作者感谢詹姆斯麦迪逊大学生物学系和詹姆斯麦迪逊大学光显微镜和成像设施的慷慨支持。Dr. 马克 l. 加布里埃尔关于年轻的产后组织准备的有用建议,和 Drs. 贾斯汀 w. 布朗和科里. 克莱兰德为外科手术材料和空间的慷慨协调。这项研究部分由 4 VA 合作研究基金资助, 这是一个促进弗吉尼亚联邦 (G.S.V.) 的合作伙伴关系, 以及弗吉尼亚科学院小项目研究补助金 (G.S.V.)。支持已慷慨提供的贝蒂祖爱管家58的本科研究奖学金 (K.M.B.), 法雷尔暑期研究奖学金 (K.M.B.), 詹姆斯麦迪逊大学第二世纪奖学金 (K.M.B.), 詹姆斯麦迪逊大学百年奖学金 (到 C.J.H.), 詹姆斯麦迪逊大学露西鲁宾逊查寻30纪念奖学金 (对 Z.L.H.) 和詹姆斯麦迪逊大学科学和数学学院协助津贴 (对 G.S.V.)。

这里描述的方法已经得到了詹姆斯麦迪逊大学动物护理和使用委员会 (ACUC) 的批准, 并符合所有相关的法规和机构准则。
1. 鼠标设置
<ol><li>房子一个年轻的 (> P60) 男性和女性合 floxed 小鼠一起建立一个饲养员对25。 注意: 一个好的负控制是建立一个额外的增殖对野生小鼠, 其中综合重组表达不会导致马赛克26。</li>
	<li>让雌鸟生下她的头, 并与雄性一起养育她的第一个垃圾。将雄性与雌性保持在一起, 以增加垃圾的存活率。断奶后的垃圾,例如在产后日 (P) 21, 分开的女性和男性。</li>
	<li>
小鼠繁殖
	<ol>
<li>设置女性和男性25之间的定时交配。使用视觉提示来确定女性的发情周期27, 并检查阴道插头在交配后的早晨25。</li>
		<li>如果找到一个插头, 分开, 并称女性。数天作为胚胎天 (E) 0.5。</li>
		<li>继续称女性每3-5 天, 以确定她是否怀孕。怀孕的女性将有10-20% 的体重增加7-10 天后受孕 (E0)。 注意: 此步骤比目视检查25更早、更可靠地确认怀孕。</li>
		<li>如果女性没有怀孕, 重复步骤 1.3. 1-1. 3.3。</li>
	</ol>
</li>
	<li>一旦确认怀孕, 请选择在子宫内电穿孔的日期。靶层 II/III 皮层锥体神经元祖细胞, 在 E15.5 进行电穿孔。</li>
</ol>2. DNA 设置
<ol><li>选择一个单一的 DNA 结构, 该编码重组以及荧光标记, 如绿色荧光蛋白 (GFP)28,29。或者, 使用 "超新星" 系统, 在以前的出版物中详细描述23,24。参见材料的名单例如构造。</li>
	<li>使用标准微生物技术30从大肠杆菌的步骤2.1 中放大 DNA 结构。</li>
	<li>按照制造商的指示, 用无内毒素质粒纯化试剂盒纯化 DNA 结构。用无内毒素水洗结构。 注: 在标准纯化试剂盒上选择无内毒素纯化试剂盒是至关重要的。</li>
	<li>根据所需的标记密度26将 DNA 洗集中到1-3 毫克/毫升。 注: 在使用新的 DNA 结构时, 必须建立一个效价, 因为它们有不同的长度和促进剂。考虑在几个不同的浓度和/或数额中注入一个结构来评估表达。例如, 注射1µL 或1.5 µL 的2.0 毫克/毫升 GFP。可以引起稀疏 (1 µL) 或致密 (1.5 µL) 标记层 II/III 视觉皮层锥体神经元26。</li>
	<li>在1x 磷酸缓冲盐水中加入0.4% 台台盼蓝 (PBS; 137 毫米氯化钠, 2.7 毫米氯化钾, 10 毫米 Na2HPO4, 1.8 mm 在 H2O 中 4, 调整为 pH 2 与 HCl) 7.4 至浓缩 DNA 溶液。</li>
	<li>在浓缩的 DNA 溶液中加入 10x PBS 1:10。</li>
	<li>将浓缩的 DNA 溶液储存在4° c。解决方案可以无限期地存储。</li>
</ol>3. 吸管设置
<ol><li>
使用玻璃毛细管拉拔器, 拉动一个非常长的 (10-15 毫米) 锥度和非常细尖端的吸管, 典型的管胚胎干细胞注射或核转移31。 注意: 为防止碎片污染管, 应在手术后2天内进行。<ol>
<li>按照制造商的指示, 通过执行斜坡测试来校准玻璃毛细管拉拔器。使用所产生的热值来编程使用以下参数的牵引协议: 热 = (斜坡试验值 + 15), 拉 = 30, 威尔 = 120, 时间 = 100, 压力 = 200。插入玻璃毛细管, 紧固在拉拔器上, 并激活程序化的牵引协议, 在玻璃毛细管上产生锥度。</li>
		<li>使用复合光显微镜 (例如10X 目标)31, 确保锥度长度介于 10-15 mm 之间。</li>
	</ol>
</li>
	<li>
打破吸管, 形成一个粗略的尖端到20-25 µm 直径。
	<ol>
<li>中心一个单任务擦拭 (见材料清单) 超过50毫升烧杯和舒展擦拭拉紧用一只手。另一方面, 按住并将吸管 (锥形侧向下) 垂直和完全通过擦拭的中心, 在锥形31中造成干净的断裂。 注: 将吸管完全通过擦拭将产生20-25 µm 直径约70% 的时间。</li>
		<li>在复合光显微镜下确认 (例如20x 目标)。 注意: 打破玻璃时要用眼睛保护。</li>
	</ol>
</li>
	<li>通过在 1x PBS 中将0.4% 台台台蓝的1µL 吸进一个部分填充的 piptte, 然后在吸管中以1µL 的高度进行1µL 标记, 使用一个细尖的耐酒精的标记来校准吸管。使用校准过的吸管在丢弃前管其他的。将管放在吸管保持器中, 不含灰尘和其他微粒。</li>
</ol>4.在子宫内电穿孔
<ol><li>将 Graefe 钳、虹膜剪刀、哈特曼蚊香钳、无纺布纱布海绵、环尖钳和培养皿放在不锈钢托盘上, 用铝箔包好。在高压釜中放置托盘和1升0.9% 盐水 (0.9% 重量/卷氯化钠)。高压釜在121° c 和3.5 人民军40分钟, 从高压釜和消毒的手术区中取出。 注意: 在手术中保持无菌状态是至关重要的。</li>
	<li>将蒸压1升盐水瓶在小水浴中加热至38° c, 至少在手术前2小时。</li>
