这项工作得到了中心的再生疗法，医学系的德累斯顿，与东风集团的合作研究中心SFB655（子项目A20）。 BA支持DIGS-BB计划，德累斯顿的奖学金。我们感谢博士。纪子大隅，野村忠，迪特尔Chichung列，和拉维Jagasia的宝贵意见期间设立在子宫穿孔和立体的病毒注射。

初步说明批准后由地方当局对动物福利的全面培训的调查人员，必须进行手术。必须采取一切预防措施，以尽量减少造成的痛苦和压力的动物，包括深度麻醉，术后镇痛和管理，业务持续时间超过10分钟，眼睛润滑剂，以防止角膜脱水和失明的应用。麻醉小鼠，必须始终保持温暖，在37°C，直到完全康复，每一个外科手术工具和解决方案必须是无菌的。虽然不是绝对必要的生物罩的使用，运营商必须采取一切合理的预防措施，以尽量减少穿一个白大褂，口罩和手套，在操作过程中感染的动物的可能性。同样，关于病毒的产生和使用的每一个步骤必须被授权，并根据地方当局的规定，必须在S2区。最后，彻底的净化和表面接触病毒可能已经在所有的工具，必须按照经批准的设施消毒的做法（艾滋病毒的70％ET-OH和/或高压灭菌擦拭）。 第1部分：扩展的胚胎NSC （1） 在子宫穿孔 <ol><li>克隆后PCMS-EGFP（Clontech公司）或pDSV-mRFPnls的载体16中的外源基因（ 即 CDK4/cyclinD1的），纯化的质粒，使用EndoFree kit和重悬在无菌PBS中的浓度为3-5微克/微升。不久，在手术前，混合质粒与在PBS 0.05％FastGreen在最后的比约。 4时04分01秒和离心处理，以使混合物在16,000 xg离心2分钟，以除去所有的析出物。 </li><li> DNA混合物加载到一个先前拉的硼硅酸盐玻璃毛细管。使用P-97移液器拉马拉参数有：拉力：200; VEL：140;时间：140。热由下式给出一个坡道测试，并取决于该规范IFIC大量的毛细血管被使用。安装在毛细管上的PicoPump的喷嘴附近的子宫穿孔平台（ 图1A），在立体显微镜下，弯曲的尖端和缺口的拐点。 </li><li>所有的手术工具消毒，在干燥的玻璃珠灭菌器和一个使用异氟醚蒸发器妊娠12-15天的孕鼠在深麻醉。将动物仰卧位，在37°C加热平台，并保持在恒定的异氟醚通过鼻锥。 </li><li>剃须腹部皮肤，消毒与Betaisodona多次，最终在两者之间擦拭用70％乙醇，并在0.1毫克/千克的浓度作为先发制人镇痛皮下注入丁丙诺啡（在PBS中稀释）。使用精细剪刀，使一个2厘米的皮肤纵向切割，随后，底层的肌肉壁进入腹膜腔。免除在腹膜腔的CA。 2IUE毫升的溶液（D-PBS中含有100 U / ml的笔/链球菌），预先加热到37℃，并保持的加热块上的解决方案。 </li><li>量的鼠标与含无菌DRAP的从将被删除的子宫的裂缝。使用钨牵引器牵开切口，确定子宫，并把它用钳子相邻的胚胎（ 图1B左），最后把它放下的无菌DRAP上。在整个操作过程中，冲洗子宫IUE解决方案，滋润器官和体腔，防止脱水的动物。 </li><li>处理子宫之间的拇指和食指小心翼翼地捧着它，直到它的头是可见的，面向运营商，并打开一个胚胎。确定在前脑半球，从后背成外侧注入其中之一。松开1-2微升DNA溶液使用脚踏开关的PicoPump直至心室概述者FastGreen（ 图1B中 ，右）。 </li><l我&gt;将注射部位和阴极上的对侧（ 图1C）上的电极的阳极和提供6个脉冲的30 V 50毫秒与950毫秒的每个脉冲之间的间隔，使用生成方形的电场的电穿孔操作通过脚踏板。避免放置电极的胎盘，这可能会导致出血和损伤胚胎。 </li><li>当所有的胚胎被注射和电穿孔，将子宫进入腹腔，关闭肌壁缝合缝合，每3-4毫米的字符串。本节应舒适的肌肉，但不要太紧，让一点点肿胀的组织。最后，关闭使用金​​属夹上覆皮肤。消毒的皮肤与Betaisodona，移开鼠标异氟醚的面罩，将其转入完全清醒时，住房笼。监测的自发行为是正常的鼠标，直到恢复。 </li></ol> 2。处理的样品PLE <ol><li>一个或多个电击后，牺牲鼠标，收集胚胎，确定正确的电穿孔用荧光体视显微镜（ 图1D）。在冰冷的PBS的大脑解剖，并解决他们在4％PFA（多聚甲醛磷酸盐缓冲液pH值7.4）过夜。最终，尿嘧啶可以牺牲前管理，根据不同的模式，以研究细胞周期的影响以及细胞增殖3。 </li><li>大脑切片的几个指标，基础的放射状胶质祖细胞和神经元（PAX6，巢，TBR2，TBR1，Tuj1等）和免疫效果进行评估的功能转基因有针对性的NSC和他们的后代，然后再处理所确定的共同电穿孔的报告基因的表达。 </li></ol><img src="/files/ftp_upload/4093/4093fig1.jpg" alt="图1" /> 图1。子宫穿孔。（A） 在子宫穿孔平台的示意图。 （B）示出的定位，在手术过程中的孕鼠（右）和注射的DNA / FastGreen的混合物（蓝色）在端脑半球的小鼠胚胎（左）的图纸。 （C）的流程示出了放置在注射部位的相邻的（蓝色）与阳极（+）与子宫壁接触的电极。箭头表示的DNA向阳极迁移。 （D）的电与GFP质粒在胚胎第13天的小鼠胚胎后24小时的图片。虚线划定的在前脑半球。一种改进的来自Calegari 等。，2004年17计划。 第2部分：扩展的成人NSC 1。艾滋病毒衍生的病毒颗粒的生产<ol><li>第1天：板5×10 6 293T细胞，在10厘米盘用8ml含10％热灭活胎牛血清，100 U / ml的笔/链球菌（培养基）的D-MEM。使用15道菜，一个病毒的准 ​​备和培养过夜，在37°C，5％CO 2。 </li><li>第2天：为每一个培养皿中，在1毫升的纯的D-MEM5μg的每个编码对于i的3个质粒稀释）VSVG（水泡性口炎病毒G型）的包膜蛋白，ⅱ）堵嘴/聚合酶（群特异性抗原/聚合酶） ，和iii）一个多顺反子构建表达的功能和记者转基因（ 即 cdk4/cyclinD1/GFP）的先前克隆在艾滋病毒为基础的转移的向量（p6NST90）9。混合此解决方案的D-MEM含45微升聚乙烯亚胺的已预先溶解在PBS中在1毫克/毫升原液，并孵育15-30分钟，在室温下用1毫升。在此期间，取出介质的细胞，每道菜的新鲜的D-MEM含15％热灭活胎牛血清，100 U / ml的笔/链球菌加4毫升。 2米升转染混合物和培养过夜。 </li><li>第3天：加120微升500毫米，每道菜，以提高转基因的表达，丁酸钠，轻轻地混合和培养6-8小时后更换转染培养基，用5毫升的培养基。 </li><li>第4天：收集上清液（约70毫升总），通过0.22μm的过滤器和转让在2 polyallomer的离心管中（35×89毫米）。超速离心机在110000×g离心90分钟，在4℃下使用的摆动转子。