This research was supported by the Robert A. and Rene E. Belfer Family Foundation, NIA R21 AG038856 (JLJ), BrightFocus Foundation Alzheimer&#8217;s Disease research grant A2010097 (JLJ), and NIA Biology of Aging Training grant T32 AG000183 (support for SDG).

执行所有的程序和涉及动物按照健康指南全国学院为护理实验动物的使用和协议。这里所描述的程序进行审查和批准医学实验动物管理和使用委员会的贝勒大学。 在啮齿类动物的脑转基因传送无法复制的腺相关病毒（AAV）载体被批准用于生物安全等级1的使用。请参阅疾病预防控制中心网站，美国政府出版的"生物安全微生物和生物医学实验室（BMBL）"，详细介绍关于妥善保护和病毒处理程序的具体要求。请与当地的兽医和环境安全的工作人员学习​​使用病毒程序制度的具体要求。关于程序的客房，检疫，鉴定和生物危害网箱条例跨机构而有所不同。 1，预削减P0小狗和养母<ol><li>提供高脂食物对所有孕妇和哺乳期的女性，以支持育种和哺乳期的能源需求。 </li><li>到了晚上，成立了养殖笼通过添加一个或两个健康的女性，以一个单一的男性居民的笼子。第二天早上，检查女性的交配塞，并从分离的男性女性，如果一个插件存在。 <ol><li>使用养母从差的产妇特征的遗传背景（例如，C57BL / 6或C3HeJ）提高幼仔。养母接受来自另一窝幼崽，如果转移幼仔在四天之内的养母自己的垃圾诞生。 </li><li>设置使用ICR或FVB雌性一起实验育种，使得两组雌性交付在大约相同的时间独立的交配笼。 </li></ol></li><li>女性将在19±1天内发货从插头日期，根据背景应变。前三天为Delivery（插头日期16天后），把怀孕的女性到一个干净的笼子里有新鲜的筑巢材料和覆盖的栖身之所。 </li><li>检查刚出生的幼崽，每天两次开始前2天预产期（插头日期17天之后）。通过底部的粉色新生儿存在偷看检查笼最小应力的母亲。一般小鼠幼崽传递在早上，但在少数情况下将提供在下午。 </li><li>计划尽快进行注射的幼仔哺乳（这将是可见的牛奶斑明显），或在6小时出生，以先到者为准。 </li></ol> 2，备好设备注射<ol><li>准备一个10微升注射器用32克的针一般P0新生儿。如果进行立体定位注射，装入注射器上的机械臂。 </li><li>如果使用的是立体框架注射，冷却新生儿阶段4-8℃，加入100％伊桑醇和干冰到储存在该块的前端。保持高于1℃的温度，以避免幼仔的冻伤。 </li></ol> 3，准备稀释病毒注射<ol><li>准备一个1％台盼蓝染料溶液（20倍的股票）。 </li><li>准备的腺相关病毒（AAV）股票5-25微升等份2级生物安全柜内。将这些等分试样在-80℃下以供将来使用。避免额外的冻融循环，因为这会导致病毒失去传导的功效。 </li><li>从-80℃冰箱中，置于冰上取下的AAV等分解冻。 </li><li>通过稀释的病毒入冰冷的1X磷酸盐缓冲盐水（PBS）中制备的病毒溶液用于注射。开始用10倍连续稀释（十月10日至8月 10日的病毒颗粒/半球）的转导模式的初始优化。 </li><li>加入20倍的台盼蓝对AAV溶液至0.05％的最终浓度。 </li></ol><pCLASS ="jove_title"&gt; 4。举办​​新生幼犬注射<ol><li>置于冰上小铝板冷却。将干燥的任务擦上盘的顶部，以保护小狗的皮肤从冰冷的金属。这就像一台冰冷的表面进行麻醉和注射的幼崽。 </li><li>准备适合饲养新生小鼠温暖注射前后变暖垫。 </li><li>一旦新生幼崽已开始吃奶，从笼子里取出大约一半的人留下的另一半与他们的母亲。将收集到的幼崽在变暖垫在等待注射。 注意:如果亲生母亲不关心幼仔，从笼子里立即删除整个垃圾，并把幼崽到暖垫等候注射。这种情况可能出现的出生对C57BL / 6的女性，或与具有较差特色养殖等近交系的第一胎。在这种情况下，牛奶斑不会对新生幼仔可见。 </li><li>传输距离变暖垫1的小狗到冰冷的金属板，诱导低温麻醉。等待2-3分钟的小狗变得完全麻醉。通过轻轻地挤压爪子确认麻醉和监测缺乏运动或呼吸。 </li></ol> 5，注射AAV到新生小鼠<ol><li>徒手颅内注射AAV到新生小鼠: <ol><li>负载5微升稀释的AAV用0.05％台盼蓝入注射器。 </li><li>轻轻擦拭麻醉的小狗的头，用棉签浸在70％的乙醇。 </li><li>确定注射部位处从拉姆达缝合到每只眼睛的距离五分之二。另一种注射部位位于约0.8-1毫米的矢状缝外侧，中途Lambda和前囟门之间。这些标志性建筑都可见通过皮肤在P0。 </li><li>马克注射部位与无毒的实验室笔。 </li><li>保持与规模MONI可见注射器toring的溶液的体积分配。 </li><li>确保拇指能达到柱塞的顶部。然后从柱塞虽然定位针，以避免意外配药病毒移除大拇指。 </li><li>找到一个舒适的位置，通过将板凳上的肘部和靠在手臂上的冰桶，以振奋的手臂注射。 </li><li>侧面朝下放置，其头部的小狗直属注射器。把小狗的头，使标记的注射部位朝上，轻轻地但坚定地保持这个姿势一条生路。 </li><li>持注射器垂直于颅骨的表面和插入针在标记的注射部位到约3mm的深度。注射针，直到阻力略有降低，表明针头已渗透到侧脑室。如果需要的参考，可3毫米针具有无毒制造的尖端。调整基于染料吨注射深度为小狗的大小和应变argeting心室。 </li><li>持注射器刚性，使柱塞可以是凹陷而不更远移动针进入大脑。