	<li>将镊子型电极和脚踏板连接到发电机 (见材料清单)。按照制造商的说明, 设置发电机, 以提供五 50 ms 脉冲 50 V (与950毫秒间隔) 时, 由脚踏板触发。消毒镊子型电极, 并将其置于消毒手术区。</li>
	<li>做一个麻醉鼻锥像以前公布的32, 但使用一个腈或乳胶手套手指的一端形成一个膜片, 适合安全地在鼻子锥开口。在膜片上切一个大的洞, 以适应老鼠的鼻子。</li>
	<li>深麻醉怀孕小鼠在 E15.5 的诱导室充满异氟醚 (2-4%) 在纯氧流动1升/分钟。1分钟后, 测试扶正反射 (轻轻地倾斜感应腔, 确保鼠标不正确)。称量小鼠以确定手术后的镇痛剂量。</li>
	<li>将鼠标转移到手术区, 并在纯氧中通过鼻锥将吸入的异氟醚 (1-2%) 输送到1升/分钟, 以维持手术平面上的小鼠。使用镊子测试踏板反射 (轻轻捏爪, 确保鼠标不返回反射) 来验证麻醉。监测呼吸率和努力, 寻找深, 定期呼吸 (〜70-100 呼吸/分钟), 粘膜是粉红色的颜色和湿润。</li>
	<li>在手术过程中, 将足够的兽医眼膏涂抹在眼睛上, 以完全遮盖, 防止干燥。</li>
	<li>用饱和盐溶液填充的密封袋的金属活化剂盘 (见材料清单), 活化盐的放热结晶。放置2单任务湿巾在袋顶部, 并把鼠标放在邮袋, 以保持体温。</li>
	<li>按摩脱毛奶油到腹部区域与棉签, 直到腹部毛皮溶化 (大约 20-40 s), 然后抹掉腹部毛皮。用70% 的乙醇和棉签仔细清洁残留的残余物和脱毛奶油。在腹部部位放置一个内皮无菌的外科悬垂。</li>
	<li>采用无菌技术, 用 Graefe 钳将皮肤从腹部向上拉出, 用虹膜剪刀在皮肤上做一个2厘米的腹中线切口, 以确保底层肌肉不被割断。 注: 用 Graefe 钳和虹膜剪刀做切口的一个可接受的替代方法是使用手术刀, 如以前发布的17。</li>
	<li>发现式阿尔巴, 以可视化的中线腹直肌。用 Graefe 钳将肌肉向上和远离腹部, 用虹膜剪刀使另一条2厘米中线切口, 露出腹腔 (子宫是半透明的, 胚胎将在下方可见)。确保下层子宫和肠道组织不被切断。使切口足够大 (典型地 ~ 2 cm) 拔出并且舒适地显露子宫。 注: 用 Graefe 钳和虹膜剪刀做切口的一个可接受的替代方法是使用手术刀, 如以前发布的17。</li>
	<li>使用无菌10µL 微提示, 免除卡洛芬 (溶于无菌0.9% 盐水) 在4毫克/千克到腹腔内镇痛。</li>
	<li>在腹直肌切口左边缘夹住一只哈特曼蚊虫钳。将培养皿上的镊子放在切口左侧, 保持切口左侧的开口。夹在腹直肌切口右侧的另一只哈特曼蚊子钳, 并将镊子放在切口右侧的翻转培养皿上, 保持切口右侧开着。将纱布海绵放在切口区域周围。</li>
	<li>使用环形钳 (没有附加的限制螺钉), 在任何两个相邻的胚胎之间的子宫上拉, 不挤压或损伤任何组织。开始通过切口取出所有的胚胎, 把它们放在纱布海绵上面。当从腹腔取出最后的胚胎时, 一定不要拉扯卵巢或子宫颈。一旦暴露, 这是至关重要的保持子宫湿润的温盐水。子宫通常包含5至8胚胎。 注: 有可能添加青霉素和链霉素到生理盐水17。</li>
	<li>将校准的吸管连接到器管组件 (见材料清单), 并将步骤2中准备的 DNA 溶液的1µL 注入到每个胚胎的侧脑室中。侧脑室出现作为两个补丁的背部端的胚胎 (补丁比周围的组织端)。使用拇指和食指在注射和电穿孔操作的胚胎, 揭示了侧脑室, 并允许胚胎被轻轻地推对子宫壁在注入期间。通过观察台盼蓝填充侧脑室, 确认成功注射。</li>
	<li>将镊子型电极的阴极 (见材料清单) 放在子宫上, 直接在内侧和尾皮层以视觉皮层为靶 (靶区的大脑需要不同的电极位置)16, 26。将阳极放在子宫上, 就在胚胎头部的前部。</li>
	<li>确认阳极和阴极在适当的位置接触到胚胎周围的子宫。用脚踏板, 在电极上触发五 50 ms 脉冲的 50 V (950 毫秒间隔) 的传递。 注: 注射后, 带负电荷的 DNA 将向阴极移动, 并将被纳入最接近阴极的心室区细胞。</li>
	<li>以同样的方向将子宫返回腹腔。使用生理盐水来润滑子宫, 同时引导它手动和极端温和, 注意不要取代胚胎从他们的位置在子宫。</li>
	<li>用可吸收的缝合线 (参见材料清单) 关闭腹部肌肉用一个简单的中断的缝合, 用外科医生的结栓末端。不要夹紧两端太近结或结将成为解开。 注意: 缝合腹部肌肉是至关重要的, 这样伤口的边缘完全闭合, 但不会引起肌肉的热烫。结应该是紧的, 所以他们不能解开。</li>
	<li>关闭皮肤层与可吸收缝合 (简单中断缝合, 外科医生的结, 如在步骤 4.19)。用少量的组织粘合剂封住伤口。将组织粘合剂涂抹在结节上以防止松开。使用微去除周围的伤口, 可以由微吸气的组织粘合剂。</li>
	<li>将异氟烷水平降低至 0.5-1.5%。一旦组织粘合剂是干的 (通过接触手套测试胶粘剂), 从异氟醚中取出, 让女性独自在一个温暖的笼子里恢复。使用1毫升注射器与 26G, ½ "针管理丁丙诺啡腹腔在0.03 毫克/千克。</li>
	<li>继续监视鼠标, 直到鼠标完全恢复并正常运行 (即梳理、探索笼子、进食或饮用)。一旦康复, 就把雌鸟放回笼子里。 注意: 如果遵循所有步骤, 鼠标通常会在2分钟内恢复知觉, 并立即开始移动笼, 显示没有任何不适的迹象。</li>
	<li>如果鼠标出现不适的迹象 (如驼背的姿势, 卟啉分泌物)33, 管理卡洛芬在0.1 毫克/毫升的供水。如果有几个不舒服的迹象存在, 或者如果不舒服持续超过4小时, 尽管卡洛芬管理, 立即安乐的老鼠通过管理腹腔内致死注射氯胺酮 (240 毫克/千克)-嗪 (48 毫克/千克)-乙酰 (1.85 毫克/千克) 鸡尾酒使用1毫升注射器与26克, ½ "针, 并遵循一个批准的第二形式的安乐死33。</li>
	<li>为了提高 electroporated 胚胎出生后的存活率, 将笼子保持在一个安静的房间里, 不受噪音和震动的束缚。用塑料圆顶、筑巢材料和诸如葵花籽等膳食补充物来丰富环境 (见材料清单)。不要移动或扰乱笼子, 特别是在分娩后的前3天。</li>
</ol>5. 荧光显微镜的组织学准备