弃去上清液，加入6毫升的PBS中在每管中，并在冰上孵育1小时。将沉淀重悬于1 polyallomer离心管中（14×95毫米）中，并将其转移。超速离心机在110000×g离心90分钟，在4℃下使用的摆动转子。弃去上清液，将沉淀重悬于50μl的PBS中，使5微升等分并存储在-80℃下病毒滴度将在10 8 -10 9菌落形成单位/毫升的范围内。 （ 注：病毒是非常敏感的温度和储存，避免ID重新冷 ​​冻，保持干冰在运输过程中解冻不久之前使用。） </li></ol> 2。立体定向注射<ol><li>用异氟烷麻醉6-10周龄的鼠标和放置动物俯卧使用耳酒吧和腭支持正确地固定在头部的立体定位框架（ 图2A）。动物被设定在37℃和下恒定的异氟醚给药上的加热垫保持温暖。 </li><li>先发制人镇痛的的丁丙诺啡工作解决方案皮下注射100μL，剃的头鼠标条纹皮肤上，desinfect在1.4。使用手术刀，加利福尼亚州。 1.5厘米长的纵行切开头部皮肤暴露颅骨矢状缝水平。收回的皮肤和骨表面用无菌棉签轻轻擦拭。 </li><li>半用石蜡油填充的玻璃毛细管（在1.2的规格），将其安装在该喷射泵插入的毛细管，直到与第二环（ 图2B）。将毛细管的毛细管的前端被定位保持器中的x，y和z轴，直到上囟，结合点之间的矢状面和横向缝合（ 图2C）（待确定与立体显微镜的帮助下），以及重新设定的x，Y，和z的值为0。 </li><li>将毛细管中的前后轴（y）的±1.6毫米的内 - 外轴（x）和-1.9毫米，使用外科记号笔骨标记。约一个洞。 0.5毫米直径的颅骨上，注意不损伤大脑的使用电动钻机。 </li><li>把一块封口膜放置在耳朵上杆5微升的液滴上的病毒悬浮液（1等分试样）和浸入的前端的毛细管中的液滴。吸加利福尼亚州。 3.5μl的病毒悬浮液的使用纳升注射器设定在55升/秒。执行此步骤，迅速的雾滴会蒸发。 </li><li>将毛细管的网站Of注射液，并将其向下移动，直到尖端触及软膜表面。背腹轴的值（Z）复位为0。此时，与毛细管-1.9毫米穿透组织和释放1.5微升病毒悬液在4升/秒的速率。等待2-3分钟，以尽量减少回流的病毒悬浮液通过毛细管轨道，然后收回毛细管。可以执行的第二注射在controlateral半球。 </li><li>缝合皮肤，消毒，并如前面所述的在子宫内的电穿孔（1.8），让动物从麻醉中恢复。 </li></ol><img src="/files/ftp_upload/4093/4093fig2.jpg" alt="图2" /> 图2。立体定向病毒注射（A和 B）的示意图，所需要的组件来执行立体定位注射液（A）和毛细管支托物的分解示出的插入约pillary，直到第二个环（B）。 （C）固定的耳朵酒吧和腭支持与皮肤的老鼠头的图片削减揭露的头骨（左）。的虚线方框的放大倍数（右）的横向和矢状缝，前囟门，注射的部位（圈）。 3。样品的处理中的<ol><li>一到三个星期后感染，灌注鼠标与CA。 100毫升的4％PFA，解剖大脑和后固定在一夜之间在4％PFA。最终，的BrdU和/或他莫昔芬可处死前给药，根据不同的范式调查细胞周期动力学和停止loxP位点两侧的病毒的转基因的表达，分别为9。 </li><li>大脑可以处理免疫组化使用干细胞和祖细胞和神经元（ 例如，Sox2基因，胶质纤维酸性蛋白（GFAP），巢，Tbr2，doublecortin等）的几个指标。 </li></ol>