如果注射移位到丘脑，病毒传播将受到严重的限制。 </li><li>开始慢慢注入病毒，同时监测从注射器分配的体积。辖2微升，最大容量为每一个心室。如果针处于正确的位置时，染料会扩散到填充心室。 </li><li>慢慢地拔出针头。 </li><li>允许第一注射部位以关闭注入另一半球之前。 注意:不要注射过一次同一站点多。重新插入针力病毒已经注入漏出，造成伤害的小狗或死亡。 </li><li>注射用同样的步骤对侧心室。 </li></ol></li><li>立体定向注射AAV到新生小鼠: <ol><li>负载5微升稀释的AAV用0.05％台盼蓝整数Ø通过立体操盘举行的注射器。 </li><li>轻轻将小狗的头新生儿帧的耳棒之间。确保磁头是通过检查拉姆达和囟门之间的线平行于台在Y轴的水平（从前到后）。确保磁头是通过检查耳朵或眼睛之间的线之间的想像线，平行于该阶段中的X轴水平（从一侧到另一侧）。 </li><li>轻轻擦拭麻醉的小狗的头，用棉签浸泡于70％乙醇。 </li><li>使用立体定向操纵器来定位上面的λ的注射器，然后调零的X和Y坐标。将立体手臂（X，Y）=（0.8，1.5）毫米标准P0幼仔。 注:小狗的尺寸和立体坐标可以通过应变和年龄而有所不同。根据需要对目标侧脑室调整基于染料注射坐标。 </li><li>慢慢放下针头插入注射部位。头骨的表面会IND耳鼻喉科，然后释放，一旦针头穿透皮肤。收回该针头至颅骨恢复其正常凹的形状，但保持所述针的斜面的皮肤下。零，在这点的Z坐标。 </li><li>将针头至Z = -1.7毫米，然后缩回至-1.5毫米。 </li><li>慢慢地注入病毒。每个半球最多可以接受2微升的解决方案。 </li><li>保持就位的注射器30-60秒完成注射后，然后缓慢缩回针超过1-2分钟。 </li><li>重复对侧半球，在X轴为注射部位使用负坐标。 请注意:用于注射的替代的坐标（X，Y，Z）=（0.8，2.0，-​​1.5）mm和（1.2，1.0，-1.5）毫米。执行第一次注射（0.8，2.0），然后移动到（0.8，-2.0），（1.2，1.0），最后（1.2，-1.0）。注入1微升的溶液在每个站点，总共每半球2微升。快速移动的注射完成在Les程序总比10分钟。 </li></ol></li></ol> 6，注射后护理<ol><li>完成注射到两个半球后，将小狗放回变暖垫，直到其体温和皮肤颜色恢复正常和小狗开始移动。 </li><li>如果使用的是亲生母亲喂养的新生儿注射，恢复注入幼崽的生母他们恢复正常运动后。将剩余的未注射的幼崽在变暖垫。重复上述步骤，直到所有的老鼠都被注射。 </li><li>如果使用的是寄养妈妈喂养的新生儿注射，注射的所有幼崽转移到养母照顾他们恢复正常运动后。 <ol><li>除去大部分或全部从笼属于养母幼仔的，以确保注射的动物的成功。 </li><li>如果寄养母亲的幼崽，并注射幼崽有相同的眼睛和皮肤的颜色，标记从养母幼仔用实验室的笔，使他们能够后来区分。 </li><li>将养母的幼崽和他们的一些床上用品一起注入幼崽。确保注入的幼崽获得她的亲生子女的香味，提高接受新的幼崽。 </li><li>将只注入幼崽放回养母的笼子。剔除从养母任何剩余的幼崽。 </li></ol></li><li>确保母亲抓和哺乳把它们入笼的注入幼仔在10分钟内。如果母亲没有在这段时间内收集的幼崽，幼崽转移到另一个的养母。 </li><li>标签笼用生物危害卡以显示它含有病毒注入动物。众议院在72小时经批准的区域内笼。 </li><li>在72小时的检疫期后，母亲和幼仔（但没有床上用品或其他物品笼）转移到一个干净的笼子。执行此转让2级生物安全柜内，以免接触到任何病毒污染的床上用品。 ŘE打开笼子的清洁与动物注射到普通的鼠标设备。 </li><li>将脏笼成生物危害袋，通过高压灭菌消毒，然后将保持架返回到动物饲养。 </li></ol> 7清理<ol><li>将剩余的病毒溶液，在4℃以备将来使用。病毒保留的转导效率为至少2个月贮存于4℃下8时。 </li><li>消毒的注射针和注射器，用2％漂白剂随后在去离子水中反复冲洗。 </li><li>清洗，用2％漂白接着用70％乙醇在工作区中。 </li><li>收集所有的一次性项目，包括吸头，管，口罩和手套在一个塑料生物危害袋。按照当地的法律和制度的政策（ 即高压灭菌或焚烧）处置废料。 </li></ol> 8，准备老鼠大脑成像<ol><li>将注入小鼠成二氧化碳安乐死室3处后4周注射。遵循正确的安乐死程序制度指引。不要灌注动物与多聚甲醛，因为这会熄灭的荧光标记。 </li><li>取下头盖骨的整个大脑，并在4℃定为3-12小时，在PBS中的4％多聚甲醛。 </li><li>固定脑转移到30％蔗糖的冷冻保护。孵育在4℃下直到大脑下沉到水底。 </li><li>收集细碎的干冰一桶。切开大脑半年下来中线。埋半脑在干冰冻结。 </li><li>通过加入干冰和100％的乙醇冷却切片机的阶段。 </li><li>保护大脑与中线向下使用PBS的阶段，并允许冻结。 </li><li>用冷冻滑动切片机通过大脑科30-45微米厚。 </li><li>处的部分为含有防冻剂溶液（250毫升甘油300毫升乙二醇500毫升的0.1M磷酸盐缓冲液，pH 7.4）的24孔板。盖板无线第锡箔，并储存在-20℃下以供将来使用。 </li><li> 1-4井收集部分，并在PBS冲洗3次。 </li><li>安装部分到显微镜载玻片和盖玻片。让幻灯片干在黑暗的位置。 </li><li>使用适当的过滤器原生YFP或tdTomato图片幻灯片，检查病毒转导的模式。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