注意: 这个组织学协议的优化, 以准备从动物的组织, 比产后日 (P) 13, 是 electroporated在子宫。为了准备来自较年轻的产后动物 (P0-P13) 的组织, 建议遵循所有步骤 (包括 transcardial 灌注), 虽然大脑应该在准备切片之前植入琼脂 (步骤 5.9)。组织甚至可以在电穿孔后1-2 天内从胚胎中制备, 使用以前描述的方法18,20。
<ol><li>
准备甲醛 (粉煤灰) 解决方案。 注意: 在实验的一天内准备新鲜的粉煤灰势在必行。 注意: 甲醛是有毒的, 应该在通风柜里用适当的个人防护设备来处理。<ol>
<li>使50毫升4% 的粉煤灰, 热40毫升水到60° c。加入2克粉煤灰时, 用玻璃棒或搅拌棒搅拌。每2分钟增加10µL 10 米氢氧化钠, 直到粉煤灰溶解。</li>
		<li>加入5毫升的 10x PBS 和带来的解决方案的最后一卷50毫升水。</li>
		<li>在50毫升的解决方案后, 根据需要调整 pH 值为 7.4, 10 M HCl. 让溶液冷却并贮存在4° c。</li>
	</ol>
</li>
	<li>安乐鼠腹腔注射氯胺酮 (240 毫克/千克)-嗪 (48 毫克/千克)-乙酰 (1.85 毫克/千克) 鸡尾酒使用1毫升注射器与 26G, ½ "针。Transcardially 灌注10或20毫升 ice-cold 1x PBS, 后跟25或40毫升新鲜制造, ice-cold 4% 粉煤灰17。使用较低体积的幼仔 (P0-P13) 小鼠和更大的体积为年长的小鼠 (P14 和以上)。</li>
	<li>立即灌注后, 斩首鼠胴体之间的枕骨和 C1 椎与大剪刀和剥离, 并丢弃大部分的皮肤周围的头骨使用虹膜剪刀, 特别是皮肤周围额, 顶叶, 和枕骨头.保持头骨和大脑完好</li>
	<li>将头骨和大脑放在10毫升4% 的粉煤灰中, 在50毫升的锥形管中放置24小时, 在4° c 至 post-fix 组织。然后添加30毫升 1x PBS 到锥形管, 以稀释解决方案, 以1% 粉煤灰为存储在4° c。 注: 为每个头骨和大脑使用一个单独的锥形管 post-fixed。不要在1% 粉煤灰中孵育超过2周的大脑, 否则荧光就会开始消退。</li>
	<li>将大脑和头骨放在一个平坦的表面上, 单任务湿巾用 1x PBS 浸湿。使用一双镊子 (见材料清单) 来去除头骨周围的任何残留的皮肤或膜。</li>
	<li>使用镊子, 首先取出枕骨, 然后小心地移除顶骨骨, 移动的镊子, 远离大脑的表面。小心地取出任何脑膜以防止损伤皮质。</li>
	<li>将脑部下的镊子的背部楔入颅骨, 切断颅神经, 并取出大脑。</li>
	<li>
年轻的产后 (P0-P13) 脑
	<ol>
<li>通过加热25毫升 1x PBS 到80° c, 使25毫升的4% 琼脂。用玻璃棒或搅拌棒搅拌和溶解1克琼脂)。</li>
		<li>冷却溶液到35° c 同时继续搅拌。把大脑放在一个小容器里 (例如一个50毫升锥形管的盖子), 然后将琼脂溶液倒在脑部。让琼脂硬化。</li>
	</ol>
</li>
	<li>用单边刀片通过大脑, 大约0.5 毫米侧到 bregma, 手工制作冠状切口。使另一冠状切口穿过大脑大约0.5 毫米的尾鳍到视觉皮层。在振动切片试样盘的中心放置一个与侧端相同直径的氰胶池。将大脑的侧端放在胶池的顶部, 确保整个侧表面的组织是绑定到标本盘与胶水。</li>
	<li>安装缓冲托盘到振动切片和安全的内置夹杆。将刀片插入刀架, 并将刀架固定在带有内置夹紧螺钉的振动切片上。使用内置夹紧装置将带脑的标本盘固定在缓冲托盘上。设置速度设置为 5.70 (= 0.285 毫米/秒), 频率设置为 5.33 (53.3 Hz)。</li>
	<li>用 1x pbs 在大脑的水平上方填充腔室, 然后将刀片式服务器降低到 pbs。开始服用100µm 冠状部分通过组织。</li>
	<li>
如果不需要进一步的处理, 如 4, 6-diamidino-2-吲 (DAPI) 核染色或免疫组织化学。
	<ol>
<li>使用精美的倾斜画笔, 直接从振动切片到显微镜幻灯片, 每张幻灯片上安装4-6 节。</li>
		<li>允许部分通过吸汗的解决方案与罚款的倾斜画笔, 使大脑切片不再漂移的幻灯片。</li>
		<li>然后, 在幻灯片的每个部分放置一滴 mountant (参见材料列表)。</li>
		<li>轻轻地放置一个 24 x 50 毫米片在上面, 确保 1) 没有气泡形成的部分, 2) 的部分不移动, 和 3) mountant 完全覆盖的区域之间的幻灯片和片。允许 mountant 治疗至少24-48 小时。</li>
	</ol>
</li>
	<li>
用 DAPI 染色核进行皮质层的组织学检查
	<ol>
<li>使用精美的倾斜画笔将每个部分转换为包含10µg/毫升 DAPI (参见材料清单) 的解决方案, 从库存解决方案中稀释的 1x PBS (20 毫克/毫升 DAPI 水)。</li>
		<li>在室温下孵育 DAPI 溶液5分钟, 然后用细尖的画笔将 DAPI 溶液中的部分转移至 1x pbs, 并在 1x pbs 中孵育5分钟室温。转移部分两次, 以新鲜 1x PBS 每5分钟。然后, 使用一个精美的倾斜的画笔, 将剖面转移到显微镜幻灯片上, 按照步骤 5.11.2-5.11.4, 然后继续步骤5.13。</li>
	</ol>
</li>
	<li>在热板上加热一个含有 Valap (等量的凡士林, 羊毛脂和石蜡) 的玻璃培养皿, 直到完全融化。通过在熔化的 Valap 上刷牙来密封幻灯片的边缘。</li>
	<li>要检查组织中的荧光, 请使用光学显微镜或共聚焦显微镜 (见材料列表) 和足够的过滤器集以查看荧光 (例如DAPI: 激发 325-375 nm, 发射 435-485 nm;GFP: 激发 450-490 nm, 发射 500-550 nm)17。共焦图像可以采取低-(4X, 数字光圈, na = 0.20), 中-(20X, na = 0.75), 和大功率 (60X, na = 1.40) 的目标。</li>
</ol>

