我们感谢劳拉 Sieber, 安娜 Neuerburg, 亚辛哈琳, 和佩特拉 Schroeter 的技术援助。我们还感谢 Drs. k. Reifenberg、k. 戴尔和 Prückl 为 DKFZ 动物实验提供帮助;成像核心设施的 DKFZ 和卡尔蔡司成像中心在 DKFZ 的共焦显微成像。这项工作得到德意志 Forschungsgemeinschaft, KA 4472/1-1 (交流) 的支持。

所有动物实验都是按照动物福利条例进行的, 并经主管当局批准 (Regierungspräsidium 卡尔斯鲁厄, 批准号: G90/13, G176/13, G32/14, G48/14 和 G133/14)。
1. 生成 pU6-sgRNA-Cbh-Cre 质粒
<ol><li>根据先前发布的协议14, 设计 sgRNA 目标为一个感兴趣的基因。 注: 在本实验中, 两个 sgRNAs 被设计为靶向小鼠Top2b基因和非靶向控制 sgRNA (表 1)。对于使用 CRISPR 技术的挖空基因, sgRNAs 需要设计为 5 ' 区域的编码序列或蛋白质的基本功能域。一个方便的起点是测试公开地可利用的预先设计的 sgRNA 序列, 象壁虎 v2 老鼠淘汰赛水池图书馆15从张实验室。或者, sgRNAs 可以使用公共可用的软件进行设计, 如以下网站: crispr.mit.edu。</li>
	<li>将 sgRNAs 克隆到 pU6-sgRNA-Cbh-Cre 向量中。 注: 本研究使用的 pU6-sgRNA-Cbh-Cre 向量12是从原 pX330 质粒16中取代 SpCas9 编码序列的 recombinase. <ol>
<li>合成两个 DNA 寡核苷酸如下。感觉: 5 '-CACCGNNNNNNNNNNNNNNNNNNNN-3 ';反义: 3 '-CNNNNNNNNNNNNNNNNNNNNCAAA-5 '。 注: 上面显示的 20 N 代表 sgRNA 序列。</li>
		<li>消化 pU6-sgRNA-Cbh-Cre 与 BbsI 30 分钟在37°c 使用以下试剂, 装载被消化的样品到1% 琼脂糖凝胶, 并且用凝胶萃取试剂盒净化它们。使用3µg 质粒;3µL BbsI;1µL FastAP;5µL 10X 消化缓冲液;添加 ddH2O 以使总容量增加到50µL。</li>
		<li>Phosphorylate 和退火每对 sgRNA 寡核苷酸使用以下试剂 (10 µL 总容量): 1 µL 自定义的感觉寡聚 (100 毫米);1µL 自定义反义寡聚 (100 毫米);1µL 10X T4 结扎缓冲器;6.5 µL ddH2O;0.5 µL T4 PNK。孵育在一个热循环仪30分钟在37°c, 和5分钟在95°c, 然后舷梯到25°c 在5°c/分钟。</li>
		<li>设置以下结扎反应, 并在室温下孵育10分钟 (10 µL 总容积): X µL BbsI 消化质粒 (50 ng);1µL 磷酸化和退火, 寡聚双相 (1:200 稀释);5µL 2X 结扎缓冲器;X µL ddH2O;1µL 快速连接酶。</li>
		<li>将结扎产物的2µL 加入50µL 的化学能力 DH5α大肠杆菌细胞中。在冰上孵化30分钟后, 热休克的样品九十年代在42摄氏度, 并孵化2分钟冰。加入100µL 的 lb 培养基, 并将其印在含有100µg/毫升氨苄西林的 lb 板上。 注: sgRNA 序列 pU6-sgRNA-Cbh-Cre 质粒的整合是根据 pX330 质粒14的克隆步骤进行的。</li>
		<li>接种两个菌落, 每2毫升的 LB 培养基在37°c 至少6小时, 并使用试剂盒进行迷你准备 DNA 隔离。</li>
		<li>利用 hU6-Forward 底漆对微型准备 DNA 进行序列化, 并通过与步骤1.2.1 中显示的序列对齐, 确定菌落的正确插入 sgRNAs。引物序列如表 1所示。本研究使用的质粒名为 pU6-sgTop2b-1-Cbh-Cre、pU6-sgTop2b-2-Cbh-Cre 和 pU6-sgControl-Cbh-Cre。</li>