我们已经描述了一种通用的程序，用于操纵使用AAV作为广泛递送到新生小鼠脑部的车辆神经元基因表达。与神经元的转基因的其它方法，如在子宫内电1或立体定向颅内注射2,3相比，新生病毒注射是比较容易和简单。基本过程可以在几分钟内完成，只有一个冰桶和微升注射器。最佳的生存和转基因表达可以通过参加几个技术细节来实现，最重要的是病毒性的股票，时机和注射的准确性，以及产后护理的质量。 病毒制剂的质量是成功转的关键。坏病毒制剂将显著减少神经细胞的感染，并产生显著星形细胞转导，可能是由非感染性颗粒的吞噬作用。许多大学有现场的核心实验室，专门从事病毒包装，和大型设施，在北卡罗莱纳州的宾夕法尼亚大学大学，并提供高品质的现成的现成的试剂中的各种血清型以更低的成本。这些设施也为载体所无法提供的预包装库存提供定制包装。一旦接收到实验室，请记住，腺相关病毒颗粒可在-80ºC稳定多年，但对温度的波动非常敏感。出于这个原因，等分应储存在-80℃，一旦解冻，不应该被再次冷冻。 对于最高的转染效率，关键是病毒幼仔交付后尽快注射。根据我们的研究，AAV8和我们的合作者，延缓不仅减少病毒从心室蔓延的注射，但也将偏压从神经元到星形胶质6,7的转导。因此，我们建议injecti纳克在诞生之日起最佳的神经元表达。在这个年龄-脑脊液-脑屏障之前已经成熟9 -注射至侧脑室会扩散到整个脑室系统，然后按照脑脊液流动到大脑4病毒颗粒。因此，侧脑室的精确定位是重要的，以最大化的病毒传播。准确的定位也最大限度地减少从快速注射相对大体积的流体的组织损伤。因此，我们强烈建议反复练习，直到针对侧脑室变得可靠和可重复性。注意，在此协议中提供的坐标是相应于一般P0幼仔并应调整为实验幼仔的应变和年龄。起初，注射剂应与染料溶液，如台盼蓝或印度墨水来进行的，所以定位和传播可以通过注射后立即摘取大脑被可视化。 íF表示侧脑室已成功锁定，染料会扩散到整个心室腔和可见的嗅球小脑一路。立体定位装置，建议如果心室不能可靠地通过有针对性的免费手注射。 如果注射被正确地执行，小鼠的比例输给注入将是微不足道的。相反，注射后的存活率是受影响最大的孕产妇保健。开始健康的动物。母亲的健康是由她的行为和她的大衣表示。她应该是整齐干净，清洁。产仔数也可以是幸福的指示。健康的年轻女性应该产生的6窝 - 8幼仔的C57BL / 6，或10 - 16个幼崽的ICR。健康的小狗应该是粉红色和蠕动。如果幼崽看起来不太好，或对自己的肚子没有牛奶斑，应立即转移到新的寄养女性。我们建议ICR或FVB为促进因它们产生足够的乳汁，易于接受新的幼崽到他们的垃圾。至关重要的是，中断对母亲之前被最小化和注射的压力后，可能会导致她的拒绝或蚕食的幼崽。通过限制噪音，光线和其他的干扰和保持架在一个安静的地方，减少压力。检查新生幼仔并在注射后最初几天时打开笼尽可能小，虽然这是必要监测母亲的注意力，以幼仔一旦被取代。母亲应显示与生俱来的行为，如聚束的幼崽在一个地方，坐在上面一堆的后代。如果母亲时，幼崽被送回笼子没有立即作出回应，再次尝试混合使用肮脏的床上用品和排料，从她的笼子里的幼崽确保他们已获得了她的气味。再后来检查的日子，幼崽仍然有牛奶斑对他们的肚子。如果可能的话，通过观察从笼要这样做下方，而不是从上方开口的保持架。第一天是生存最关键的，而且当阴将最敏感中断。 如果用心做，脑室注射AAV提供了一个快速和容易操作体内神经细胞基因表达没有传统的转基因胚系的成本和时间的手段。病毒转导的天然镶嵌可以被利用来控制表达的密度，使其成为实验分离转基因的细胞的内在和细胞外在后果的理想方法。此外，两种病毒可以是共注射使得能够表达在任一重叠或不同的神经元群体的多种蛋白质。这些操作凸显了该方法的灵活性，但我们相信我们才刚刚触及其潜在的表面。对断开的，现成的试剂的日益普及将使它更容易开发病毒注射˚F或新的实验需求，同时混合血清型有明显的趋性的出现可扩展蜂窝剧目，可以有针对性的10,11。