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作者没有利益冲突透露。

在这里, 我们介绍了在子宫内电穿孔与重组在 floxed 小鼠的组合, 以产生马赛克脑组织。这种方法的优点是, 每次要针对不同的蜂窝子类型时都不需要生成新的鼠标线:在子宫内电穿孔可用于针对兴奋性神经元、抑制性神经元或胶质细胞, 这取决于时间以及电穿孔的位置15,16,17,18,<sup c...

生成一个遗传马赛克是一个经典的实验范式, 以了解的功能基因的兴趣。为了确定基因是否是细胞表型的必要条件, 最简单的方法是在整个生物体中导致基因功能的丧失 (例如击倒)。然而, 要确定某一基因是否需要特定的细胞类型, 基因在整个生物体中的击倒并不是一种有效的方法。相反, 一个方法是需要的, 这将导致在一个给定的细胞中的基因功能丧失, 而它是由野生 (即不受基因干扰) tissue-in 换句话说, 创造马赛克组织。如果突变细胞显示出突变的表型, 但周围的野生细胞没有, 该基因功能的细胞自主的方式。分析的马赛克组织, 其中突变细胞周围的野生组织, 是理想的理解细胞自主功能的基因, 特别是在大脑中的神经元和神经胶质形成一个巨大的互联网络的组织。
多种形式的马赛克脑组织为研究基因的细胞自主功能提供了强有力的模型。研究的重点是神经元移植1, 女性 X 链接嵌2,3,4, 以及内生体细胞嵌5,6根据马赛克得出了他们的结论脑组织。有条件地删除基因通过 "氧合酶复合系统" 是一种充分利用转基因小鼠品系的方法。在这个方法中, 两个 loxP 站点被引入到一个必需的基因序列的两侧 (如外显子), 它的两侧是 loxP 的站点, 它们都朝同一方向 ("floxed")。重组将 loxP 站点之间的顺序7。通过将 floxed 小鼠转到另一条表达重组的老鼠线上, 并在细胞子集 ("记者行") 中使用荧光标记, 可以实现对重组的再组合。这已被证明在各种方法, 以揭示的功能, 一个基因的细胞子集, 如兴奋性神经元或胶质细胞8。记者线可以表达 CreER 的T2 , 以允许由药物诱导的重组 (神经元标记与诱导的综合治疗的淘汰赛, 或光滑)9。在另一种称为 "镶嵌分析双标记 (决策)"10,11的策略中, interchromosomal 重组允许与杂组织一起创建一个纯合突变体。在这些方法中, 每个候选基因或细胞亚型都需要在每次测试时产生一条新的小鼠。另外, 通过导入12或通过病毒载体 (例如腺相关病毒13或慢14携带特定于细胞的子类型, 可以引入后天重组。发起人)。这一策略产生强烈的产后标记。为靶向发育的脑皮质神经元稀少和产前, 一个理想的策略是宫内电穿孔重组与荧光标记。
除了通过在子宫内电穿孔, 通过在体内生成马赛克组织, 我们还对其他已发布的协议15、16中的过程进行了一些改编. 17,18,19,20,21。我们提供信息, 以提高成功繁殖定时怀孕的女性。我们还概述了在皮层组织中引入稀疏和明亮的神经元标记的两个策略: 一种策略是滴定重组的单个构造编码的级别和一个荧光标记22。另一种策略是使用 "超新星" 系统, 这些参数专门设计为23,24。此外, 我们提供了改进的生产一致的显微注射管和简化的在子宫内电穿孔手术。最后, 我们概述的关键步骤, 简化组织学准备, 允许分析树突棘和树突和轴突乔木, 没有进一步染色或免疫组化。