		<li>用试剂盒 (例如从 Qiagen) 接种正确识别的菌落, 并纯化子宫内电穿孔中无内毒素的质粒 DNA。洗脱在 1:10 (10% TE 缓冲器) 中, 用无内毒素的特缓冲液稀释 ddH2O 的 DNA。质粒的 DNA 浓度需要高于2毫克/毫升。 注意: 不要使用常规的最大准备试剂盒进行 DNA 制备。贮存质粒-溶液在4°c 定期短期使用 (最多4周), 或整除适当的体积约25µL, 并保持在-20 摄氏度, 长期储存。</li>
	</ol>
</li>
</ol>2. 用 EGxxFP 质粒系统检测 sgRNAs 的效率
注: sgRNAs 的效率通常由验船师或 T7E1 (T7 限制性 I) 化验检验。在本协议中, 使用一种简单有效的替代方法。这一方法的关键是使用 pCAG EGxxFP 质粒, 其中包含重叠的 EGFP 片段分离的 DNA 序列, 其中包含 sgRNA 靶点17。在 pCAG EGxxFP 与转染细胞中的 sgRNA 和 Cas9 的表达后, 在目标序列中的 Cas9-mediated 双绞线断裂 (争端处理) 由内生同源依赖性机制修复, reconstitutes 表达磁带。
<ol><li>设计引物以放大以 sgRNA 靶序列为中心的基因组区域。PCR 扩增子大约是 500 bp。在本实验中, 应用 DNA 组装将 PCR 扩增片段克隆成 pCAG-EGxxFP 质粒。 注: 或者, 适当的限制点可以添加到 5 ' 末端的 PCR 引物。pCAG-EGxxFP 向量中可用的限制站点是 BamHI、NheI、PstI、贝里沙、EcoRI 和 EcoRV。</li>
	<li>使用下面的表单和 PCR 参数, 用设计的引物放大基因组 DNA。装载样品 (19.7 µL H2O; 0.3 µL 10 µL 小鼠基因组 DNA; 2.5 µL 10 µM EGxxFP-Top2b-F; 2.5 µL 10 µM EGxxFP-Top2b-R; 25 µL 2X PCR 主混合; 50 µL 总容积) 到1% 琼脂糖凝胶和凝胶-用凝胶萃取试剂盒纯化 PCR 片段。使用以下反应 conditions:95 °c 为3分钟;然后35x 周期98°c 为二十年代, 60 °c 十五年代, 72 °c 为二十年代和72°c 为1分钟;4摄氏度, 无限期。 注: 在本实验中, Top2b外显子1中的一个区域被放大;在表 1中找到引物序列。</li>
	<li>消化 pCAG-EGxxFP 载体与限制酶 BamHI 和 EcoRI 2 小时在37°c, 然后装载样品到1% 琼脂糖凝胶和凝胶-净化载体骨干使用凝胶萃取试剂盒。使用以下试剂 (50 µL 总容积): X µL 质粒 (3 µg);2µL BamHI;2µL EcoRI;5µL 10X 缓冲器;X µL ddH2O。</li>
	<li>根据制造商的指示建立 DNA 组装反应18 , 转化为 DH5α的大肠杆菌。</li>
	<li>接种两个殖民地, 每在2毫升的 LB 培养基在37°c 至少6小时, 执行一个迷你准备 DNA 隔离使用套件,例如, 从 Qiagen, 并确定殖民地与正确的插入 (以下简称为 pCAG-EG-Top2b-FP) 使用桑格测序与 EGxxFP-Top2b-F 底漆。</li>
	<li>染 HEK293T 细胞。<ol>
<li>在转染前将 HEK293T 细胞种子成24井板24小时。 注: 细胞培养在37°c 细胞孵化器与 5% CO2,在 DMEM 与谷氨酰胺补充含有10% 血清和1% 青霉素/链霉素。确保细胞在转染当日60% 汇合。在本实验中, 我们利用自制的 HEK293T 细胞来稳定地表达 SpCas9 来检测 pU6-sgRNA-Cbh-Cre。但是, 如果提供了 SpCas9 表达式向量, 则可以使用 wildtype HEK293T 单元格。</li>
		<li>染用聚乙烯亚胺 (PEI) 试剂 HEK293T 细胞。染 pCAG-EG-Top2b-FP 质粒12和120的 pU6-sgTop2b-1-Cbh-Cre 或 pU6-sgTop2b-2-Cbh-Cre 在24井板中的 120 ng/井的细胞。在80µL unsupplemented DMEM 培养基中用谷氨酰胺补充剂和1.8 µL, 在涡流后室温下进行15分钟混合。</li>
		<li>转染后6小时, 取出培养基, 用新鲜培养基替换。</li>