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J., Gaiano, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-Guided+Microinjection+into+the+Mouse+Forebrain+In+Utero+at+E9.5.">Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5.</a> J. Vis. Exp. (45), e2047 (2010).</li><li>Osumi, N., Inoue, T. G. e. n. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=transfer+into+cultured+mammalian+embryos+by+electroporation.">transfer into cultured mammalian embryos by electroporation.</a> Methods. 24, 35-42 (2001).</li></ol>

BA和FC已经提交了专利申请描述​​的条件扩大的成年体干细胞由CDK4/cyclinD1的病毒（EP 10160288.6）。

在子宫穿孔的一个关键步骤是因为它的定向迁移在分娩过程中的电场取决于它的电荷的DNA的质量和纯度。 DNA纯化的协议“，不包括额外电荷的分子，如内毒素的除去，可能会导致掩蔽的天然DNA的负电荷导致出货不畅。显然，尽可能接近以有针对性的面积与电极接触子宫壁将提高电穿孔效率。同样重要的是使用，因为这是基本为提供最佳的电场的电极的质量。微划痕甚至没有可见的眼和/或有机残基，如血清痕迹，脂类，等等，将不可避免地随时间而累积，导致失去它们的属性的电极。 ，老和/或脏electropes的最可能的原因突然下降，电效率，因此，应该是replaced，因为这是发现。虽然比较简单， 子宫穿孔 ，通常需要几个星期的训练，以达到满意的和可重复性的结果。这种技术是非常通用的，因为它可以被应用，实际上，任何器官和组织的胚胎发育。当然，含有的空腔，如脑，器官是特别容易的目标，并允许由于易于注射相对大体积的DNA的转染细胞的比例很高。 设立立体定向病毒注射过程中遇到的一个常见的​​问题是很少有感染的细胞和/或不期望的区域为目标。第一个问题主要是与注射低滴度的病毒。 293T细胞数量较少的通道，文化的增长速度和较高的病毒滴度。使用细胞的年纪比约。 20代不是推荐的装置，与小于80％的效率的转染将不能保证是好的病毒的产生。再悬浮的病毒颗粒也很关键，病毒悬浮液出现同质和病毒的团块可能阻塞毛细管有要避免。病毒是非常敏感的温度下降和长期贮存。出于这个原因，等分试样需要被永久保存在-80℃下，并不能再冷冻。即使在这些条件下，可观察到减少传染性6-12个月后的存储。在学习阶段，我们建议，以测试每个病毒制备的病毒滴度，并做了一遍后长期储存。感染的细胞中存在的不想要的大脑区域可以依赖于一个次优的定位鼠标的头部立体定位框架，这需要一些练习。此外，它是开放颅骨上的孔的同时，不要损坏大脑表面的关键，因为否则它不会有可能正确地复位0上的软膜的表面上，导致不精确的注射。坐标我们在本协议中被称为C57/BL6 6-10周岁女性，我们建议优化他们针对不同的应变/年龄/性别。这项技术的收购，可能需要更多的时间比在子宫穿孔阶段的时间越长，需要准备的病毒和分析样本。我们强烈建议，病毒只用鼠标手术，立体定向注射后，已成为可靠和可重复性。实现这一目标的第一个步骤是注入细胞穿透物DNA Hoechst 33342荧光染料，例如，在实施安乐死的小鼠，并立即分析大脑。 要敏锐地是两个强大的系统和组织，特别是中枢神经系统内的控制基因的表达在哺乳动物发育和成年期的子宫穿孔和立体的病毒注射。尽管他们的目标只有一个细胞亚群的限制，这些系统提供了前所未有的速度和易用性相比更传统的方法，我Ň尤其是费时费力代的转基因小鼠。然而，尽管这种相似性，存在着一些不同的两种技术之间的，是值得一提。首先，针对不同类型的细胞， 在子宫内的电穿孔方面最初导致转染适当的干细胞（也称为放射状胶质细胞），因为这些是唯一的细胞限定的DNA被注入脑室。作为细胞分裂的结果，质粒将随后被从针对性，母亲干细胞继承其祖细胞和/或神经元的子细胞，作为时间的函数的整个皮质导致转染细胞的再分配。与此相反，在成人大脑使用艾滋病毒慢病毒导致的任何细胞类型，包括间充质干细胞，前体细胞，神经元，以及成熟的神经胶质细胞的同时感染。值得注意的是，关于使用艾滋病毒慢病毒，而不是更常用的逆转录病毒是事实慢病毒的整合将独立地发生从有丝分裂的细胞的状态，从而使成人，静态干细胞的定向也。 其次，电穿孔和病毒感染之间的另一个重要的区别是，一个暂时的，游离表达来实现，而不是由前者在后者所取得的受感染的细胞的基因组中的DNA的不可逆结合。作为一个结果，连续的细胞分裂，将电穿孔的质粒稀释，和异位cyclinD1蛋白的降解，就会随之于各细胞周期。因此，扩大胚胎发育过程中的NSC大约两天15只，而在成人大脑中发现这种效果持续数月（结果未显示）。 第三，因为我）cdk4/cyclinD1具有较强的影响对细胞具有较长的G1及ii）致力于神经祖细胞发育过程中有较长的细胞周期于干细胞，而在成年期则相反，我们的操作触发的扩张，主要是在开发和干细胞在成年神经祖细胞。在任一情况下，在神经元的增加这两种方法已取得了。 第四，病毒注射可以很容易地进行，可靠和可重复的，在许多不同的区域的成年脑立体定位坐标通过简单地改变。与此相反，精确定位的中枢神经系统的某些区域，如脑干，海马阵型，或者脊髓， 在子宫穿孔可能会对更难以实现再现性。此外，可以执行对任何年龄的小鼠而立体定位注射， 子宫穿孔的最佳性能是有限的约E11至E16。至E11之前阶段，将需要使用的超声波后向散射显微镜18,19或全胚胎邪教余吕杏茜系统17,20。 近日，成体干细胞的基础细胞生物学的兴趣越来越被推广，因为他们的学习和操作被认为是对开发新的基于细胞的再生疗法的基础。特别是过度的CDK4/cyclinD1的，我们的协议提供的装置，一过性增加扩展的NSC 在体内 （ 图3B和C和图4B和C），以增加在成人大脑产生的神经元的数目。我们认为，这些操作可能是很重要的作用NSC在不同的上下文，以便更好地了解大脑发育，演化和平衡，以及他们的认知功能或神经系统的退化或损伤的恢复。