<table><tbody><tr><td>C57BL/6J mice</td><td>The Jackson Laboratory</td><td>#000664</td><td>See &quot;1. Mouse set-up&quot; (step 1.1, &quot;wildtype mice&quot;)</td></tr><tr><td>GFP.Cre empty vector</td><td>AddGene</td><td>#20781</td><td>See &quot;2. DNA set-up&quot; (step 2.1 &quot;single DNA construct that codes for Cre recombinase as well as a fluorescent marker&quot;). GFP.Cre empty vector was a gift from Tyler Jacks.</td></tr><tr><td>pK029.CAG-loxP-stop-loxP-RFP-ires-tTA-WPRE (Supernova)</td><td>AddGene</td><td>#69138</td><td>See &quot;2. DNA set-up&quot; (step 2.1 &quot;Supernova&quot; system) and http://snsupport.webcrow.jp/. pK029.CAG-loxP-stop-loxP-RFP-ires-tTA-WPRE (Supernova) was a gift from Takuji Iwasato.</td></tr><tr><td>pK031.TRE-Cre (Supernova)</td><td>AddGene</td><td>#69136</td><td>See &quot;2. DNA set-up&quot; (step 2.1 &quot;Supernova&quot; system) and http://snsupport.webcrow.jp/. pK031.TRE-Cre (Supernova) was a gift from Takuji Iwasato.</td></tr><tr><td>pK038.CAG-loxP-stop-loxP-EGFP-ires-tTA-WPRE (Supernova)</td><td>AddGene</td><td>#85006</td><td>See &quot;2. DNA set-up&quot; (step 2.1 &quot;Supernova&quot; system) and http://snsupport.webcrow.jp/. pK038.CAG-loxP-stop-loxP-EGFP-ires-tTA-WPRE (Supernova) was a gift from Takuji Iwasato.</td></tr><tr><td>EndoFree Plasmid Maxi Kit (10)</td><td>Qiagen</td><td>#12362</td><td>See &quot;2. DNA set-up&quot; (step 2.3 &quot;endotoxin-free plasmid purification kit&quot;)</td></tr><tr><td>Trypan Blue powder, BioReagent grade</td><td>Sigma</td><td>T6146-5G</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;trypan blue&quot;)</td></tr><tr><td>Sodium Chloride, ACS, 2.5 kg</td><td>VWR</td><td>BDH9286-2.5KG</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;NaCl&quot;)</td></tr><tr><td>Potassium Chloride, ACS, 500 g</td><td>VWR</td><td>#97061-566</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;KCl&quot;)</td></tr><tr><td>Sodium phosphate dibasic, ReagentPlus, 100 g</td><td>Sigma-Aldrich</td><td>S0876-100G</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&quot;)</td></tr><tr><td>Potassium phosphate monobasic, ReagentPlus, 100 g</td><td>Sigma-Aldrich</td><td>P5379-100G</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&quot;)</td></tr><tr><td>Hydrochloric acid, ACS reagent, 500 mL</td><td>Fisher Scientific</td><td>A144-500</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;HCl&quot;)</td></tr><tr><td>P-97 Micropipette Puller</td><td>Sutter Instrument</td><td>P-97</td><td>See &quot;3. Pipette set-up&quot; (step 3.1 &quot;glass capillary puller&quot;)</td></tr><tr><td>3.0 mm wide trough filament</td><td>Sutter Instrument</td><td>FT330B</td><td>See &quot;3. Pipette set-up&quot; (step 3.1 &quot;glass capillary puller&quot;)</td></tr><tr><td>Thin Wall Glass Capillaries, 4&quot;, 1 / 0.75 OD/ID</td><td>World Precision Instruments</td><td>TW100-4</td><td>See &quot;3. Pipette set-up&quot; (step 3.1.1 &quot;glass capillary&quot;)</td></tr><tr><td>Single Ply Soft-Tech Wipes, 4.5&quot;</td><td>Phenix</td><td>LW-8148</td><td>See &quot;3. Pipette set-up&quot; (step 3.2.1 &quot;single-ply task wipe&quot;); other single-ply wipes (e.g. Kimwipes) can be used.</td></tr><tr><td>Graefe Forceps, 7 cm, Straight, 0.7 mm 1x2 Teeth</td><td>World Precision Instruments</td><td>#14140</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;Graefe forceps&quot;)</td></tr><tr><td>Iris Scissors, 11.5 cm, Straight, 12-pack</td><td>World Precision Instruments</td><td>#503708-12</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;iris scissors&quot;)</td></tr><tr><td>Hartman Mosquito Forceps, 9 cm, Straight, 12-pack</td><td>World Precision Instruments</td><td>#503728-12</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;Hartman mosquito forceps&quot;)</td></tr><tr><td>General Purpose Non-Woven Sponges, 2&quot; x 2&quot;, 4-ply</td><td>Medrepexpress</td><td>#2204-c</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;non-woven gauze sponges&quot;)</td></tr><tr><td>Ring Tipped Forceps, 10 cm, Straight, 2.2mm ID</td><td>World Precision Instruments</td><td>#503203</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;ring-tipped forceps&quot;)</td></tr><tr><td>Pyrex petri dishes complete, O.D. &amp;times; H 100 mm &amp;times; 20 mm</td><td>Sigma-Aldrich</td><td>CLS3160102-12EA</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;Petri dishes&quot;)</td></tr><tr><td>Flat Type Instrument Tray, Stainless Steel, 13-5/8&quot; x 9-3/4&quot; x 5/8&quot;</td><td>Amazon</td><td>B007SHGAHA</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;stainless steel tray&quot;)</td></tr><tr><td>Platinum Tweezertrode, 5 mm</td><td>BTX</td><td>#45-0489</td><td>See &quot;4. In utero electroporation&quot; (step 4.3 and 4.16 &quot;tweezer-type electrodes&quot;)</td></tr><tr><td>ECM 830 Foot Pedal</td><td>BTX</td><td>#45-0211</td><td>See &quot;4. In utero electroporation&quot; (step 4.3 and 4.17 &quot;foot pedal&quot;)</td></tr><tr><td>ECM 830 Generator</td><td>BTX</td><td>#45-0052</td><td>See &quot;4. In utero electroporation&quot; (step 4.3 &quot;generator&quot;)</td></tr><tr><td>Single Animal Isoflurane Anesthesia System with Small Induction Box</td><td>Harvard Apparatus</td><td>#72-6468</td><td>See &quot;4. In utero electroporation&quot; (step 4.4 and 4.6 &quot;nose cone&quot;, step 4.4 &quot;induction