		<li>使用荧光显微镜在20x 放大后监测活 GFP+细胞的存在48小时。 注意: GFP+单元格的数量表明 sgRNA 的目标效率。有效和非有效的 sgRNAs (sgTop2b-1 和 sgTop2b-2 的结果分别) 见图 2A。</li>
	</ol>
</li>
</ol>3.在宫内电穿孔中进行
<ol><li>繁殖6到8周大的老鼠, 并准备在子宫电穿孔的工具。杂交雄性纯合Rosa26-CAG-LSL-Cas9-P2A-EGFP小鼠与雌性 CD-1 小鼠。时间怀孕的 CD-1 小鼠从当天阴道插头检测 (E0.5)。胚胎在 E13.5 天转。<ol>
<li>拉硼玻璃毛细血管 (内径: 0.6-0.8 毫米) 与微拉出。使用以下设置: P = 500, 热 = 560, 拉 = 150, 威 = 75, 时间 = 250。用黑色墨水标记毛细管尖端, 以便在注射过程中更好地可视化。用尺子和锋利的针镊子修剪显微镜下的毛细管锥度, 在长度约6毫米的情况下修剪笔尖。</li>
		<li>保持毛细血管的统一性, 保持实验的重现性。在修剪过程中创建单面斜面边缘。提示应尽可能尖锐, 以便于穿透子宫壁和避免组织损伤的胚胎。 注: 或者, 使用微磨床调整35°角度19。毛细血管可以储存在室温下, 在10厘米培养皿下无病原条件。</li>
		<li>高压釜手术器械。</li>
		<li>将 sgRNA 质粒在同等部位与 pT2K-IRES-Luc 混合, 最终浓度至少为每粒1µg/µL, 并以快速绿色 (最终浓度为 0.05%) 来着色质粒溶液。用无内毒素蒸馏水制备1% 快速绿色库存溶液, 通过0.22 µm 过滤器过滤, 从溶液中去除染料微粒。 注: pT2K IRES 的联合转染是可选的, 但允许在出生后使用生物发光成像识别可行的转动物。请参阅步骤3.5。准备足够数量的质粒混合 , 使怀孕小鼠的总数量作。每只老鼠使用大约15-20 µL 混合体 (12 胚)。立即进行手术。</li>
	</ol></li>
	<li>准备小鼠做手术。
	<ol>
<li>麻醉怀孕 CD-1 小鼠与异氟醚。在一个3-4 卷的异氟烷 (氧流速: 1.5 升/分) 的盒子中诱导麻醉, 并在手术通过鼻锥时保持2卷-%。 注: 完整手术不应超过30分钟, 如有必要, 减少注射和转胚胎的数量。使用无菌手套进行手术。</li>
		<li>将鼠标放在加热垫上, 调整麻醉。</li>
		<li>注射镇痛剂 (Metamizol, 800 毫克/千克体重, 皮下 (南卡罗来纳州), 24 h 车厂)。 注: 您可以使用其他授权止痛药比 Metamizol。</li>
		<li>在手术中应用眼膏防止眼部干燥。</li>
		<li>用胶带固定四肢, 用消毒剂消毒腹部。用纱布遮住老鼠, 只留下手术部位。在手术区和纱布上传播70% 乙醇。</li>
		<li>使皮肤切口长度约2厘米, 用直的手术剪刀轻轻地扩大间隙。用钝尖的组织剪刀定位 linea, 通过腹膜稍微缩小第二切口。</li>
		<li>用预预热的无菌 1x PBS 滋润开腹腔。</li>
		<li>用环钳仔细提取腹腔内的子宫角。轻轻地抓住子宫壁, 只在两个相邻胚胎的蛋黄囊之间的间隙。避免对胚胎的任何压力, 同时拉它通过切口。必要时扩大切口, 并格外小心, 将子宫角放到腹部, 同时不压缩血流通过子宫动脉。 注意: 在某些情况下, 建议一次提取一个子宫角, 并在继续第二个子宫角之前替换它。</li>
	</ol></li>
	<li>质粒 dna 的注射和电穿孔
	<ol>
<li>用制备的玻璃毛细管吸出大约15µL 的彩色质粒溶液。在这个过程中避免任何气泡。 注: 质粒溶液如果浓度高, 可以很容易堵塞尖端。在注射前释放一些 DNA 对毛细血管进行测试。</li>
		<li>用环钳轻轻地抱住胚胎, 找到颈部区域。 注: 如果胚胎处于难以注射的位置, 跳过它, 然后进入下一个胚胎。增加胚胎的重新定位可能会增加对其造成伤害的机会。</li>
		<li>慢慢刺穿前准备的玻璃毛细管的尖端进入第四脑室通过穿透背后脑, 随后注入约1µL 的质粒混合。确认染色的 DNA 不是从大脑中泄露出来的, 它是一个钻石形状的结构。 注: 作为一种选择, 使用2.5 倍放大镜, 以更好地可视化第四脑室和周围血管。注射后立即提供电脉冲, 以防止质粒溶液向其它心室扩散。</li>
