在体外和体内生物发光记者人类胚胎干细胞的基因显像
Summary
分子成像技术在干细胞疗法的兴趣与日俱增，是理想的监测干细胞移植后的行为。荧光素酶基因已启用非侵入性，重复评估细胞的存活，位置，并在体内扩散。本视频将演示如何跟踪生活在一个小鼠胚胎干细胞增殖。
Abstract
人类胚胎干细胞（胚胎干细胞）的发现大大增加了在再生医学感兴趣的医学科学家可用的工具。然而，直接注射胚胎干细胞，并从人类胚胎干细胞分化的细胞进入生物体，迄今已阻碍了显着的细胞死亡，畸胎瘤的形成，与宿主的免疫排斥反应。了解在体内的人类胚胎干细胞移植后行为，需要新的成像技术的纵向监测人类胚胎干细胞的本地化，增殖和活力。分子成像已发出超过体内细胞的增殖天，星期，甚至几个月的跟踪调查一个高通量，价格低廉，而敏感的手段。这种进步已经大大提高了人类胚胎干细胞植入，增殖和生活科目的畸胎瘤形成的时空动力学的理解。
分子成像中的一个重大进步，已经从分子和细胞生物学的无创性的记者基因检测在体内的多模态成像平台的延伸。这些记者的基因，控制工程的推动者和促进剂，利用宿主细胞转录机制的优势下，引入使用各种向量和非向量方法的细胞。一旦在细胞中，记者的基因可以被转录组成或只在特定的生物或细胞的条件，对启动子的类型而定。成具有生物活性的蛋白质，记者基因的转录和翻译是那么敏感的，非侵入性的仪器使用探针信号产生的D -荧光素等（例如，CCD摄像头）检测。
为了避免兴奋的光需要跟踪干细胞在体内荧光成像，生物发光报告基因成像系统的要求只是一个外生管理探针诱导光发射。萤火虫荧光素酶，从萤火虫Photinus pyralis的，编码一种酶，它催化D -荧光素的光学活性的代谢产物，oxyluciferin。光学活性，然后可以与外部的CCD摄像头监视。稳定转染细胞进行的记者在他们的染色体DNA的构造，将通过记者构造子细胞的DNA，使人类胚胎干细胞在体内的生存和扩散的纵向监测。此外，记者基因产物的表达，因为信号产生所需，将创建唯一可行的父母和女儿细胞生物发光信号;凋亡或死亡的细胞不会。
在这个视频，跟踪干细胞的增殖和畸胎瘤的形成与生物发光成像所需的具体材料和方法进行说明。
Protocol
建设双融合报告基因 为了进行人类胚胎干细胞的生物发光成像，你首先需要获得稳定表达如像泛素或EF1a构推动者驱动的萤火虫荧光素酶的荧光素酶记者基因的细胞。 本协议的重点是对报告基因的应用，所以这里不提供详细的程序。然而，我们实验室的总体战略是使用双重融合构建载体，包含萤火虫荧光素酶（fluc）和增强型绿色荧光蛋白（EGFP）间隔内pCDNA 3.1 +分隔。 简而言?…
Discussion
相比其他方式，如PET和MRI，生物发光有限的空间分辨率和降低组织的渗透，由于相对薄弱的发射光子的能量（2-3 EV）;，由于这些原因，迄今尚未适用于大型动物。然而，生物发光的优点是低成本，高通量，和非侵入性，使其具有很高的理想，在体内的干细胞在小动物的跟踪。非生物发光记者的基因，如PET和荧光结构，可用于荧光素酶的结合，创造一个融合记者基因，包含个别记者基因的不同的域组成。例如，本…
Acknowledgements
添多伊尔博士和斯坦福大学在体内与生物发光成像援助的成像中心。同时也感谢银黄，博士分享她的技术，干细胞共同注射矩阵解。最后，感谢史蒂夫认为，博士协助兽医照顾动物。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Dulbecco’s Modified Eagle’s Medium (DMEM) | HyClone | |||
| BD Matrigel™ Basement Membrane Matrix | Growth factor reduced (optional: phenol-red free) | BD Biosciences | ||
| mTeSR1 Maintenance Medium for Human Embryonic Stem Cells | StemCell Technologies | |||
| Phosphate Buffered Saline (PBS) | ||||
| D-Luciferin Firefly, potassium salt | Biosynth AG | |||
| Collagenase IV solution | Dissolve 30 mg Collagenase Type IV in 30 mL DMEM-F12 media. Sterile filter and store at 4 degrees (Celsius). | |||
| Baked Pasteur pipets | ||||
| 6-well tissue culture-treated plates | TPP | 92006 |
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Tags
In VitroIn VivoBioluminescenceReporter Gene ImagingHuman Embryonic Stem CellsHESCsRegenerative MedicineCell DeathTeratoma FormationHost Immune RejectionTransplantationImaging TechniquesHESC BehaviorLocalizationProliferationViabilityMolecular ImagingSpatio-temporal KineticsEngraftmentTeratoma-formationNoninvasive Reporter Gene AssaysMulti-modality Imaging PlatformsEngineered Promoters And Enhancers
Cite This Article
Wilson, K., Yu, J., Lee, A., Wu, J. C. In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells. J. Vis. Exp. (14), e740, doi:10.3791/740 (2008).