here we present a regioselective biolistic delivery method to label and genetically manipulate these slices. 

视频: Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

可伸缩流控喷油器阵列病毒完整的3 - D的大脑回路为目标

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

科研

神经科学

Organotypic Culture of Adult Rabbit Retina

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with imaging techniques, genetic manipulation, and electrophysiological recording. Here we present a simple interphase tissue culture system for adult rabbit retina that requires no specialized equipment and very little maintenance. We demonstrate the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells. Rabbit retinas cultured this way can be kept alive for up to 6 days with very little changes of the overall anatomical structure or the morphology of individual ganglion- and amacrine cells.

This article demonstrates the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells.

器官型培养的成年兔视网膜

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with ...

生物学

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

从大脑皮层的器官型切片制备全细胞记录

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.  

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

基因枪子弹的制备和神经元的基因枪转染切片文化

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed ...

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls.
The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of ~50-150 &#181;m and extends over ~15 &#181;m around the mean penetration depth, which is adjustable between 0 and 50 &#181;m. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches.
For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery.
When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.

In this video, we show a procedure for an accurate biolistic delivery of reagents into live tissue with a novel miniature gene gun. We are knocking down the expression of the axon guidance molecule Netrin in leech embryos by delivering molecules of dsRNA into the ventral body wall and ganglia of single segments.

本地化的RNAi和药用水蛭异位基因表达

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to ...

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits&#160;in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations&#160;in vitro.

Optogenetically有针对性的前脑电路的激光扫描光刺激

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the ...

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

激光制导神经跟踪在脑外植体

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

<meta charset="utf8"></head><body>通过电穿孔在切片的人皮质类器官中敲除 CRISPR/Cas9 基因

Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function

Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. Studies analyzing gene function by overexpression or knockout have been instrumental in animal models to provide mechanistic insights into the regulation of neocortex development. Here, we present a detailed protocol for CRISPR/Cas9-mediated acute gene knockout by electroporation of sliced human cortical organoids. The slicing of cortical organoids aids the identification of ventricle-like structures for injection and subsequent electroporation, making this a particularly well-suited model for acute genetic manipulation during human cortical development. We describe the design of guide RNAs and the validation of targeting efficiency in vitro and in cortical organoids. Electroporation of cortical organoids is performed at mid-neurogenic stages, enabling the targeting of most major cell classes in the developing neocortex, including apical radial glia, basal progenitor cells, and neurons. Taken together, the electroporation of sliced human cortical organoids represents a powerful technique to investigate gene function, gene regulation, and cell morphology during cortical development.

<meta charset="utf8"></head><body>切片人皮质类器官的电穿孔用于基因功能研究

Here, we present the acute genetic manipulation of sliced human cortical organoids by electroporation. These cortical organoid models are particularly amenable to injection as ventricle-like structures can be readily identified after slicing, enabling the functional investigation of human cortical development, neurodevelopmental disorders, and cortical evolution.

切片人皮质类器官的电穿孔用于基因功能研究

Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. ...

区域选择性基因枪瞄准的器官脑片用改进的基因枪

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此视频中的章节

可伸缩流控喷油器阵列病毒完整的3 - D的大脑回路为目标

器官型培养的成年兔视网膜

从大脑皮层的器官型切片制备全细胞记录

基因枪子弹的制备和神经元的基因枪转染切片文化

本地化的RNAi和药用水蛭异位基因表达

Optogenetically有针对性的前脑电路的激光扫描光刺激

激光制导神经跟踪在脑外植体

切片人皮质类器官的电穿孔用于基因功能研究

切片人皮质类器官的电穿孔用于基因功能研究

切片人皮质类器官的电穿孔用于基因功能研究