		<li>应用电平方脉冲 (5 脉冲, 32 mV, 50 ms, 950 毫秒) 与钳状铂镊子 (见材料表)。将电极侧面与覆盖耳朵的负极和位于小脑普利摩顿的正杆置于子宫壁上。使用5毫米直径的电极板, 有效地电穿孔 E13.5 胚胎。 注: 通过操纵每座大坝的胚胎总数量少于 2/3路, 提高幼崽的存活率。避免胚胎离宫颈太近。如果控 , 他们可能会在分娩过程中造成并发症 , 并危及整个垃圾。如果电极太靠近胚胎的心脏, 这可能会导致心脏骤停。</li>
	</ol></li>
	<li>电穿孔术后护理
	<ol>
<li>小心地将子宫角放回腹腔, 填上预热后的无菌 1x PBS。让子宫角滑入一个自然的位置。</li>
		<li>用简单的连续缝合法分别闭合腹膜和皮肤。 注意: 在缝合腹膜时, 要格外小心, 不要刺穿子宫壁。</li>
		<li>关闭麻醉, 把动物腹部放到一个新的笼子里。</li>
		<li>在手术后, 在清醒阶段和24小时内对动物进行监测。把笼子放在暖气灯下, 如果颤抖在15分钟的时间内没有改善, 动物也不会开始梳理。</li>
		<li>确认成功的电穿孔荧光素酶成像。<ol>
<li>确保作的水坝按时下降胚胎 ( 在 E19 . 5 ) 。 注: 由于手术的缘故, 生日可能会推迟到第二天。</li>
			<li>麻醉转小鼠 (P5-P7) 与2.5% 异氟醚和注射 (腹腔 (ip)) 与 d-荧光素 (3 毫克/千克体重) 5 分钟之前的成像。</li>
			<li>使用生物发光成像仪检测转幼崽中的荧光素酶信号。 注: 1 分钟曝光时间足以检测荧光素酶信号。辐射 (p/秒/厘米2/sr) 大约 > 1 x 106。</li>
		</ol>
</li>
	</ol></li>
</ol>4. 准备 Cryosections 从转小脑
<ol><li>弄死转 P7 幼崽并解剖出小脑。 注意: 可以使用 epifluorescent 立体镜观察 GFP 信号。</li>
	<li>固定的小脑在5毫升每脑4% 粉煤灰在 PBS 4 摄氏度。</li>
	<li>将固定小脑一夜之间转移到10毫升每脑30% 蔗糖在 PBS 4 摄氏度。 注: 在蔗糖溶液孵化后, 组织应达到15毫升管的底部。</li>
	<li>用一张纤维素滤纸浸泡剩下的蔗糖溶液后, 将小脑浸泡在一次性塑料模具中的最佳切削温度 (OCT) 化合物中, 并将其冷冻在干冰上。低温块可以储存在-80 °c, 直到使用。 注意: 协议可以在这里暂停。</li>
	<li>使用恒温器将低温块切成10µm 的截面, 并将节保持在-80 摄氏度, 直到使用。 注: 在将 OCT 化合物与 PBS 清洗后, 可以在切片中确认 GFP 表达式。</li>
</ol>5. Cryosections 的染色
<ol><li>在室温下将幻灯片干燥30分钟, 在 PBS 中清洗两次, 10 分钟。</li>
	<li>用液体阻挡笔圈出切片, 并在室温下在阻塞溶液 (PBS 中含有 0.1% Triton-X100 的10% 正常驴子血清) 中孵化出1小时。</li>
	<li>在阻断溶液中添加对感兴趣分子 (例如GFP 和拓扑异构酶 II β (Top2B)) 的主要抗体, 并在4摄氏度的一夜之间孵化。 注: 所用抗体的名称和浓度列于材料表中。为了增强 gfp 信号, 抗 gfp 抗体可以选择与其他抗体一起使用。</li>
	<li>用含有 DAPI 的阻塞溶液中稀释的荧光共轭二次抗体, 在室温下将0.1% 的含海卫 PBST 3X 的切片冲洗成1小时。</li>
	<li>在 PBS 3X 中清洗幻灯片10分钟, 装入幻灯片并保持在4摄氏度直到成像。 注意: 协议可以在这里暂停。</li>
</ol>6. 成像和分析
<ol><li>使用共聚焦显微镜在20x 放大图像的部分。</li>
	<li>计算荧光蛋白阳性细胞的数量, 从而失去目标蛋白质的表达。图 2B显示了一个代表性图像。</li>
	<li>通过 immuostaining12检查大亚湾及其后代的靶基因消融后的分子表型。</li>
</ol>

