通过检测全小鼠胚胎β乳糖酶活性追踪基因表达
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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) 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)。 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…
Representative Results
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…
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| REAGENTS | |||
| 2-Propanol | SIGMA-ALDRICH | 24137-1L-R | |
| Agarose | SCHARLAU | 50004/ LE3Q2014 | |
| Aqueous mounting medium | VECTOR LABS | H-5501 | |
| Synthetic mounting media | MERCK | 100579 | |
| 96% Ethanol | PROLABO | 20824365 | |
| 99.9% Ethanol absolute | SCHARLAU | ET00021000 | |
| 50% Glutaraldehyde solution | SIGMA-ALDRICH | G6403-100ml | |
| 85% Glycerol | MERCK | 104094 | |
| 99.9% Glycerol | SIGMA-ALDRICH | G5516 | |
| Magnesium chloride hexahydrate | SIGMA-ALDRICH | 63064 | |
| Nonionic surfactant (Nonidet P-40) | SIGMA-ALDRICH | 542334 | |
| Nuclear Fast Red counterstain | SIGMA-ALDRICH | N3020 | |
| Paraffin pastilles | MERCK | 111609 | |
| Paraformaldehyde | SIGMA-ALDRICH | 158127-500g | |
| Phosphate buffered saline (tablets) | SIGMA-ALDRICH | P4417-50TAB | |
| Potassium ferrocyanate | MERCK | 1049840500 | |
| Potassium ferrocyanide | MERCK | 1049731000 | |
| Sodium azide | SIGMA-ALDRICH | S8032 | |
| Sodium deoxycholate | SIGMA-ALDRICH | 30970 | |
| Sodium dihydrogen phosphate monohydrate | SIGMA-ALDRICH | 106346 | |
| Sodium phosphate dibasic dihydrate | SIGMA-ALDRICH | 71638 | |
| Thymol | SIGMA-ALDRICH | T0501 | |
| Tris hydrochloride (Tris HCl) | SIGMA-ALDRICH | 10812846001 (Roche) | |
| X-GAL | VENN NOVA | R-0004-1000 | |
| Xylene | VWR CHEMICALS | VWRC28973.363 | |
| EQUIPMENT | |||
| Disposable plastic cryomolds 15x15x5 mm | SAKURA | 4566 | |
| Rotatory Microtome | Leica | RM2235 | |
| Cassettes | Oxford Trade | OT-10-9046 | |
| Microscope Cover Glasses 24×60 mm | VWR | ECN631-1575 | |
| Microscope slides | Thermo Scientific, MENZEL-GLÄSER | AGAA000001#12E | |
| Adhesion microscope slides | Thermo Scientific, MENZEL-GLÄSER | J1820AMNZ | |
| Flotation Water bath | Leica | HI1210 | |
| Disposable Low Profile Microtome Blades | Feather | UDM-R35 | |
| Paraffin oven | J.R. SELECTA | 2000205 | |
| Wax Paraffin dispenser | J.R. SELECTA | 4000490 | |
| Stereomicroscope | Leica | DM500 | |
| Polypropylene microcentrifuge tubes 2.0 mL | SIGMA-ALDRICH | T2795 | |
| Polypropylene microcentrifuge tubes 1.5 mL | SIGMA-ALDRICH | T9661 | |
| Orbital shaker | IKA Labortechnik | HS250 BASIC | |
| Stirring Hot Plate | Bibby | HB502 | |
| Vortex Shaker | IKA Labortechnik | MS1 | |
| Laboratory scale | GRAM | FH-2000 | |
| Precision scale | Sartorius | ISO9001 | |
| pHmeter | Crison | Basic 20 | |
| Optic fiber | Optech | PL2000 |
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Citer Cet Article
Blanco, M. J., Learte, A. I., Marchena, M. A., Muñoz-Sáez, E., Cid, M. A., Rodríguez-Martín, I., Sánchez-Camacho, C. Tracing Gene Expression Through Detection of β-galactosidase Activity in Whole Mouse Embryos. J. Vis. Exp. (136), e57785, doi:10.3791/57785 (2018).