<table border="1"><tbody><tr><td> 材料/设备 </td><td> 提供者 </td><td> 产品编号 </td><td></td></tr><tr><td> Endofree马克西质粒试剂盒</td><td> Qiagen公司</td><td> 12362 </td><td></td></tr><tr><td> FastGreen </td><td>西格玛</td><td> F7258 </td><td></td></tr><tr><td> P-97火焰山/褐色吸管拉马</td><td>萨特仪器</td><td></td><td></td></tr><tr><td>高硼硅毛细管长丝</td><td>萨特仪器</td><td> BF120-69-10 </td><td></td></tr><tr><td>桌面实验动物麻醉系统</td><td> VetEquip </td><td> 901806 </td><td></td></tr><tr><td>异氟醚</td><td>巴克斯特</td><td>热浸镀锌板9623 </td><td></td></tr><tr><td>种子发芽玻璃珠灭菌器500 </td><td> SouthPointe手术</td><td> GRM5-1460 </td><td></td></tr><tr><td>虹膜镊子</td><td> WPI </td><td> 15917 </td><td></td></tr><tr><td> Betaisodona解决方案</td><td> Mundipharma </td><td> 1931491 </td><td></td></tr><tr><td>笔克泵</td><td> WPI </td><td> PV820 </td><td></td></tr><tr><td> ECM 830方波电穿孔</td><td>哈佛设备</td><td> 450052 </td><td></td></tr><tr><td>圆镊子电极</td><td> NEPAGENE </td><td> CUY650P1 </td><td></td></tr><tr><td>缝合材料</td><td>爱惜</td><td> V493H </td><td></td></tr><tr><td>反射伤口愈合的施夹</td><td> WPI </td><td> 500345 </td><td></td></tr><tr><td> Temgesic注射液</td><td>埃塞克斯制药公司</td></tr><tr><td> DMEM瓦特/ Glutamax的-I </td><td> Invitrogen公司</td><td> 31966021 </td><td></td></tr><tr><td>胎儿鲍文血清</td><td> Invitrogen公司</td><td> 1027016 </td><td></td></tr><tr><td>青霉素/ Streptomicin </td><td> Gibco公司</td><td> 15140 </td><td></td></tr><tr><td>组织培养皿</td><td>鹰</td><td> 353003 </td><td></td></tr><tr><td>聚乙烯亚胺250毫升</td><td> Sigma-Aldrich公司</td><td> 40872-7 </td><td></td></tr><tr><td>正丁酸，钠盐</td><td>西格玛</td><td>乙5887 </td><td></td></tR&gt; <tr><td>管，薄壁，polyallomer </td><td>贝克曼</td><td> 331374分之326823 </td><td></td></tr><tr><td> 900型小动物立体定位仪</td><td> KOPF工具</td><td></td><td></td></tr><tr><td> OPMI微型显微镜</td><td>蔡司</td><td></td><td></td></tr><tr><td> SYS-MICRO4控制器和Nanoliter2000的喷油器</td><td> WPI </td><td> B203MC4 </td><td></td></tr><tr><td>玻璃更换3.5纳升</td><td> WPI </td><td> 4878 </td><td></td></tr><tr><td> SR钻一般的应用</td><td> Foredam </td><td> K2272 </td><td></td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td><strong&gt;抗体</td><td> 提供者 </td><td> 产品编号 </td><td> 稀释 </td></tr><tr><td> Doublecortin </td><td>圣克鲁斯</td><td> SC8066 </td><td> 1:100 </td></tr><tr><td> GFAP </td><td> Chemicon公司</td><td> MAB3404 </td><td> 1:500 </td></tr><tr><td>巢</td><td> BD Biosciences公司</td><td> 556309 </td><td> 1:1000 </td></tr><tr><td> S100β </td><td> Abcam公司</td><td> Ab868 </td><td> 1:1000 </td></tr><tr><td> Sox2基因</td><td>密理博</td><td> AB-5603 </td><td> 1:500 </td></tr><tr><td> TBR2 </td><td> Abcam公司</td><td> Ab23345 </td><td> 1:400 </td></tr><tr><td colspan="4"> 抗体的表。 </td></tr></tbody>
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expansion of embryonic and adult neural stem cells by in utero electroporation or viral stereotaxic injection