传统的方法用于修改神经基因表达需要耗时和昂贵的种系操作。另一种从头方法，如在子宫内电穿孔或立体定位慢病毒注射液产生更快的结果，而且成本较低，但有需要复杂的外科手术1-3的劣势。此外，转基因表达具有有限的空间范围使用这些方法。在此，我们通过脑室注射腺相关病毒（AAV）到新生小鼠大脑描述了一种快速，简便，经济的方法进行广泛的神经元处理。该方法最初是由约翰·沃尔夫和Marco Passini于2001年，在那里他们建议的小粒径的AAV允许它在脑脊髓液中的扩散，因为它从侧脑室穿过未成熟的室管膜屏障并进入脑实质4描述的， 5。脑室注射腺相关病毒的F内IRST的出生国债收益率神经亚群跨越大脑的各个区域，从嗅球脑干6,7普遍病毒转导后24小时。病毒交付的转基因表达，并在注射几天活跃，持续长达一年后转。因此，这种多功能的操作使研究从出生后早期大脑发育到衰老和退变的成人。 在适应的技术，我们的具体实验需要，我们主要侧重于AAV8血清型，因为它是最有效的传感神经元6。我们表明，病毒滴度可以稀释，以控制转导神经细胞的遗传操作的实验检测细胞内在后果的密度。此外，我们证明，这两种病毒可以被共注射，以产生被偏向不同的或重叠的集合的神经元的表达模式，这取决于所选择的血清型病毒包装。我们的工作扩展这种技术在范围广泛的实验神经科学设置使用的通用性。