chamber&quot;)</td></tr><tr><td>Ophthalmic ointment</td><td>Hanna Pharmaceutical Supply Co</td><td>#0536108691</td><td>See &quot;4. In utero electroporation&quot; (step 4.7 &quot;veterinary ophthalmic ointment&quot;)</td></tr><tr><td>Space Gel (AIMS)</td><td>VWR</td><td>#95059-640</td><td>See &quot;4. In utero electroporation&quot; (step 4.8 &quot;sealed pouch filled with supersaturated salt solution&quot;)</td></tr><tr><td>Hair Remover Gel Cream, Sensitive Formula</td><td>Veet</td><td>#062200809951</td><td>See &quot;4. In utero electroporation&quot; (step 4.9 &quot;depilatory cream&quot;)</td></tr><tr><td>10ul Low Retention Tip Starter (960 tips/pk)</td><td>Phenix Research Products</td><td>TSP-10LKIT</td><td>See &quot;4. In utero electroporation&quot; (step 4.12 &quot;sterile 10 &amp;micro;L micropipette tip&quot;)</td></tr><tr><td>Aspirator tube assemblies for calibrated microcapillary pipettes</td><td>Sigma-Aldrich</td><td>A5177</td><td>See &quot;4. In utero electroporation&quot; (step 4.15 &quot;aspirator tube assembly&quot;)</td></tr><tr><td>Braided Absorbable Suture, 4-0, Needle NFS-2(FS-2), 27&quot;</td><td>Medrepexpress</td><td>MV-J397</td><td>See &quot;4. In utero electroporation&quot; (step 4.19 &quot;absorbable sutures&quot;)</td></tr><tr><td>&amp;ldquo;LiquiVet Rapid&amp;rdquo; Tissue Adhesive</td><td>Medrepexpress</td><td>VG3</td><td>See &quot;4. In utero electroporation&quot; (step 4.20 &quot;tissue adhesive&quot;)</td></tr><tr><td>Hypodermic syringes, polypropylene, Luer lock tip, capacity 1.0 mL</td><td>Sigma-Aldrich</td><td>Z551546-100EA</td><td>See &quot;4. In utero electroporation&quot; (step 4.21 &quot;1 mL syringe&quot;)</td></tr><tr><td>BD Precisionglide syringe needles gauge 26, L 1/2 in.</td><td>Sigma-Aldrich</td><td>Z192392-100EA</td><td>See &quot;4. In utero electroporation&quot; (step 4.21 &quot;26G, &amp;frac12;&amp;rdquo; needle&quot;)</td></tr><tr><td>Nestlets Nesting Material</td><td>Ancare</td><td>NES3600</td><td>See &quot;4. In utero electroporation&quot; (step 4.24 &quot;nesting materials&quot;)</td></tr><tr><td>Sunflower Seeds, Black Oil, Sterile</td><td>Bio-Serv</td><td>S5137</td><td>See &quot;4. In utero electroporation&quot; (step 4.24 &quot;sunflower seeds&quot;)</td></tr><tr><td>Paraformaldehyde, 97%</td><td>Alfa Aesar</td><td>A11313</td><td>See &quot;5. Histology&quot; (step 5.1.1 &quot;PFA&quot;)</td></tr><tr><td>Economy Tweezers #3, 11 cm, 0.2 x 0.4 mm tips</td><td>World Precision Instruments</td><td>#501976</td><td>See &quot;5. Histology&quot; (step 5.5 &quot;tweezers&quot;)</td></tr><tr><td>Agar powder</td><td>Alfa Aesar</td><td>#10752</td><td>See &quot;5. Histology&quot; (step 5.8.1 &quot;agar&quot;)</td></tr><tr><td>Single-edge razor blades, #9 blade</td><td>Stanley Tools</td><td>#11-515</td><td>See &quot;5. Histology&quot; (step 5.9 &quot;single-edge razor blade&quot;)</td></tr><tr><td>Specimen disc S D 50 mm</td><td>Leica</td><td>#14046327404</td><td>See &quot;5. Histology&quot; (step 5.9 &quot;vibrating microtome specimen disc&quot;)</td></tr><tr><td>Buffer tray S assembly</td><td>Leica</td><td>#1404630132</td><td>See &quot;5. Histology&quot; (step 5.10 &quot;buffer tray&quot;)</td></tr><tr><td>VT1000 S Vibratome</td><td>Leica</td><td>#14047235612</td><td>See &quot;5. Histology&quot; (step 5.10 &quot;vibrating microtome&quot;)</td></tr><tr><td>Double Edge Razor Blades</td><td>Personna</td><td>BP9020</td><td>See &quot;5. Histology&quot; (step 5.10 &quot;blade&quot;)</td></tr><tr><td>Knife Holder S</td><td>Leica</td><td>#14046230131</td><td>See &quot;5. Histology&quot; (step 5.10 &quot;knife holder&quot;)</td></tr><tr><td>Studio Elements Golden Taklon Short Handle Round Brush Set</td><td>Amazon</td><td>B0089KU6XE</td><td>See &quot;5. Histology&quot; (step 5.12.1 &quot;fine tipped paintbrush&quot;)</td></tr><tr><td>Superfrost Plus Slides</td><td>Electron Microscopy Services</td><td>#71869-11</td><td>See &quot;5. Histology&quot; (step 5.12.1 &quot;microscope slide&quot;)</td></tr><tr><td>ProLong Diamond Antifade Mountant, 10 ml</td><td>Thermofisher</td><td>P36970</td><td>See &quot;5. Histology&quot; (step 5.12.3-5.12.4 &quot;mountant&quot;)</td></tr><tr><td>Cover Glass, 24 x 50 mm, No. 1</td><td>Phenix Research Products</td><td>MS1415-10</td><td>See &quot;5. Histology&quot; (step 5.12.4 &quot;coverslip&quot;)</td></tr><tr><td>4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td><td>Sigma-Aldrich</td><td>D9542</td><td>See &quot;5. Histology&quot; (step 5.13.1 &quot;DAPI&quot;)</td></tr><tr><td>Fixed Stage Upright Microscope</td><td>Olympus</td><td>BX51WI</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;light microscope&quot;)</td></tr><tr><td>Laser Scanning Confocal Microscope</td><td>Nikon</td><td>TE2000/C2si</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;confocal microscope&quot;)</td></tr><tr><td>4x objective, NA = 0.20</td><td>Nikon</td><td>CFI Plan Apo Lambda 4X</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;low-power objective&quot;)</td></tr><tr><td>20x objective, NA = 0.75</td><td>Nikon</td><td>CFI Plan Apo Lambda 20X</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;medium-power objective&quot;)</td></tr><tr><td>60x objective, NA = 1.40</td><td>Nikon</td><td>CFI Plan Apo VC 60X Oil</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;high-power objective&quot;)</td></tr></tbody></table>