成体干细胞可以分化以产生额外的干细胞（膨胀）或多个不同类型的细胞（分化），这是根本胚胎发育过程中的组织的形成和组织的平衡，在成年期1。目前，下大力气投资对控制扩展到分化的成体干细胞的开关，因为这被认为是发展新的战略再生医学1,2的基础。然而，成体干细胞的研究和利用的一大挑战是，他们的扩张已被证明是非常难以控制的。 在这里，我们描述了一个系统，允许通过操纵CDK4/cyclinD1的复杂的表达，G1期的主要调节控制神经干/祖细胞在小鼠胚胎皮质或成年海马（共简称为NSC）扩展的细胞周期和体干细胞差异erentiation 3,4。具体地说，两种不同的方法，其中所述的cdk4/cyclinD1复杂的是过度的NSC 体内 。通过第一种方法，是通过以下方式获得注入质粒编码CDK4/cyclinD1的在管腔中的鼠标端脑在子宫内的电穿孔后跟交付给NSC的横向皮质，从而，引发的游离表达过度的细胞周期调节转基因5-8。由所述第二方法中，高度浓缩的艾滋病毒衍生的病毒立体定位注射在成年小鼠海马齿状回，因此，触发整合后的病毒构建体在感染的细胞的基因组中9的组成型表达的细胞周期调节。这两种方法，其基本原则已经其他视频协议10-14优化为i）减少组织损伤，二宽的部分）的目标非常特定的大脑区域选择性ns中，ⅲ）获得高操纵细胞数目在每个区域内，和iv）触发在每个小区之内的外源基因的高表达水平。瞬时过量表达的转基因使用这两种方法是通过不同的方式，即通过自然稀释的电穿孔质粒由于细胞分裂或他莫昔芬的管理Cre重组酶表达NSC感染病毒携带CDK4/cyclinD1的两侧loxP位点，分别为9,15 。 这些方法提供了一个非常强大的平台，急性和任何基因的表达，在小鼠大脑组织专门的操作。通过操纵的CDK4/cyclinD1的复杂的表达，特别是，我们的系统允许的时间控制的NSC扩展及其分化中的开关，从而，最终提高哺乳动物大脑中产生的神经元的数目。我们的方法是非常重要的基础研究和使用成体干细胞治疗干细胞组织的形成作出贡献的哺乳动物的中枢神经系统，同时提供更好地了解我）在开发过程中，二）组织的平衡在成年期，三）成人神经认知功能的作用，也许四）更好地利用成体干神经退行性疾病的模型中的细胞。