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作者没有什么可透露的。

使用外子宫内电穿孔, 我们以前报告 siRNA在体内功能分析的 Atoh1 在早期的小脑颗粒细胞分化8。由于 siRNA 稀释/降解和子宫壁外的胚胎暴露, 转颗粒细胞的表型分析仅限于胚胎阶段。然而, 目前的方法能够分析产后动物的表型。
我们以前的研究表明,在宫内电穿孔中抑癌Ptch1的 Cas9-mediated 敲除成功诱导髓母细胞2。相比...

脑部疾病是最可怕的致命疾病之一。它们通常是由基因突变和随后的失调引起的。为了了解脑部疾病的分子机制, 不断努力破译人类的基因组, 发现了一些潜在的致病基因。到目前为止, 生殖基因工程动物模型已被用于体内增益功能 (GOF) 和丧失功能 (LOF) 分析这些候选基因。由于功能验证研究的加速发展, 一种更可行、更灵活的体内基因检测系统对基因功能的研究是可取的。
应用体内电穿孔基因转移系统对小鼠脑发育有较好的作用。事实上, 在子宫电穿孔中使用的几项研究显示了它们在发育中的大脑1、2、3进行功能分析的潜力。实际上, 老鼠大脑的几个区域, 如大脑皮层4, 视网膜5, 间脑6, 后脑7, 小脑8, 脊髓9已被体细胞基因交付的目标方法, 到目前为止。
事实上, 胚胎小鼠脑内电穿孔的瞬态基因表达长期以来一直被用于 GOF 分析。最近基于座子的基因组集成技术进一步启用了10、11感兴趣基因的长期和/或有条件表达, 这有利于在空间和时间上解剖基因功能, 在发展。与 GOF 分析相比, LOF 分析更具挑战性。在对 siRNAs 和 shRNA 质粒进行瞬态转染的同时, 由于外部引入的核酸 (如质粒和 dsRNAs) 的最终降解, 不能保证基因 LOF 的长期影响。然而, CRISPR/Cas 技术在 LOF 分析中提供了突破。基因编码的荧光蛋白 (如GFP) 或生物发光蛋白 (如萤火虫荧光素酶) 已与 CRISPR-Cas9 和 sgRNAs, 以标签的细胞暴露在 CRISPR-Cas9-mediated 体细胞突变。然而, 这种方法在增殖细胞的功能研究中可能有局限性, 因为外源标记基因在长期增殖后会被稀释和降解。当转染细胞及其子细胞在基因组中经历 CRISPR 诱导的突变时, 它们的脚印可能会随着时间而消失。因此, 基因标记方法适合于解决这一问题。
我们最近开发了一种基于 CRISPR 的 LOF 方法在小脑颗粒细胞中经历长期增殖在他们的分化12。为了对转染细胞进行基因标记, 我们构建了一种携带 sgRNA 的质粒, 并将质粒引入 Rosa26-CAG-LSL-Cas9-P2A-EGFP 小鼠13 在宫内电穿孔中的小脑。与常规质粒载体编码 EGFP 不同, 这种方法成功地标记了转染的颗粒神经元前体 (大亚湾) 及其子细胞。该方法在正常脑发育和肿瘤易发背景下对增殖细胞感兴趣基因的体内功能有很大的支持作用。

