Name: tracing gene expression through detection of β-galactosidase activity in whole mouse embryos
Uploaded: 2018-06-26T14:00:00.000+00:00
Duration: 8 min 42 s
Description: Watch this Scientific Journal Video about Tracing Gene Expression Through Detection of Î²-galactosidase Activity in Whole Mouse Embryos at JoVE.com

我们要感谢病理学服务在 Investigaciones Cardiovasculares (CNIC) 中心的技术援助。我们还感谢 Motoharu 博士精机为 Mt4-mmpLacZ小鼠和阿罗约博士提供支持我们的项目和她对手稿的批判性阅读。我们要感谢彼得邦尼对这篇文章的校对。这项工作得到马德里大学欧洲大学的支持, 2017UEM01 授予 C.S.C。

所有实验程序都得到了 CNIC 动物实验伦理学委员会 (Investigaciones Cardiovasculares) 和马德里 Comunidad 国立的批准, 以确保最小的动物痛苦。
1. 从怀孕小鼠中采集胚胎 (从 E8.5 到 E12.5)
<ol><li>通过宫颈脱位或一氧化碳2吸入来牺牲怀孕的老鼠。第一个被观察的阴道插头的天被考虑了胚胎天 0.5 (E0.5)。</li>
	<li>将动物放在吸水垫的仰卧位上, 用70% 乙醇清洁老鼠的腹部皮肤。 注意: 以下步骤不要求不育。</li>
	<li>用小鼠牙钳捏起腹部皮肤。</li>
	<li>切口通过皮肤和腹壁, 2 到4厘米垂直横跨中线的腹部使用手术剪刀。</li>
	<li>用手术剪刀将子宫内弯曲的血管切开, 从母亲身上取出子宫角。</li>
	<li>去掉胚胎的字符串, 将其转移到含有10毫升冰冷0.01 米磷酸盐缓冲盐水 pH 值 7.4 (PBS) 的冰上的培养皿中。</li>
	<li>仔细解剖胚胎从子宫下的显微镜在一个培养皿与10毫升新鲜冰冷 PBS。用制表钳抓住肌壁以暴露羊膜囊, 然后用镊子的提示撕去羊膜以除去封闭的胚胎和蛋黄囊。</li>
	<li>从怀孕小鼠早期 (从 E8.5 到 E12.5) 收集 littermate 胚胎 (图 2)。</li>
	<li>用弯曲钳将胚胎移植到10毫升冷 1x PBS 的新鲜菜肴上, 或者, 对于非常小的胚胎 (E8.5-E9.5), 使用塑料转移吸管收集没有胚胎损伤的样品。</li>
	<li>在开始固定之前, 在离心管中取一个胚胎样本进行基因分型, 方法是切割一小部分胚胎, 或将最小胚胎中的羊膜 (从 E8.5 到 E9.5) 与制表钳结合。 注: LacZ 基因分型由聚合酶链反应 (PCR) 引物确定: 向前, 5´-ATCGTGCGGTGGTTGAACTG 3´;反转, 5´-CGAGGCGTTAACCGTCAGCA-3´。</li>
</ol>2. 小鼠胚胎的固定
<ol><li>将每个胚胎移植到一个2毫升的离心管中, 其中包含1毫升 1x PBS 的圆形底座。 注意: 使用滚动振动筛在这些管道中执行所有步骤。</li>
	<li>在室温下将 1x PBS 中的胚胎洗净1分钟, 以消除剩余的血液。使用塑料转移吸管改变管中的液体。</li>
	<li>在冰上固定1毫升0.125% 戊二醛的胚胎。 注: 固定时间取决于胚的大小: E8.5 E9.5 胚20分钟, E10.5 胚30分钟, E12.5 胚1小时。</li>