<ol><li>总体而言， 在子宫内的电穿孔，得到60-80％的电穿孔的胚胎显示外侧皮质的广泛区域（ 图3A）中的报告基因的强表达。荧光蛋白可以很容易地检测到整个安装胚胎的只要3-6个小时后，手术持续了一个多星期的信号。在目标区域内，通常约30-50％的细胞表达转基因的细胞的比例，共表达的两个（或多个）通常在90％以上的共电穿孔转基因（ 图3B，左）6,8（s）与，15。最低程度的细胞凋亡（约2.0-2.5％），虽然可以观察到电约2小时后，该值将返回到生理水平（约0.5〜1.0％）后约6小时（FC，未发表的数据）。由于手术的胚胎或孕鼠死亡的比例通常是可以忽略不计（&lt;5％）。在这些条件下电与CDK4/cyclinD1的，然后48小时的发展触发增加神经祖在膨胀30％（ 图3B，右，和C）15。 </li></ol><img src="/files/ftp_upload/4093/4093fig3.jpg" alt="图3" /> 图3。 （A）的荧光图像的截面通过在胚胎第13天与GFP和RFP质粒电穿孔后24小时的小鼠胚胎的外侧皮质神经祖细胞CDK4/cyclinD1的过度膨胀 。 （B）在合作电穿孔与cdk4/GFP和cyclinD1/RFP质粒和为基底祖标记TBR2的免疫标记A 48小时后的荧光照片。荧光记者与：DAPI counterstainig（左）和Tbr2（右）所示。线表示的顶端表面，心室区（VZ;白线）和脑室下区（SVZ;黄色虚线）的边界。请注意，增加的THI在将电穿孔的部分的皮质（白色箭头）由于增加过度的CDK4/cyclinD1的后基底祖细胞的产生和扩展的SVZ ckness。 B.酒吧= SD，P &lt;0.05，N = 3（C）所示的效果的量化。在C条形图Lange 等人，2009年15。 <ol start="2"><li>立体定位注射病毒后，约80％的操作小鼠表现出很强的外源基因的表达，在整个齿状回。绿色荧光蛋白基因的组成型表达，可以容易地检测出在40μm切片尽快手术后4天。沿着吻端到尾鳍轴的齿状回细胞，约10-30％（相当于共约10,000-30,000细胞），通常表达的转基因（S）（ 图4）。小鼠死于手术的比例是可以忽略的（&lt;5％）。三个星期的CDK4/cyclinD1的过度表达在这些条件下触发增加NSC超过两褶皱（ 图4B），同时降低了70％（ 图4C）9神经元。 </li></ol><img src="/files/ftp_upload/4093/4093fig4.jpg" alt="图4" /> 图4。病毒感染的影响。（A）三维重建的海马状突起沿吻端到尾鳍轴的荧光拍摄的照片从40微米厚连续冠状切片（收集每6）。 GFP +感染的细胞（绿色）和DAPI核染色（蓝色）的齿状回（顶部）所示。明视场的冠状部分，分别对应A（中）和三维表示（底部）与海马中强调绿色伪（修改艾伦大脑图谱<a href="http://www.brain-map.org" target="_blank">www.brain-map.org的</a>整个大脑半球的图片） 。白色箭头指向注射部位。横向（L），延髓头端（R），和多尔表示萨尔（D）的轴（的x，y，和z立体定向坐标）（右）。 （B和C）的巢+ NSC（B）和DCX +（C）新生神经元内的人口GFP +立体定向注射3周后的GFP（白色）或cdk4/cyclinD1/GFP（黑色）病毒感染的细胞的比例。酒吧= SD，P &lt;0.005，N = 3。图B和C Artegiani 等，2011。

Watch this Scientific Journal Video about Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection at JoVE.com

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

控制扩展成体干细胞是一个重要的因素，阻碍了他们的研究，并在治疗中使用。在这里，我们描述了一种系统，在时间控制在开发过程中膨胀和成年期神经干细胞，可用于增加在小鼠大脑产生的神经元的数目。

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 ...

