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>

<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。相比之下, 目前的方法有两大优点: 1) Cas9 不需要与质粒一起交付, 允许多种基因靶向通过携带多个 sgRNA 表达盒在一个单一的质粒代替;和 2) 目标细胞及其后代被永久标记为 GFP, 使活细胞可视化, 并分析在体内转化肿瘤细胞的行为。
本议定书最关键的步骤是适当地将质粒注入到正确的位置, 并将电脉冲传送到大脑中适当的地方。小脑神经元是出生于小脑 neuroepithelium 按出生日期依赖性的方式。并非所有类型的小脑普利摩顿的细胞都可以通过电穿孔的目标, 因为只有一个特定的时间窗口在 E12.5-14.5 是技术上可行的。深小脑核神经元和浦肯野细胞发生在 E10.5-11.5, 当额外的胚胎膜是不透明的, 胚胎是不清楚地可见的 DNA 注射。后来, 单极刷细胞来源于 E17.5 上 RL (url), 当第四脑室太窄, 无法在 uRL 附近注入足够数量的 DNA。因此, 我们的方法仅适用于 E12.5-14.5, 主要针对中后期出生的祖细胞, 如大亚湾和抑制中间神经元。该方法的另一个缺点是, 当与 LoxP 介导的生殖 GEMMs 相比, 完全挖空表型可能是不可行的, 因为只有数以千计的小脑细胞可以被电穿孔在每个胚胎的靶向。
在早期的研究中, 大约80% 的 GFP 阳性的小脑颗粒细胞失去了目标基因12的表达。虽然分子标记及其分布证实了颗粒细胞的特性, 但这种识别方法可能并不总是适用的, 因为感兴趣的基因可以参与标记表达和神经元迁移。使用特定于细胞的启动子进行条件激活, 可以解决这种情况下的问题。因此, pU6-sgRNA-Cbh-Cre 质粒中目前无处不在的 Cbh 启动子可以用细胞特异启动子代替。