	<li>在处理 X-加仑染色样品前, 取出固定剂并在 1x PBS 中清洗两次, 室温为10分钟。</li>
</ol>3. 小鼠胚胎全芒乳糖酶组织化学研究
<ol><li>在开始操作之前, 准备好 x-加仑冲洗缓冲液和 x-gal 染色溶液 (见表 1和材料表)。使用野生型 (littermate) 胚胎作为对酶反应的阴性对照。</li>
	<li>在室温下以10分钟的温度 (图 2) 在 X-加仑中孵化胚胎。</li>
	<li>在 X-加仑染色溶液中孵化胚胎, 从2小时到隔夜, 在37摄氏度保护免受光照。通过解剖显微镜定期监测颜色反应, 直到在没有胚胎背景的情况下观察到足够的蓝色染色强度。保持溶液 pH 值介于8和9之间, 以减少整个染色过程中的背景。如果染色溶液开始变亮, 用新鲜的基质溶液取代, 以延长酶的反应。</li>
	<li>当所需信号通过在室温下每两次1毫升 1x PBS 的离心管中的10分钟内洗涤时, 停止反应。</li>
	<li>将染色的胚胎转移到1毫升4% 多聚甲醛 (粉煤灰) 重新修复, 1 小时至一夜4摄氏度。 注: 胚胎可贮存在4% 粉煤灰/PBS 中, 较长时间为4摄氏度, 或可立即加工切片。这一最后的固定步骤是长期保存染色模式的关键。</li>
	<li>在室温下, 将1毫升 1x PBS 中的胚胎清洗两次, 10 分钟。</li>
	<li>备选方案 1: 通过浸泡在一系列解决方案中, 清除胚胎, 增加甘油浓度 (20%、40%、60% 和80% 甘油 1x PBS), 每种溶液室温下为1小时。 注: 在80% 甘油中洗涤胚胎, 时间长, 甚至数天, 直到清除, 然后在80% 甘油4摄氏度储存在这个步骤 (图 2)。建议在甘油或 1x PBS 中添加极少量的叠氮化钠麝香晶体, 以防止霉菌生长。清除后, 全芒胚可用于拍照 (步骤 4)。</li>
	<li>选项 2: 使用切片进行石蜡嵌入和切片的加工胚 (图 2)。在切片前记录整个染色胚的表达模式 (步骤4和 5)。</li>
</ol>4. 全芒胚摄影
<ol><li>要在切片前拍摄全芒染色的胚胎, 将其转移到一个培养皿中, 并在 1x PBS 中固化1-2% 琼脂糖。</li>
	<li>完全覆盖胚胎与10毫升 1x PBS 在琼脂糖涂层的菜肴, 以防止脱水和反射, 而在拍摄的液体。</li>
	<li>用镊子将胚胎浸入 1x PBS 中。对于年长的胚胎 (E10.5-E12.5), 用镊子在琼脂糖中做一个小孔, 以不同的角度放置和放置标本, 避免摄影过程中的自由漂浮。</li>
	<li>将盘子放在显微镜台上, 使用透射 (下) 光。改变 "黑暗场" 和 "光明场" 的调整, 以设定在摄影过程中所需的照明和优化样品的半透明性。 注: 对后期胚胎使用光纤照明, 以避免胚胎表面的反射。当拍摄被清除的胚胎, 遵循相同的程序, 但转移到一个含有100% 甘油的培养皿, 并充分浸泡他们。</li>
</ol>5. X-加仑染色胚的石蜡嵌入和切片
<ol><li>石蜡嵌入<ol>
<li>从4°c (步骤 3.5) 中取出 X-加仑染色的胚胎, 并用1毫升 1x PBS 清洗它们, 以消除多余的固定剂。</li>
		<li>用镊子将每个胚胎移植到组织学盒中。</li>
		<li>将含有胚胎的卡带放入烧杯中, 然后在室温下通过分级乙醇系列将其脱水: 70% 乙醇, 一次为30分钟;96% 乙醇, 每两次30分钟;100% 乙醇, 每两次30分钟。 注: 胚胎可以储存在70% 乙醇的不确定时间在4摄氏度, 直到开始脱水过程。</li>