<table><tbody><tr><td>ICR outbred mice</td><td>Harlan</td><td>Hsd:ICR (CD-1)</td><td>This strain is also known as CD-1</td></tr><tr><td>FVB inbred mice</td><td>The Jackson Laboratory</td><td>1800</td><td>5-6 weeks of age</td></tr><tr><td>Nestlets</td><td>Lab Supply</td><td>NESTLETS</td><td></td></tr><tr><td>Shepherd shacks</td><td>Lab Supply</td><td>SS-mouse</td><td></td></tr><tr><td>High fat rodent chow</td><td>Purina Mills</td><td>PicoLab Mouse diet 20, #5058</td><td>This is our standard breeder chow</td></tr><tr><td>High fat rodent chow (alternative)</td><td>Harlan Laboratories</td><td>Teklad Global 19% protein rodent diet #2019S</td><td>If low phytoestrogen, autoclavable diet is needed</td></tr><tr><td>Injection syringe</td><td>Hamilton</td><td>7653-01</td><td>10 ml syringe</td></tr><tr><td>Injection needles</td><td>Hamilton</td><td>7803-04, RN 6PK PT4, 0.375&quot;</td><td>32 G, for standard P0 injections</td></tr><tr><td>Metal plate for cryoanesthesia</td><td>McMaster Carr</td><td>8975K439</td><td>Raw aluminum plate, 6&amp;rdquo; x 12&amp;rdquo;, 0.25&amp;rdquo; thick, will need to be cut into 3 equal pieces and edges sanded by local machine shop</td></tr><tr><td>Small animal stereotaxic device with digital readout</td><td>David Kopf Instruments</td><td>Model 940</td><td></td></tr><tr><td>Universal syringe holder with needle support foot</td><td>David Kopf Instruments</td><td>Model 1772-F1</td><td></td></tr><tr><td>Neonatal frame</td><td>Stoelting</td><td>51625</td><td>Officially called a mouse and neonatal rat adaptor</td></tr><tr><td>Biohazard disposal bags with sterile indicator</td><td>VWR</td><td>14220-030</td><td>Important! &amp;ndash; Check with local veterinary and environmental safety staff to learn your institute&amp;rsquo;s protocol for biohazard waste disposal</td></tr></tbody></table>

intracerebroventricular viral injection of the neonatal mouse brain for persistent and widespread neuronal transduction

随着科技进步的步伐迅速加快，需要对实验神经科学的新方法，能够快速，轻松地操纵基因表达的小鼠大脑。在这里，我们描述了一种技术，最早由Passini和沃尔夫介绍了颅内直接供货病毒编码的转基因到新生小鼠大脑。在其最基本的形式中，该过程仅需要一个冰桶和微升注射器。然而，该协议也可适于与立体定位框架上使用，以提高用于研究新的技术一致。该方法依赖于重组腺相关病毒（AAV），以从脑室自由移动进入脑实质，而室管膜衬里仍然是在出生后的第12-24小时期间，未成熟的能力。脑室注射的AAV在这个年龄导致的神经元在整个大脑的普遍转导。在注射几天表达开始，持续了lifetimE中的动物。病毒滴度可以调节，以控制转导神经元的密度，而共表达的荧光蛋白的规定转导的细胞的一个重要的标签。随着病毒核心设施的上升可用性，以提供现成的货架，预包装试剂和自定义病毒制剂，这种方法提供了及时的方法在小鼠大脑中快速，方便，便宜得多操纵基因表达比传统的生殖工程。