inducing cre-lox recombination in mouse cerebral cortex through in utero electroporation

基因的细胞自主神经元功能可以通过在小的和稀疏的神经元中造成基因功能的丧失或增益而显露出来。要做到这一点, 就需要生成一个镶嵌体, 其中基因的功能丧失或增益的神经元被不受基因扰动的组织包围。在这里, 我们结合了综合考试-液氧复合系统与在宫内电穿孔, 以产生马赛克脑组织, 可用于研究细胞自主功能的基因在神经元。DNA 结构 (可通过存储库提供), 编码的荧光标签和重组, 被引入到开发的皮层神经元含有基因的 loxP 部位的大脑中的小鼠胚胎使用在子宫穿孔.此外, 我们描述了各种适应的子宫内电穿孔方法, 提高生存能力和再现性。该方法还包括建立一个在稀疏或稠密的神经元中的重组的效价。标记脑组织的组织学准备不需要 (但可以适应) 免疫组化。该结构的使用保证了荧光标记神经元携带的基因重组。组织学准备可以通过对树突和轴突和树突棘的共聚焦成像对神经元进行形态学分析。由于稀疏镶嵌组织中功能的丧失或增益达到了目的, 因此, 该方法允许研究基因产品的细胞自主性和足够度在体内。

单一结构的 GFP。electroporated 在 E15.5 和可视化的 P14。根据结构的浓度和注入量, 可以获得稀疏或稠密的结果22,26。例如, 注射1µL 2 毫克/毫升 GFP。考试结果在稀疏分布的标记细胞, 其中一些可以是明亮的 (图 1A), 并本地化第二层/III (图 1B)。由于组织是100µm 厚, 大多数树突乔木保存 (<strong...