		<li>在室温下, 在异丙醇上孵化整盒30分钟。</li>
		<li>使用镊子, 将盒式磁带转移到含有预热异丙醇的玻璃染色槽中, 并在烤箱中留出60摄氏度, 30 分钟。</li>
		<li>将卡带放在一个新的玻璃染色槽中, 其中含有50% 异丙醇/50% 熔融石蜡, 并在60摄氏度烤箱中离开4小时。</li>
		<li>用两个熔融石蜡变化的胚胎孵化盒, 每30分钟60摄氏度。</li>
		<li>最后石蜡冲洗结束后, 打开卡带, 将每个胚转移到一个单独的组织嵌入模具中, 以查看显微镜下的样品。</li>
		<li>用60摄氏度的熔融石蜡填充井。使用加热钳保持蜡熔化, 并仔细定位在模具的胚胎。 注意: 样品的正确方向是在所需平面切片胚胎的关键步骤。</li>
		<li>在霉菌凝固之前, 放置一个没有盖子的盒式卡带, 用熔融石蜡填充。让剩余的蜡冷却, 直到一夜之间室温凝固。</li>
		<li>石蜡完全固化后, 从模具中取出实心块, 在室温或4摄氏度前将石蜡块贮存至切片14。</li>
	</ol>
</li>
	<li>石蜡切片<ol>
<li>将刀片定向到切片上, 并设置5-7 µm 的厚度。5.2.2. 冷却冰上的石蜡块, 修剪它以产生立方体或金字塔。</li>
		<li>使用少量的熔融蜡将块连接到切片的支架上。</li>
		<li>将块定向到最佳切割。用标准切片在室温下将石蜡块分成5-7 µm 厚片。</li>
		<li>使用精美的画笔, 将部分放置在室温下的水浴中, 用于操作和选择切片。</li>
		<li>从水浴中拿起显微镜滑动的部分, 并将它们转移到42摄氏度的水浴中伸展。</li>
		<li>从水浴中移除部分, 然后轻轻地将它们放到粘附显微镜幻灯片上。</li>
		<li>干燥的幻灯片在一个烤箱在37°c 过夜, 使各部分完全坚持, 否则, 他们可能会在染色过程中丢失。 注: 将幻灯片保存在4摄氏度, 直到开始染色程序。</li>
	</ol>
</li>
	<li>Deparaffinization 和 Counterstaining 染色石蜡切片的研究<ol>
<li>将幻灯片放在显微镜下, 在60摄氏度的烤箱中, 至少45分钟, 使石蜡蜡更流畅。</li>
		<li>将幻灯片转移到机架上, 并使用玻璃染色碟执行以下步骤: 将机架浸入含有减少浓度醇的染色罐中, 以完成石蜡去除和组织水化: 5 分钟的异丙醇60°c;异丙醇3分钟;100% 乙醇2分钟;96% 乙醇1分钟;70% 乙醇在室温下1分钟。</li>
		<li>将蒸馏水中的部分冲洗两次, 以确保没有酒精残留物。</li>
		<li>Counterstain 部分与污点 (例如核快速的红色解答) 为5分钟 (通常在3-8 分钟之间) 在室温 (图 2)。 注意: 这种染色有助于揭示切片的整体组织结构。</li>
		<li>染色后, 将蒸馏水中的滑梯清洗1分钟, 并在显微镜下检查切片染色。 注: 如果所需染色太弱, counterstain 节再长一段时间。</li>
		<li>脱水部分在以下系列以增加的乙醇的浓度: 二洗涤在95% 乙醇为2分钟每, 二洗涤在100% 乙醇为2分钟每, 并且, 为了避免溶化蓝色沉淀, 仅一个快速洗涤在二甲苯。</li>
		<li>将幻灯片从机架中一个一个地取出, 然后放在一张纸上。然后安装和盖玻片每张幻灯片使用合成, 永久安装介质。 注: 二甲苯是一种易燃产品, 其蒸气对人体健康和水生生物有害。它需要在引擎盖内操作, 并需要使用适当的保护设备。</li>
	</ol>
</li>
</ol>