成功脑室病毒注射液产量普遍和强大的神经元表达。在这里，我们评估病毒转导使用YFP或鸡β肌动蛋白启动子（CBA子）的控制下tdTomato荧光基因。这些构建体包装到AAV8和注入新生（P0），ICR小鼠的侧脑室。高病毒效价（每半球10 10个粒子）导致致密标签嗅球，纹状体，皮层，海马和小脑（ 图1A，左）。标签也是在小脑浦肯野神经元明显，但小脑颗粒神经元的尚未出现大量的P0明显缺席。更少的病毒颗粒注射液（10 7）导致稀疏标签和较低强度的表达（ 图1A，右图）。我们通常使用AAV8的浓度范围内10 7 10 10颗粒之间的每个半球的<html> Ñ ​​简单和可靠的方式来控制通过注射（ 图1B）产生的转基因嵌合的程度。我们还适于通过共注射两种或更多种病毒，以表达多种外源基因的技术。共注射的两种病毒包装成相同的血清型（ 即两个AAV8）施力将所得转导到重叠的神经元群体，从而使许多细胞表达两种转基因（ 图2A）。在较低的浓度下，表达模式变得更加独立，并共标记的细胞的百分比降低（ 图2B）。非重叠的表达也可通过共注射不同的AAV血清型的实现。联合注射AAV1和AAV8编码不同的荧光报告产生的双嵌合体（ 图2C）一个基本上独立的格局。 ig1highres.jpg"宽度="600"/&gt; 图1中的病毒滴度可用于控制病毒转导的密度。 （A）中的广泛范围的转基因嵌合体可以通过稀释从10 10病毒溶液倒至10 7个/半球来实现。矢状部分代表图像显示了基于病毒滴度两种截然不同的传导模式。图片取自收获注射用AAV8-YFP 3周后，以提供小鼠2.0×10 10（左）或5.0×10 7个（右）颗粒/半球（B）皮质（上排）和小脑的高倍图像（下排）示出了由AAV8的连续稀释来实现转基因嵌合体的光谱。转由本地YFP的荧光显现。曝光时间为全脑图像进行皮层和海马，其最明亮的荧光来确定，而曝光时间为放大板分别被调节至各R内显示的模式egion。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51863/51863fig1highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图2" fo:content-width="6in" src="/files/ftp_upload/51863/51863fig2highres.jpg" width="600" /> 图2滴度和血清型独立地控制病毒的共表达的程度。代表性的图像显示在大脑皮质（A，B）和小脑（C）病毒注射3周后传导模式（A），共注射2 AAV8含有不同的荧光记者（tdTomato或YFP）病毒产生整个大脑广泛传递。大多数表达两种转基因转导细胞（B）共同注射时产生的每个蛋白的稀疏表达低滴度，其中较少的细胞通过两种病毒转导相同的病毒（C）共同注射腺相关病毒的离子打包成不同的血清型（AAV8和AAV1），即使在适度的高滴度，产生病毒表达的主要是不重叠的图案。每个荧光蛋白的图案是几乎相同的表达时单独注射，表示1的AAV转导是独立于其它的。显示为红色，YFP在绿色TdTomato荧光。传导是由天然荧光显现。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51863/51863fig2highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction at JoVE.com

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

intracerebroventricular-viral-injection-neonatal-mouse-brain-for

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

58.9K 次观看.  Baylor College of Medicine. Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

视频: Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

Intracranial Injection of Adeno-associated Viral Vectors

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

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

颅内注射腺相关病毒载体

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

科研

神经科学

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders 1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid 3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS 5. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.

Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

交付的治疗药物通过侧脑室（ICV）和静脉（IV）注射液在小鼠

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most ...

医学

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

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

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

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

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

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

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 &#181;l of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-&#181;l syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 &#181;l of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization.

