Name: in vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells
Uploaded: 2008-05-02T17:59:00
Description: Watch this Scientific Journal Video about In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells at JoVE.com

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。

<ol><li> 建设双融合报告基因 <ol><li>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。 </li><li>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。 </li><li>简而言?...

<ol>
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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...

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Dulbecco's Modified Eagle's Medium (DMEM)</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>BD Matrigel&#x2122; Basement Membrane Matrix</td>
<td>Growth factor reduced (optional: phenol-red free)</td>
<td>BD Biosciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>mTeSR1 Maintenance Medium for Human Embryonic Stem Cells</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffered Saline (PBS)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>D-Luciferin Firefly, potassium salt</td>
<td>&nbsp;</td>
<td>Biosynth AG</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Collagenase IV solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Dissolve 30 mg Collagenase Type IV in 30 mL DMEM-F12 media. Sterile filter and store at 4 degrees (Celsius).</td>
</tr>
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<td>Baked Pasteur pipets</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>6-well tissue culture-treated plates</td>
<td>&nbsp;</td>
<td>TPP</td>
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in vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells

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。

Watch this Scientific Journal Video about In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells at JoVE.com

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

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。

在体外和体内生物发光记者人类胚胎干细胞的基因显像

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

Research

JoVE Journal

Biology

Derivation of Human Embryonic Stem Cells by Immunosurgery

人类胚胎干细胞的推导Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

科研

生物学

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

在人类胚胎干细胞的线粒体复杂的活动初探

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

在人类胚胎干细胞体外培养的标签用于磁共振成像

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Culture and Maintenance of Human Embryonic Stem Cells  

文化和维护人类胚胎干细胞

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Freezing and Thawing Human Embryonic Stem Cells

人类胚胎干细胞冷冻和解冻

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

Ferumoxides植入人类间质干细胞在软骨缺损的标记

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

视频人类胚胎干细胞克隆生长的生物信息学分析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

胚胎干细胞分化成少突胶质前体

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

人类体细胞重编程为诱导多能干细胞（iPS细胞）用绿色荧光蛋白逆转录病毒载体

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...