<table><tbody><tr><td>Alexa 488 Goat anti-Chicken</td><td>ThermoFisher</td><td>A11039</td><td>1:400 dilution</td></tr><tr><td>Alexa 568 Donkey anti-Mouse</td><td>Life Technologies</td><td>A-10037</td><td>1:400 dilution</td></tr><tr><td>Alexa 594 Donkey anti-Rabbit</td><td>ThermoFisher</td><td>A21207</td><td>1:400 dilution</td></tr><tr><td>Alexa 647 Donkey anti-Rabbit</td><td>Life Technologies</td><td>A31573</td><td>1:400 dilution</td></tr><tr><td>Alkaline Phosphatase (FastAP)</td><td>ThermoFisher</td><td>EF0654</td><td></td></tr><tr><td>Autoclave band</td><td>Kisker Biotech</td><td>150262</td><td></td></tr><tr><td>BamHI (HF)</td><td>NEB</td><td>R3136S</td><td></td></tr><tr><td>BbsI (FastDigest)</td><td>ThermoFisher</td><td>FD1014</td><td></td></tr><tr><td>Cellulose Filter Paper (Whatman)</td><td>Sigma-Aldrich</td><td>WHA10347525</td><td></td></tr><tr><td>Cloth</td><td>Tork</td><td>530378</td><td></td></tr><tr><td>Confocal laser scanning microscope</td><td>Zeiss</td><td>LSM800</td><td></td></tr><tr><td>D-Luciferin</td><td>biovision</td><td>7903-1</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td>1:1000 dilution</td></tr><tr><td>Disposable plastic molds (Tissue-Tek Cyromold)</td><td>VWR</td><td>4566</td><td></td></tr><tr><td>DMEM Glutamax</td><td>ThermoFisher</td><td>31966047</td><td></td></tr><tr><td>Donkey serum</td><td>Sigma-Aldrich</td><td>D9663</td><td></td></tr><tr><td>EcoRI (HF)</td><td>NEB</td><td>R3101S</td><td></td></tr><tr><td>Electro Square Porator</td><td>BTX</td><td>ECM830</td><td></td></tr><tr><td>Endofree Maxi Kit</td><td>Qiagen</td><td>12362</td><td></td></tr><tr><td>Ethanol</td><td>Merck</td><td>107017</td><td></td></tr><tr><td>Eye ointment (Bepanthen)</td><td>Bayer</td><td>81552983</td><td></td></tr><tr><td>Fast Green</td><td>Merck</td><td>104022</td><td></td></tr><tr><td>FBS</td><td>ThermoFisher</td><td>10270-016</td><td></td></tr><tr><td>Filter (0.22 &amp;micro;m)</td><td>Merck</td><td>F8148</td><td></td></tr><tr><td>Fluorescent cell imager (ZOE)</td><td>Biorad</td><td>1450031</td><td></td></tr><tr><td>Forceps straight</td><td>Fine Science Tools</td><td>91150-20</td><td></td></tr><tr><td>Gauze (X100 ES-pads 8f 10 x 10 cm)</td><td>Fisher Scientific</td><td>15387311</td><td></td></tr><tr><td>GFP antibody</td><td>Abcam</td><td>ab13970</td><td>1:1000 dilution</td></tr><tr><td>Gibson Assembly Master Mix</td><td>NEB</td><td>E2611S</td><td></td></tr><tr><td>Glass Capillary with Filament</td><td>Narishige</td><td>GD1-2</td><td></td></tr><tr><td>Heating Pad</td><td>ThermoLux</td><td>463265 / -67</td><td></td></tr><tr><td>Image Processing software (ImageJ and Fiji)</td><td>NIH</td><td>-</td><td></td></tr><tr><td>Insulin syringe (B. Braun OMNICAN U-100)</td><td>Carl Roth</td><td>AKP0.1</td><td></td></tr><tr><td>Isoflurane</td><td>Zoetis</td><td>TU061219</td><td></td></tr><tr><td>IVIS Lumina LT Series III Caliper</td><td>Perkin Elmer</td><td>CLS136331</td><td></td></tr><tr><td>Kalt Suture Needles</td><td>Fine Science Tools</td><td>12050-02</td><td></td></tr><tr><td>KAPA HIFI HOTSTART READY mix</td><td>Kapa Biosystems</td><td>KK2601</td><td></td></tr><tr><td>Ki67 antibody</td><td>Abcam</td><td>ab15580</td><td>1:500 dilution</td></tr><tr><td>Light Pointer</td><td>Photonic</td><td>PL3000</td><td></td></tr><tr><td>Liquid blocker pen</td><td>Kisker Biotech</td><td>MKP-1</td><td></td></tr><tr><td>Metamizol</td><td>WDT</td><td>-</td><td></td></tr><tr><td>Microgrinder</td><td>Narishige</td><td>EG-45</td><td></td></tr><tr><td>Microinjector</td><td>Narishige</td><td>IM300</td><td></td></tr><tr><td>Micropipette Puller</td><td>Sutter Instrument Co.</td><td>P-97</td><td></td></tr><tr><td>Microscope software ZEN</td><td>Zeiss</td><td>-</td><td></td></tr><tr><td>Non-sterile Silk Suture Thread (0.12 mm)</td><td>Fine Science Tools</td><td>18020-50</td><td></td></tr><tr><td>O.C.T. Compound (Tissue-Tek)</td><td>VWR</td><td>4583</td><td></td></tr><tr><td>p27 antibody</td><td>BD bioscience</td><td>610241</td><td>1:200 dilution</td></tr><tr><td>Paraformaldehyde</td><td>Roth</td><td>335.3</td><td></td></tr><tr><td>PBS (1x)</td><td>Life Technologies</td><td>14190169</td><td></td></tr><tr><td>pCAG-EGxxFP</td><td>Addgene</td><td>50716</td><td></td></tr><tr><td>Polyethylenimine</td><td>Sigma-Aldrich</td><td>408727</td><td></td></tr><tr><td>pX330 plasmid</td><td>Addgene</td><td>42230</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td></td></tr><tr><td>Quick Ligation Kit</td><td>NEB</td><td>M2200S</td><td></td></tr><tr><td>Ring Forceps</td><td>Fine Science Tools</td><td>11103-09</td><td></td></tr><tr><td>Slides (SuperFrost)</td><td>ThermoFisher</td><td>10417002</td><td></td></tr><tr><td>Software for biostatistics (Prism 7)</td><td>GraphPad Software, Inc</td><td>-</td><td></td></tr><tr><td>Spitacid</td><td>EcoLab</td><td>3003840</td><td></td></tr><tr><td>Stereomicroscope</td><td>Nikon</td><td>C-PS</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S5016</td><td></td></tr><tr><td>Surgical scissors</td><td>Fine Science Tools</td><td>91460-11</td><td></td></tr><tr><td>Surgical scissors with blunt tip</td><td>Fine Science Tools</td><td>14072-10</td><td></td></tr><tr><td>Suture (Supramid schwarz DS 16, 1.5 (4/0))</td><td>SMI</td><td>220340</td><td></td></tr><tr><td>T4 DNA Ligation Buffer</td><td>NEB</td><td>B0202S</td><td></td></tr><tr><td>T4 PNK</td><td>NEB</td><td>M0201S</td><td></td></tr><tr><td>Tissue scissors Blunt (11.5 cm)</td><td>Fine Science Tools</td><td>14072-10</td><td></td></tr><tr><td>TOP2B antibody</td><td>Santa Cruz</td><td>sc13059</td><td>1:200 dilution</td></tr><tr><td>Trypsin (2.5 %)</td><td>ThermoFisher</td><td>15090046</td><td></td></tr><tr><td>Tweezers w/5mm &amp;Oslash; disk electrodes Platinum</td><td>Xceltis GmbH</td><td>CUY650P5</td><td></td></tr><tr><td>Vaporizer</td><td>Dr&amp;auml;gerwerk AG</td><td>GS186</td><td></td></tr></tbody></table>