病毒颗粒和荧光微球侧脑室和血管内注入新生儿脑

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

新生小鼠脑巴细胞基因调控的立体定向手术

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Targeting the Corticospinal Tract in Neonatal Rats with a Double-Viral Vector using Combined Brain and Spine Surgery

Successfully tackling the obstacles that constrain research on neonatal rats is important for studying the differences in outcomes seen in pediatric spinal cord injuries (SCIs) compared to adult SCIs. In addition, reliably introducing therapies into the target cells of the central nervous system (CNS) can be challenging, and inaccuracies can compromise the efficacy of the study or therapy. This protocol combines viral vector technology with a novel surgical technique&#160;to accurately introduce gene therapies into neonatal rats at postnatal day 5. Here, a virus engineered for retrograde transport (retroAAV2) of Cre is introduced at the axon terminals of corticospinal neurons in the spinal cord, where it is subsequently transported to the cell bodies. A double-floxed&#160;inverted orientation (DIO) designer receptor exclusively activated by designer drug(s) (DREADD) virus is then injected into the somatomotor cortex of the brain. This double-infection technique promotes the expression of the DREADDs only in the co-infected corticospinal tract (CST) neurons.&#160;Thus, the simultaneous co-injection of the somatomotor cortex and cervical CST terminals is a valid method for studying the chemogenetic modulation of recovery following cervical SCI models in neonatal rats.

This protocol demonstrates a novel method for applying gene therapies to subpopulations of cells in neonatal rats at postnatal ages 5-10 days by injecting an anterograde chemogenetic modifier into the somatomotor cortex and a retrogradely transportable Cre recombinase into the cervical spinal cord.

使用脑和脊柱联合手术用双病毒载体靶向新生大鼠的皮质脊髓束

Successfully tackling the obstacles that constrain research on neonatal rats is important for studying the differences in outcomes seen in pediatric ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

在新生小鼠静脉注射

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

生物学

在新生小鼠中进行鞘内注射的综合方案

Intrathecal Injection of Newborn Mouse for Genome Editing and Drug Delivery

Intrathecal injection is a commonly employed procedure in both pediatric and adult clinics, serving as an effective means to administer medications and treatments. By directly delivering medications and treatments into the cerebrospinal fluid of the central nervous system, this method achieves higher localized drug concentrations while reducing systemic side-effects compared to other routes such as intravenous, subcutaneous, or intramuscular injections. Its importance extends beyond clinical settings, as intrathecal injection plays a vital role in preclinical studies focused on treating neurogenetic disorders in rodents and other large animals, including non-human primates. However, despite its widespread application, intrathecal injection in young, particularly neonatal pups, poses significant technical challenges due to their small size and fragile nature. Successful and reliable administration of intrathecal injections in newborn mice requires meticulous attention to detail and careful consideration of various factors. Thus, there is a crucial need for a standardized protocol that not only provides instructions but also highlights key technical considerations and good laboratory practices to ensure procedural consistency, as well as the safety and welfare of the animals.
To address this unmet need, we present a detailed and comprehensive protocol for performing intrathecal injections specifically in newborn pups on postnatal day 1 (P1). By following the step-by-step instructions, researchers can confidently perform intrathecal injections in neonatal pups, enabling the accurate delivery of drugs, antisense oligos, and viruses for gene replacement or genome editing-based treatments. Furthermore, the importance of adhering to good laboratory practices is emphasized to maintain the well-being of animals and ensure reliable experimental outcomes. This protocol aims to address the technical challenges associated with intrathecal injections in neonatal mice, ultimately facilitating advances in the field of neurogenetic research that aims to develop potential therapeutic interventions.

作者聚焦: 鞘内注射 – 一种高效可靠的递送方法，用于测试新生小鼠大脑中基因编辑的功效

The present protocol outlines step-by-step instructions for performing intrathecal injections in neonatal mice for gene editing and drug delivery.

新生小鼠鞘内注射用于基因组编辑和药物递送

Intrathecal injection is a commonly employed procedure in both pediatric and adult clinics, serving as an effective means to administer medications and ...

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

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交付的治疗药物通过侧脑室（ICV）和静脉（IV）注射液在小鼠

扩展胚胎及成体神经干细胞

病毒颗粒和荧光微球侧脑室和血管内注入新生儿脑

新生小鼠脑巴细胞基因调控的立体定向手术

使用脑和脊柱联合手术用双病毒载体靶向新生大鼠的皮质脊髓束

在新生小鼠静脉注射

新生小鼠鞘内注射用于基因组编辑和药物递送

新生小鼠鞘内注射用于基因组编辑和药物递送

新生小鼠鞘内注射用于基因组编辑和药物递送