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 &#181;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 &#216; disk electrodes Platinum</td>
			<td>Xceltis GmbH</td>
			<td>CUY650P5</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaporizer</td>
			<td>Dr&#228;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。
有代表性的结果如图 2所示。质粒结构的功能测试通过瞬变转染 pU6-sgTop2b-Cbh-Cre 和 pCAG-EG-Top2b-FP17, 携带 sgTop2b 目标序列, 进入 HEK293T 细胞稳定表达 SpCas9 (图 2A)。sgRNA 制导 Cas9 限制性活动诱导 EGFP 表达盒的同源定向修复。因此, 通过对 GFP 表达的检测, 直接分析了 sgRNA 的作用。根据 Mashiko 等, 30% 以上的转染效率决定了 sgRNA 的有效性17。
大亚湾起源于菱形唇 (RL), 这是一个与第四脑室顶部毗邻的 E12.5 胚胎阶段的区域, 直到 E16.5。已知这些细胞在外外部颗粒层 (oEGL) 出生后会发生大量增殖, 并在退出细胞周期20后转移到内 EGL (研究所)。因此, 大亚湾是一个适当的例子来检验我们的基因标记方法的优势。
通过遵循这个协议,在子宫内电穿孔的大亚湾允许追踪的编辑细胞与 GFP。我们介绍了 pU6-sgRNA-Cbh-Cre 质粒表达 sgControl 的小脑普利摩顿Rosa26-CAG-LSL-Cas9-P2A-EGFP小鼠在胚胎阶段 E13.5。小鼠在产后 P7 的时候被牺牲, 小脑组织切片的矢状切片被免疫组化染色。外部和内部 EGL 分别由 Ki67 表达式 (oEGL) 和 p27 表达式 (研究所) 定义。尽管有增殖和成熟, 成熟的小脑颗粒神经元 (CGNs) 仍然保留强烈的 GFP 表达 (图 2B)。
在强调本议定书的效用的同时, 我们还利用 sgRNA 靶向 DNA Top2b 作为一个代表性的例子。实验过程如上文所述 (见图 2B)。大多数的 GFP 阳性细胞转染 sgRNAs 为Top2b显示 Top2b 表达在内部颗粒层 (IGL), 而引入控制 sgRNAs 并没有导致明确减少其表达 (图 2C)。图 2D显示了此结果的量化。这些数据为在发育或肿瘤形成过程中长时间追踪编辑过的细胞提供了宝贵的工具。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57311/57311fig1.jpg"> 图 1: 在增殖和非增殖细胞中 GFP 表达的示意图.(A) 当一种外源基因编码gfp (例如, pCAG-EGFP) 在非增殖细胞中转染时, 细胞可能长时间 (上车道) 表达 gfp。同时, 由于外源gfp (中间车道) 的稀释, gfp 表达可能消失于增殖细胞中。相反, 携带LoxP-LoxP-gfp (LSL gfp) 转基因的细胞可以保持 gfp 在增殖过程中, 在基因转染后, recombinase (下巷)。(二) 在 Rosa26-CAG-LSL-Cas9-P2A-EGFP 小鼠 sgRNA 转染后, SpCas9-mediated 基因沉默和 GFP 表达的原则。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57311/57311fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57311/57311fig2.jpg"> 图 2: 本议定书结果的代表性图像.(A) HEK293T 细胞稳定表达 SpCas9, 转染 pU6-sgTop2b-Cbh-Cre 和 pCAG-EG-Top2b-FP 质粒。用荧光细胞成像仪 (见材料表) 对活细胞中 GFP 的表达进行监测, 转染后48小时。左、右面板分别显示了基于 GFP 表达式的有效和非有效的 sgRNA 序列。刻度条: 100 µm (B) 染色的 GFP, Ki67 和 p27 P7 小脑小鼠在 Rosa26-CAG-LSL-Cas9-P2A-EGFP 的电穿孔 E13.5 与 pU6-sgRNA-Cbh-Cre 质粒结构表示控制 sgRNA。该部分与 DAPI (蓝色) 复染。刻度杆:50 µm;20x 放大倍数。注意 GFP 表达的细胞在 oEGL 标记的 Ki67。(C) 在 Rosa26-CAG-LSL-Cas9-P2A-EGFP 转小鼠 P7 小脑上染色的 GFP (绿色) 和 Top2b ( 洋红) 的 E13.5 与 pU6-sgRNA-Cbh-Cre sgRNA 的质粒结构表示 Top2b (sgTop2b) 和控制序列 (sgControl)。箭头指示在同一字段中 GFP+单元格中的 Top2b 表达式。刻度杆:20 µm;20x 放大倍数。(D) 在 sgControl 和 sgTop2b 实验中定量测定转 (GFP 阳性) 细胞中的 Top2b。分别对三只小鼠进行了 sgControl 和 sgTop2b 的研究, 并对各脑25个细胞进行了分析。误差条表示 "平均值" 标准误差 (SEM), p值由不配对 t检验计算, *p = 0.0002。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57311/57311fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>名字</td>
			<td colspan="2">序列</td>
			<td>应用</td>
		</tr>
<tr>
<td>sgTop2b-1</td>
			<td colspan="2">CTTCGTCCTGATACATACAT</td>
			<td>sgRNA 目标序列</td>
		</tr>
<tr>
<td>sgTop2b-2</td>
			<td colspan="2">AGCTGTCCAAAAATTAAAGC</td>
			<td>sgRNA 目标序列</td>
		</tr>
<tr>
<td>sgControl</td>
			<td colspan="2">GCGACCAATACGCGAACGTC</td>
			<td>sgRNA 目标序列</td>
		</tr>
<tr>
<td>EGxxFP-Top2b-F</td>
			<td colspan="2">gctgcccgacaaccactgagTACCTTGATATCTTAGAGAGCTG</td>
			<td>克隆成 pCAG-EGxxFP</td>
		</tr>
<tr>
<td>EGxxFP-Top2b-R</td>
			<td colspan="2">gggtcagcttgccgatatcgCTCGCGCATTGTCTTAGC</td>
			<td>克隆成 pCAG-EGxxFP</td>
		</tr>
<tr>
<td>hU6-F</td>
			<td colspan="2">GAGGGCCTATTTCCCATGATT</td>
			<td>sgRNA 测序</td>
		</tr>
</tbody></table>表 1: 本研究中使用的寡核苷酸序列。

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

Nanyang Technological University

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

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

科研

发育生物学

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 ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

损失 - 和增益的功能角度进行调查研究早期细胞命运决定在植入前小鼠胚胎

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

基因转移到鸡听觉器官通过<em&gt;在卵</em&gt;微电

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

在Active ERK的时空分析<em&gt;℃。线虫</em&gt;生殖系

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological studies to depict intricate morphologies and to manipulate the function of genes in subsets of neurons within otherwise unmarked and unperturbed organisms. Genetic mosaics generated in the MARCM system are mediated through site-specific recombination between homologous chromosomes within dividing precursor cells to produce both marked (MARCM clones) and unmarked daughter cells during mitosis. An extension of the MARCM method, called twin-spot MARCM (tsMARCM), labels both of the twin cells derived from a common progenitor with two distinct colors. This technique was developed to enable the retrieval of useful information from both hemi-lineages. By comprehensively analyzing different pairs of tsMARCM clones, the tsMARCM system permits high-resolution neural lineage mapping to reveal the exact birth-order of the labeled neurons produced from common progenitor cells. Furthermore, the tsMARCM system also extends gene function studies by permitting the phenotypic analysis of identical neurons of different animals. Here, we describe how to apply the tsMARCM system to facilitate studies of neural development in Drosophila.

Here, we present a protocol for a mosaic labeling technique that permits the visualization of neurons derived from a common progenitor cell in two distinct colors. This facilitates neural lineage analysis with the capability of birth-dating individual neurons and studying gene function in the same neurons of different individuals.

细胞谱系分析和基因功能研究使用双点MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological ...

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

在小鼠胚胎干细胞结构功能研究使用重组酶介导的盒式交换

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Pax7 和层粘连蛋白抗体免疫荧光法鉴定骨骼肌卫星细胞

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...