提交人宣布, 他们没有竞争的经济利益。

大肠杆菌LacZ 基因因其灵敏度高、检测方便, 在基因表达模式研究中得到了广泛的应用。本议定书描述了一种基于酶反应的检测β-gal 的经典方法, 它既简单又快速, 而且价格低廉。这种方法也可以应用在整个芒胚, 完整的器官, 恒温器组织切片或培养细胞没有重大的修改。
这种方法的准确应用导致了解释染色的鲁棒性。但是, 强烈建议同时控制和随后的验证步骤。在这方?...

为了描述特定的基因表达模式, 使用报告基因作为标记是最重要的从果蝇到哺乳动物。在涉及转基因和击倒动物的实验中, 大肠杆菌 (大肠杆菌) 的细菌β -乳糖酶基因(LacZ) 是其中最广泛使用的1、2、3、4. β-乳糖酶 (β-加仑) 催化β-galactosides (如乳糖) 的水解成其单糖 (葡萄糖和半乳糖)5。它最常用的基质是 X-加仑 (5-溴-4-氯-3-哚β D-galactopyranoside), 一种由β-乳糖酶水解而成的糖苷, 它能产生 5-溴-4-氯-3-羟基吲哚和半乳糖。第一种被氧化成二聚体, 当与费里钾和氰化物结合使用时, 产生一种特征不溶的蓝色沉淀 (图 1)6。
在三十年前的7,8, LacZ基因开始被用作一个记者基因。通常, LacZ是插入在一个内源启动子下游的开放阅读框架, 所以它可以用于细菌和细胞培养可视化的细胞含有特定的插入物, 以及在转基因动物作为内源性的示踪剂基因表达模式在发展期间9。在这方面, β-乳糖酶活性的可视化在果蝇中得到了广泛的应用, 以了解从单细胞到整个组织的发育和细胞过程。果蝇遗传学倾向于稳定线的生成 , 其中一个含有报告基因LacZ的修饰 P 元素结构入到基因组的随机位置。因此, 当置于增强因子的影响下, 它可能会以组织特定的方式驱动其表达, 这使得在过去的两年中, 对许多基因的表达模式进行系统分析10。此外, 利用转基因小鼠监测LacZ基因的表达也可以通过 loxP 介导的重组来检测基因重组事件, 并在嵌合体分析中定位突变胚胎干细胞衍生物11, 促进LacZ表达的控制在特定组织并且世俗地。此外, 在整个胚胎中, β-乳糖酶活性的检测可能产生不同强度的差异染色模式, 可以在不同的发育阶段方便地观察, 以分析基因表达的时间变化。8,12。
在这篇文章中, 我们提出了一个协议, 通过 X-gal 染色在小鼠胚胎发育阶段的整个组织中对基因表达进行可视化。我们提出这种组织化学的方法是一种高度敏感和廉价的技术, 有利于准确检测的标记细胞, 无论是在整个装载标本或在细胞水平后石蜡嵌入组织或胚胎。该方法可使小鼠组织中染色的直接可视化与其他方法13相比具有最小背景。