crispr-mediated loss of function analysis in cerebellar granule cells using in utero electroporation-based gene transfer

大脑畸形通常是由基因突变引起的。破译患者衍生组织中的突变, 确定了疾病的潜在致病因素。为了验证突变基因功能失调对疾病发展的贡献, 携带突变的动物模型的生成是一个明显的方法。虽然生殖基因工程小鼠模型 (GEMMs) 是流行的生物工具和展示重现性的结果, 它受到时间和成本的限制。同时, 非生殖 GEMMs 往往能更可行地探索基因功能。由于某些脑部疾病 (如脑肿瘤) 似乎是由躯体而不是生殖突变引起的, 非生殖嵌合体小鼠模型, 正常和异常细胞共存, 可能有助于疾病相关的分析。本研究报告了一种诱导小脑 CRISPR 介导的躯体突变的方法。具体地说, 我们利用条件敲鼠, 其中Cas9和GFP是长期激活的CAG (CMV 增强/鸡ß肌动蛋白) 启动后的基因组重组。自行设计的单导 rna (sgRNAs) 和 recombinase 序列, 都编码在一个单一质粒结构, 被交付到小脑茎/祖细胞在胚胎阶段使用的子宫电穿孔。因此, 转染细胞及其子细胞被标记为绿色荧光蛋白 (GFP), 从而促进进一步的表型分析。因此, 该方法不仅显示了以电穿孔为基础的基因传递到胚胎小脑细胞, 而且还提出了一种新的定量方法来评估 CRISPR 介导的丧失功能表型。

在体内功能分析中, 发现外源基因的细胞被引入是至关重要的。当一个标记的表达, 如 GFP 在非增殖细胞可以长期随访时, 信号在增殖细胞中依次消失。图 1显示了这种效果的说明。为了规避在 LOF 分析中丢失转染细胞的足迹, 我们开发了一种新的方法, 将电穿孔基因的传递与 CRISPR/Cas9 技术相结合。
有...

Watch this Scientific Journal Video about CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer at JoVE.com

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

传统的功能丧失基因的研究使用挖空动物往往是昂贵和耗时。基于电穿孔的 CRISPR 介导体细胞诱变是了解体内基因功能的有力工具。在这里, 我们报告了一个方法来分析的淘汰赛表型的小脑增殖细胞。

CRISPR 介导的小脑颗粒细胞在宫内电穿孔基因转移中的功能分析损失

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

crispr-mediated-loss-function-analysis-cerebellar-granule-cells-using

Research

JoVE Journal

Developmental Biology

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

9.4K 次观看.  German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance. 传统的功能丧失基因的研究使用挖空动物往往是昂贵和耗时。基于电穿孔的 CRISPR 介导体细胞诱变是了解体内基因功能的有力工具。在这里, 我们报告了一个方法来分析的淘汰赛表型的小脑增殖细胞。

视频: CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

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 Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

在子宫内的电穿孔其次原发性神经文化为研究基因的功能在皮层神经元的一个子集

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

神经科学

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

鸡小脑切片的体外培养和空间上有针对性的颗粒细胞前体电穿孔

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

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 ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