Watch this Scientific Journal Video about Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation at JoVE.com

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

在稀疏的细胞群中, 可以通过诱导功能的丧失或增益来研究脑内基因的细胞自主功能。在这里, 我们描述在子宫内电穿孔, 以提供重组到稀疏的人群发育皮层神经元与 floxed 基因, 导致功能丧失在体内。

宫内电穿孔诱导小鼠大脑皮质中氧合酶的重组

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...

inducing-cre-lox-recombination-mouse-cerebral-cortex-through-utero

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

11.6K 次观看.  James Madison University. 在稀疏的细胞群中, 可以通过诱导功能的丧失或增益来研究脑内基因的细胞自主功能。在这里, 我们描述在子宫内电穿孔, 以提供重组到稀疏的人群发育皮层神经元与 floxed 基因, 导致功能丧失在体内。

视频: Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific Cre models, deletion of a protein throughout a tissue or cell population often leads to complex phenotypes resulting from multiple interacting mechanisms. Determining whether a phenotype results from disruption of a cell autonomous mechanism, which is intrinsic to the cell in question, or from a non-cell autonomous mechanism, which would result from impairment of that cell&#8217;s environment, can be difficult to discern. To gain insight into protein function in an in vivo context, in utero electroporation (IUE) enables gene deletion in a small subset of cells within the developing cortex or some other selected brain region. IUE can be used to target specific brain areas, including the dorsal telencephalon, medial telencephalon, hippocampus, or ganglionic eminence. This facilitates observation of the consequences of cell autonomous gene deletion in the context of a healthy environment. The goal of this protocol is to show how IUE can be used to analyze a defect in radial migration in a floxed transgenic mouse line, with an emphasis on distinguishing between the cell autonomous and non-cell autonomous effects of protein deletion. By comparing the phenotype resulting from gene deletion within the entire cortex versus IUE-mediated gene deletion in a limited cell population, greater insight into protein function in brain development can be obtained than by using either technique in isolation.

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment.

诱导蛋白缺失的通过<em&gt;在子宫内</em&gt;电穿孔来定义赤字的神经细胞迁移的转基因模型

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific ...

科研

发育生物学

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

E15小鼠胚胎在子宫内侧脑室注射和电穿孔

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

生物学

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

高效的基因传递到多个中枢神经系统使用新界在子宫内电穿孔

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

神经科学

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

在子宫内电穿孔：鼠标控制时空基因Transefection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

神经元形态的研究方法：<em&gt;体外</em&gt; RNAi技术在胚胎小鼠大脑皮质的电穿孔

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

Cortex-，Hippocampus-，Thalamus-，下丘脑 - ，横向隔核 - 纹状体专用<em&gt;在子宫内</em&gt;电穿孔的C57BL / 6小鼠

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

在子宫内电途径研究神经元亚群的兴奋性和单细胞连接

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Double In Utero Electroporation to Target Temporally and Spatially Separated Cell Populations

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian corticogenesis. This procedure takes advantage of the brain ventricles to allow the introduction of DNA of interest and uses a pair of electrodes to direct the entrance of the genetic material into the cells lining the ventricle, the neural stem cells. This method allows researchers to label the desired cells and/or manipulate the expression of genes of interest in those cells. It has multiple applications, including assays targeting neuronal migration, lineage tracing, and axonal pathfinding. An important feature of this method is its temporal and regional control, allowing circumvention of potential problems related with embryonic lethality or the lack of specific CRE driver mice. Another relevant aspect of this technique is that it helps to considerably reduce the economic and temporal limitations that involve the generation of new mouse lines, which become particularly important in the study of interactions between cell types that originate in distant areas of the brain at different developmental ages. Here we describe a double electroporation strategy that enables targeting of cell populations that are spatially and temporally separated. With this approach we can label different subtypes of cells in different locations with selected fluorescent proteins to visualize them, and/or we can manipulate genes of interest expressed by these different cells at the appropriate times. This strategy enhances the potential of in utero electroporation and provides a powerful tool to study the behavior of temporally and spatially separated cell populations that migrate to establish close contacts, as well as long-range interactions through axonal projections, reducing temporal and economic costs.

Double in utero electroporation allows targeting cell populations that are spatially and temporally separated. This technique is useful to visualize interactions between those cell populations using fluorescent proteins in normal conditions but also after functional experiments to perturb genes of interest.

双电穿孔，以定时和空间分离的细胞群

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian ...

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines.

In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes

Astrocytes tile the cerebral cortex uniformly, making the analysis of their complex morphology challenging at the cellular level. The protocol provided here uses multicolor labeling based on in utero electroporation to single out cortical astrocytes and analyze their volume and morphology with a user-friendly image analysis pipeline.

在多地址基因组集成颜色 （MAGIC） 标记的 Utero 电冲中， 以个性化皮质小鼠星形细胞

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at ...

宫内电穿孔诱导小鼠大脑皮质中氧合酶的重组

摘要

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此视频中的章节

诱导蛋白缺失的通过

E15小鼠胚胎在子宫内侧脑室注射和电穿孔

高效的基因传递到多个中枢神经系统使用新界在子宫内电穿孔

在子宫内电穿孔：鼠标控制时空基因Transefection

神经元形态的研究方法：

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘

Cortex-，Hippocampus-，Thalamus-，下丘脑 - ，横向隔核 - 纹状体专用

在子宫内电途径研究神经元亚群的兴奋性和单细胞连接

双电穿孔，以定时和空间分离的细胞群

在多地址基因组集成颜色（MAGIC）标记的 Utero 电冲中，以个性化皮质小鼠星形细胞

宫内电穿孔诱导小鼠大脑皮质中氧合酶的重组

摘要

探索更多视频

此视频中的章节

诱导蛋白缺失的通过

E15小鼠胚胎在子宫内侧脑室注射和电穿孔

高效的基因传递到多个中枢神经系统使用新界在子宫内电穿孔

在子宫内电穿孔：鼠标控制时空基因Transefection

神经元形态的研究方法：

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘

Cortex-，Hippocampus-，Thalamus-，下丘脑 - ，横向隔核 - 纹状体专用

在子宫内电途径研究神经元亚群的兴奋性和单细胞连接

双电穿孔，以定时和空间分离的细胞群

在多地址基因组集成颜色 （MAGIC） 标记的 Utero 电冲中， 以个性化皮质小鼠星形细胞

在多地址基因组集成颜色（MAGIC）标记的 Utero 电冲中，以个性化皮质小鼠星形细胞