<table><tbody><tr><td>&lt;strong&gt;REAGENTS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2-Propanol</td><td>SIGMA-ALDRICH</td><td>24137-1L-R</td><td></td></tr><tr><td>Agarose</td><td>SCHARLAU</td><td>50004/ LE3Q2014</td><td></td></tr><tr><td>Aqueous mounting medium</td><td>VECTOR LABS</td><td>H-5501</td><td></td></tr><tr><td>Synthetic mounting media</td><td>MERCK</td><td>100579</td><td></td></tr><tr><td>96% Ethanol</td><td>PROLABO</td><td>20824365</td><td></td></tr><tr><td>99.9% Ethanol absolute</td><td>SCHARLAU</td><td>ET00021000</td><td></td></tr><tr><td>50% Glutaraldehyde solution</td><td>SIGMA-ALDRICH</td><td>G6403-100ml</td><td></td></tr><tr><td>85% Glycerol</td><td>MERCK</td><td>104094</td><td></td></tr><tr><td>99.9% Glycerol</td><td>SIGMA-ALDRICH</td><td>G5516</td><td></td></tr><tr><td>Magnesium chloride hexahydrate</td><td>SIGMA-ALDRICH</td><td>63064</td><td></td></tr><tr><td>Nonionic surfactant (Nonidet P-40)</td><td>SIGMA-ALDRICH</td><td>542334</td><td></td></tr><tr><td>Nuclear Fast Red counterstain</td><td>SIGMA-ALDRICH</td><td>N3020</td><td></td></tr><tr><td>Paraffin pastilles</td><td>MERCK</td><td>111609</td><td></td></tr><tr><td>Paraformaldehyde</td><td>SIGMA-ALDRICH</td><td>158127-500g</td><td></td></tr><tr><td>Phosphate buffered saline (tablets)</td><td>SIGMA-ALDRICH</td><td>P4417-50TAB</td><td></td></tr><tr><td>Potassium ferrocyanate</td><td>MERCK</td><td>1049840500</td><td></td></tr><tr><td>Potassium ferrocyanide</td><td>MERCK</td><td>1049731000</td><td></td></tr><tr><td>Sodium azide</td><td>SIGMA-ALDRICH</td><td>S8032</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>SIGMA-ALDRICH</td><td>30970</td><td></td></tr><tr><td>Sodium dihydrogen phosphate monohydrate</td><td>SIGMA-ALDRICH</td><td>106346</td><td></td></tr><tr><td>Sodium phosphate dibasic dihydrate</td><td>SIGMA-ALDRICH</td><td>71638</td><td></td></tr><tr><td>Thymol</td><td>SIGMA-ALDRICH</td><td>T0501</td><td></td></tr><tr><td>Tris hydrochloride (Tris HCl)</td><td>SIGMA-ALDRICH</td><td>10812846001 (Roche)</td><td></td></tr><tr><td>X-GAL</td><td>VENN NOVA</td><td>R-0004-1000</td><td></td></tr><tr><td>Xylene</td><td>VWR CHEMICALS</td><td>VWRC28973.363</td><td></td></tr><tr><td>&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Disposable plastic cryomolds 15x15x5 mm</td><td>SAKURA</td><td>4566</td><td></td></tr><tr><td>Rotatory Microtome</td><td>Leica</td><td>RM2235</td><td></td></tr><tr><td>Cassettes</td><td>Oxford Trade</td><td>OT-10-9046</td><td></td></tr><tr><td>Microscope Cover Glasses 24x60 mm</td><td>VWR</td><td>ECN631-1575</td><td></td></tr><tr><td>Microscope slides</td><td>Thermo Scientific, MENZEL-GL&amp;Auml;SER</td><td>AGAA000001#12E</td><td></td></tr><tr><td>Adhesion microscope slides</td><td>Thermo Scientific, MENZEL-GL&amp;Auml;SER</td><td>J1820AMNZ</td><td></td></tr><tr><td>Flotation Water bath</td><td>Leica</td><td>HI1210</td><td></td></tr><tr><td>Disposable Low Profile Microtome Blades</td><td>Feather</td><td>UDM-R35</td><td></td></tr><tr><td>Paraffin oven</td><td>J.R. SELECTA</td><td>2000205</td><td></td></tr><tr><td>Wax Paraffin dispenser</td><td>J.R. SELECTA</td><td>4000490</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>DM500</td><td></td></tr><tr><td>Polypropylene microcentrifuge tubes 2.0 mL</td><td>SIGMA-ALDRICH</td><td>T2795</td><td></td></tr><tr><td>Polypropylene microcentrifuge tubes 1.5 mL</td><td>SIGMA-ALDRICH</td><td>T9661</td><td></td></tr><tr><td>Orbital shaker</td><td>IKA Labortechnik</td><td>HS250 BASIC</td><td></td></tr><tr><td>Stirring Hot Plate</td><td>Bibby</td><td>HB502</td><td></td></tr><tr><td>Vortex Shaker</td><td>IKA Labortechnik</td><td>MS1</td><td></td></tr><tr><td>Laboratory scale</td><td>GRAM</td><td>FH-2000</td><td></td></tr><tr><td>Precision scale</td><td>Sartorius</td><td>ISO9001</td><td></td></tr><tr><td>pHmeter</td><td>Crison</td><td>Basic 20</td><td></td></tr><tr><td>Optic fiber</td><td>Optech</td><td>PL2000</td><td></td></tr></tbody></table>

tracing gene expression through detection of &#946;-galactosidase activity in whole mouse embryos