通过基于电穿孔的质粒 DNA 递送进行体内脑肿瘤建模

Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures

Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in immunotherapy, it is even more critical to have a consistent, clinically pertinent, immunocompetent mouse model to examine the tumor and immune cell populations in the brain and their response to treatment. While most preclinical models utilize orthotopic transplantation of established tumor cell lines, the modeling system presented here allows for a "personalized" representation of patient-specific tumor mutations in a gradual, yet effective development from DNA constructs inserted into dividing neural precursor cells (NPCs) in vivo. DNA constructs feature the mosaic analysis with the dual-recombinase-mediated cassette exchange (MADR) method, allowing for single-copy, somatic mutagenesis of driver mutations. Using newborn mouse pups between birth and 3 days old, NPCs are targeted by taking advantage of these dividing cells lining the lateral ventricles. Microinjection of DNA plasmids (e.g., MADR-derived, transposons, CRISPR-directed sgRNA) into the ventricles is followed by electroporation using paddles that surround the rostral region of the head. Upon electrical stimulation, the DNA is taken up into the dividing cells, with the potential of integrating into the genome. The use of this method has successfully been demonstrated in developing both pediatric and adult brain tumors, including the most common malignant brain tumor, glioblastoma. This article discusses and demonstrates the different steps of developing a brain tumor model using this technique, including the procedure of anesthetizing young mouse pups, to microinjection of the plasmid mix, followed by electroporation. With this autochthonous, immunocompetent mouse model, researchers will have the ability to expand preclinical modeling approaches, in efforts to improve and examine efficacious cancer treatment.

作者聚焦:使用基于电穿孔的质粒 DNA 递送代表患者突变特征的体内脑肿瘤建模  

Utilizing an immunocompetent, autochthonous tumor model driven by common patient mutations for preclinical testing is critical for immunotherapeutic testing. This protocol describes a method to generate brain tumor mouse models using electroporation-based delivery of plasmid DNA that represent common patient mutations, thus providing an accurate, reproducible, and consistent mouse model.

使用基于电穿孔的质粒DNA递送对 体内 脑肿瘤进行建模，以代表患者突变特征

Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in ...

癌症研究

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of intellectual disability and account for 20-40% of drug-resistant epilepsy in childhood. High-resolution brain imaging has facilitated in vivo identification of a large group of MCD phenotypes. Despite the advances in brain imaging, genomic analysis and generation of animal models, a straightforward workflow to systematically prioritize candidate genes and to test functional effects of putative mutations is missing. To overcome this problem, an experimental strategy enabling the identification of novel causative genes for MCD was developed and validated. This strategy is based on identifying candidate genomic regions or genes via array-CGH or whole-exome sequencing and characterizing the effects of their inactivation or of overexpression of specific mutations in developing rodent brains via in utero electroporation. This approach led to the identification of the C6orf70 gene, encoding for a putative vesicular protein, to the pathogenesis of periventricular nodular heterotopia, a MCD caused by defective neuronal migration.

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo.

一种新的结合阵列全息、全外测序和宫内电穿孔的方法来识别脑畸形的致病基因

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of ...

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

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 do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.

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

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in vivo.

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 ...

CRISPR 介导的小脑颗粒细胞在宫内电穿孔基因转移中的功能分析损失

摘要

探索更多视频

此视频中的章节

诱导蛋白缺失的通过

在子宫内的电穿孔其次原发性神经文化为研究基因的功能在皮层神经元的一个子集

鸡小脑切片的体外培养和空间上有针对性的颗粒细胞前体电穿孔

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

小脑颗粒神经元的遗传操作

使用基于电穿孔的质粒DNA递送对体内脑肿瘤进行建模，以代表患者突变特征

使用基于电穿孔的质粒DNA递送对体内脑肿瘤进行建模，以代表患者突变特征

使用基于电穿孔的质粒DNA递送对体内脑肿瘤进行建模，以代表患者突变特征

一种新的结合阵列全息、全外测序和宫内电穿孔的方法来识别脑畸形的致病基因

宫内电穿孔诱导小鼠大脑皮质中氧合酶的重组

CRISPR 介导的小脑颗粒细胞在宫内电穿孔基因转移中的功能分析损失

摘要

探索更多视频

此视频中的章节

诱导蛋白缺失的通过

在子宫内的电穿孔其次原发性神经文化为研究基因的功能在皮层神经元的一个子集

鸡小脑切片的体外培养和空间上有针对性的颗粒细胞前体电穿孔

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

小脑颗粒神经元的遗传操作

使用基于电穿孔的质粒DNA递送对 体内 脑肿瘤进行建模，以代表患者突变特征

使用基于电穿孔的质粒DNA递送对 体内 脑肿瘤进行建模，以代表患者突变特征

使用基于电穿孔的质粒DNA递送对 体内 脑肿瘤进行建模，以代表患者突变特征

一种新的结合阵列全息、全外测序和宫内电穿孔的方法来识别脑畸形的致病基因

宫内电穿孔诱导小鼠大脑皮质中氧合酶的重组

使用基于电穿孔的质粒DNA递送对体内脑肿瘤进行建模，以代表患者突变特征

使用基于电穿孔的质粒DNA递送对体内脑肿瘤进行建模，以代表患者突变特征

使用基于电穿孔的质粒DNA递送对体内脑肿瘤进行建模，以代表患者突变特征