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

16.6K 次观看.  DFG - Research Center and Cluster of Excellence for Regenerative Therapies Dresden, Germany. 控制扩展成体干细胞是一个重要的因素，阻碍了他们的研究，并在治疗中使用。在这里，我们描述了一种系统，在时间控制在开发过程中膨胀和成年期神经干细胞，可用于增加在小鼠大脑产生的神经元的数目。

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

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

成年鼠神经干细胞的分离和扩大使用神经干含量

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

科研

神经科学

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

从小鼠胚胎神经管的神经嵴细胞的分离和培养

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

外用转基因大鼠通过代<em&gt;在子宫内</em&gt;电穿孔和<em&gt;在体内</em&gt;生物发光筛选

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

立体定向注射病毒载体的基因操作在小鼠脊髓条件

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

人类神经的聚集体cGMP的适用展开法干细胞和祖细胞来源从多能干细胞或胎儿脑组织

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

胚胎小鼠神经干细胞的分离与培养

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

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 ...

In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian development. In utero electroporation is a key technique for the manipulation of gene expression in the embryonic mammalian brain in vivo. A protocol for in utero electroporation of the embryonic neocortex of ferrets, a small carnivore, is presented here. The ferret is increasingly being used as a model for neocortex development, because its neocortex exhibits a series of anatomical, histological, cellular, and molecular features that are also present in human and nonhuman primates, but absent in rodent models, such as mouse or rat. In utero electroporation was performed at embryonic day (E) 33, a midneurogenesis stage in ferret. In utero electroporation targets neural progenitor cells lining the lateral ventricles of the brain. During neurogenesis, these progenitor cells give rise to all other neural cell types. This work shows representative results and analyses at E37, postnatal day (P) 1, and P16, corresponding to 4, 9, and 24 days after in utero electroporation, respectively. At earlier stages, the progeny of targeted cells consists mainly of various neural progenitor subtypes, whereas at later stages most labeled cells are postmitotic neurons. Thus, in utero electroporation enables the study of the effect of genetic manipulation on the cellular and molecular features of various types of neural cells. Through its effect on various cell populations, in utero electroporation can also be used for the manipulation of histological and anatomical features of the ferret neocortex. Importantly, all these effects are acute and are performed with a spatiotemporal specificity determined by the user.

Presented here is a protocol to perform genetic manipulation in the embryonic ferret brain using in utero electroporation. This method allows for targeting of neural progenitor cells in the neocortex in vivo.

通过子宫电位对脑神经祖细胞进行神经祖细胞的振动定位

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian ...

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages over conventional multi-photon calcium imaging systems: (1) compact; (2) light-weighted; (3) affordable; and (4) allows recording from freely behaving animals. This protocol describes brain surgeries for deep brain in vivo calcium imaging using a custom-developed miniscope recording system. The preparation procedure consists of three steps, including (1) stereotaxically injecting the virus at the desired brain region of a mouse brain to label a specific subgroup of neurons with genetically encoded calcium sensor; (2) implantation of gradient-index (GRIN) lens that can relay calcium image from deep brain region to the miniscope system; and (3) affixing the miniscope holder over the mouse skull where miniscope can be attached later. To perform in vivo calcium imaging, the miniscope is fastened onto the holder, and neuronal calcium images are collected along with simultaneous behavior recordings. The present surgery protocol is compatible with any commercial or custom-built single-photon and two-photon imaging systems for deep brain in vivo calcium imaging.

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

Miniscope in vivo calcium imaging is a powerful technique to study neuronal dynamics and microcircuits in freely behaving mice. This protocol describes performing brain surgeries to achieve good in vivo calcium imaging using a miniscope.

立体定向病毒注射和梯度指数晶状体植入术用于深部脑 体内 钙成像

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

成人皮层的器官型培养作为人类干细胞移植和验证的 离体 模型

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...