大肠杆菌 LacZ基因, 编码β-乳糖酶, 主要是作为一个记者的基因表达和作为示踪剂在细胞谱系研究。经典的组织化学反应是基于基片 X-gal 与铁和铁离子的水解, 产生了一种易于可视化的不溶性蓝色沉淀。因此, β-乳糖酶活动作为发展过程中感兴趣基因表达模式的标志。在这里, 我们描述了检测早期全小鼠胚胎β-乳糖酶活性的标准协议, 以及随后的石蜡切片和 counterstaining 方法。此外, 还提供了一个澄清整个胚胎的程序, 以更好地可视化 X-gal 染色在更深的区域的胚胎。通过执行此过程获得一致的结果, 尽管需要优化反应条件以最小化背景活动。还应考虑试验中的局限性, 特别是关于整个芒染色中胚胎的大小。我们的协议为在小鼠发育过程中的β乳糖酶检测提供了一种灵敏和可靠的方法, 可进一步应用于恒温器切片以及整个器官。因此, 在整个胚胎中使用该协议可以很容易地分析整个发育过程中的动态基因表达模式, 而且在石蜡切片后可以评估细胞水平的详细表达。

在这里, 我们显示了应用标准协议的β-乳糖酶组织化学反应使用 X-gal 作为基质在整个小鼠胚胎 (图 1和图 2)。利用该协议, 在不同胚胎发育阶段 (E9.5、E11.5 和 E12.5) 上, 利用 Mt4-mmp 突变小鼠, 在内源性控制下, 对膜型 4-基质金属蛋白酶 (Mt4-mmp) 的表达进行了研究。Mt4-mmp 启动子 (图 3,图 4,<s...

Watch this Scientific Journal Video about Tracing Gene Expression Through Detection of Î²-galactosidase Activity in Whole Mouse Embryos at JoVE.com

Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

在这里, 我们描述了检测早期全小鼠胚胎β乳糖酶活性的标准协议, 以及石蜡切片和 counterstaining 的方法。这是一个简单和快速的过程, 以监测基因表达在发展期间, 也可以应用于组织切片, 器官或培养细胞。

通过检测全小鼠胚胎β乳糖酶活性追踪基因表达

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. ...

tracing-gene-expression-through-detection-galactosidase-activity

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

26.1K 次观看.  Universidad Europea de Madrid. 在这里, 我们描述了检测早期全小鼠胚胎β乳糖酶活性的标准协议, 以及石蜡切片和 counterstaining 的方法。这是一个简单和快速的过程, 以监测基因表达在发展期间, 也可以应用于组织切片, 器官或培养细胞。

视频: Tracing Gene Expression Through Detection of β-galactosidase Activity in Whole Mouse Embryos

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

蛋白表达的定量分析来研究宗族规范小鼠植入前胚胎

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

科研

发育生物学

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 ...

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

慢病毒载体介导的转基因小鼠生产: 一种简单高效的创始人直接研究方法

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

遗传学

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

全山荧光在小鼠胚胎发生过程中心脏室发育的分析

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

毛因神经发生期间诺奇信号动力学的实时生物发光成像

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

小鼠胚胎感觉轴突预测整个安装成像

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

神经科学

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

有条件的遗传跨突触标记在胚胎小鼠脑

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

使用早期的小鼠胚胎组织特异性基因的染色质免疫沉淀检测

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

生物学

用于单细胞测序的原肠胚胚分离和基因分型

Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, a robust pipeline was designed to perform single-cell and nuclei analysis on mouse embryos from embryonic day E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to wild-type perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, a methodology is presented designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with a same-day genotyping protocol (3 h) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. This method further includes guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate that this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.

作者聚焦：以单细胞分辨率分析谱系特异性突变胚胎的管道

This paper establishes a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.

突变全小鼠胚胎的单细胞 RNA 测序：从上胚层到原肠胚形成结束

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states ...

通过检测全小鼠胚胎β乳糖酶活性追踪基因表达

摘要

探索更多视频

此视频中的章节

蛋白表达的定量分析来研究宗族规范小鼠植入前胚胎

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

慢病毒载体介导的转基因小鼠生产: 一种简单高效的创始人直接研究方法

全山荧光在小鼠胚胎发生过程中心脏室发育的分析

毛因神经发生期间诺奇信号动力学的实时生物发光成像

小鼠胚胎感觉轴突预测整个安装成像

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