Name: a cre-lox p recombination approach for the detection of cell fusion in vivo
Uploaded: 2012-01-04T12:00:00
Description: Watch this Scientific Journal Video about A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo at JoVE.com

作者想感谢慷慨提供H1的干细胞，并添哈克博士，博士Gouqing宋和吉尔科克女士威斯康星大学心血管生理学博士Peiman Hemmati（医学系，威斯康星大学麦迪逊分校）执行鼠标手术的核心设施。这项工作是由国家科学基金会通过一个研究生研究奖学金布莱恩弗里曼和美国国立卫生研究院R21 HL089679的支持。

1。捐赠者细胞转染<ol><li> 丰年间质干细胞（MSCs的，从H1胚胎干细胞Peiman Hematti博士惠赠捐赠所得;或者任何假设保险丝在体内的任何物种的细胞类型可受聘）在70 - 80％与1X胰蛋白酶合流（Mediatech 马纳萨斯VA 5分钟）。失活胰蛋白酶，α- MEM完全培养基（无抗生素，Invitrogen公司，卡尔斯巴德CA）18 。在300 XG离心5分钟。 </li><li>小心吸上清和重新暂停颗粒在1毫升...

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这里的方法允许的话，第一次，在生物识别离散细胞融合和时空分析，包括小动物。后续的生物光子图像分析的方法相结合的Cre - LoxP位重组。跟踪不仅是细胞与细胞融合的方法是适合，而且病毒的细胞融合等可以跟踪病毒感染的证明。图像分析是快速，有可能图像的多个小动物的同时，融合检测融合在其相应的微环境，特定的细胞伙伴的频率和LoxP位停止LoxP位的萤光素酶质粒转染效?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>霓虹灯转染系统</td><td> Invitrogen公司，卡尔斯巴德，CA </td><td> MPK5000 </td><td></td></tr><tr><td>霓虹灯100μL，包</td><td> Invitrogen公司，卡尔斯巴德，CA </td><td> MPK10025 </td><td>包含R和发送缓冲区</td></tr><tr><td> A - MEM粉末</td><td> Invitrogen公司，卡尔斯巴德，CA </td><td> 12000-022 </td><td></td></tr><tr><td>胎牛血清（FBS） </td><td> Hyclone公司，UT斯达康洛根</td><td> SH30070.03 </td><td></td></tr><tr><td> B6.C - TG（CMV - CRE）1Cgn / J </td><td>巴尔港杰克逊实验室，ME </td><td> 006054 </td><td></td></tr><tr><td>胰蛋白酶10X </td><td>费希尔科学，森林草坪，NJ </tD&gt; <td> MT - 25 - 054 - CL </td><td></td></tr><tr><td> L -谷氨酰胺</td><td>费希尔科学，森林草坪，NJ </td><td> 25030-081 </td><td></td></tr><tr><td> D -荧光素</td><td>卡尺生命科学，霍普金顿，马</td><td> 122796 </td><td></td></tr><tr><td>精诺真生物光子成像系统</td><td>卡尺生命科学，霍普金顿，马</td><td> IVIS频谱</td><td></td></tr><tr><td>钠Biocarbonate </td><td>西格玛Aldrich公司，圣路易斯，密苏里州</td><td> S6014 - 500G </td><td></td></tr><tr><td>非必需氨基酸</td><td> Invitrogen公司，卡尔斯巴德，CA </td><td> 11140-050 </td><td></td></tr></tbody></table>

a cre-lox p recombination approach for the detection of cell fusion in vivo

已使用两个或两个以上的相同类型的细胞融合的能力，在整个进化的后生动物，形成了许多复杂的器官，包括骨骼肌肉，骨骼和胎盘。现代研究表明，同一类型的细胞融合赋予了增强的功能。例如，当胎盘保险丝的滋养层细胞形成的合体，合体能够更好地运输营养和激素跨比 1-4非稠合滋养的母胎屏障。最近的研究表明，不同类型的细胞融合可以直接细胞的命运。曾经被认为“回归”或修改融合细胞的命运是有限的细胞培养系统。但干细胞移植的到来我们发现，干细胞可以在体内与体细胞融合，融合促进干细胞分化 5-7 。因此，细胞融合是一个监管过程中Capable促进细胞的存活和分化，从而可以发展，修复组织和疾病的发病机制的极端重要性。 限制细胞融合的研究，是缺乏适当的技术：1）准确地识别融合的产品，以及2）随着时间的推移跟踪融合产品。这里我们提出一种新的方法来解决双方的局限性，通过融合后诱导生物发光（图1）;可生物发光检测灵敏度高，在 体内 8-15，我们利用一个构造编码萤火虫荧光素酶基因（Photinus pyralis）放在相邻一个终止密码子两侧LoxP位序列的细胞与细胞表达Cre重组酶蛋白基因的保险丝时，LoxP位网站裂解，使荧光素酶转录停止信号切除，由于信号是induciBLE，假阳性信号的发病率是很低的。与现有方法利用CRE / LoxP系统16，17不同的是，我们已成立了一个“活”的探测信号，从而承受的首次机会跟踪体内细胞融合的动力学。 为了证明这种方法，无处不在表达Cre重组酶的小鼠干细胞转染与构造来表达一个floxed终止密码子的荧光素酶的下游接受者。干细胞移植通过内注射和移植活体图像分析后进行跟踪存在随着时间的推移的心脏和周围组织的融合产品。这种方法可以适应在任何组织类型分析，在任何阶段的发展，疾病或成人组织修复细胞融合。

Watch this Scientific Journal Video about A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo at JoVE.com

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

一个在生活随着时间的推移的生物体细胞融合的方法来跟踪描述。该方法利用CRE -<em&gt; LoxP位</em&gt;重组荧光素酶表达诱导细胞融合后。产生荧光信号可以检测到使用生物光子成像系统的检测〜1000细胞周围组织的敏感性，在生活的有机体。

阿的Cre - LOX P为检测细胞融合重组方法在体内</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

Research

JoVE Journal

Bioengineering

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

生物分子检测的干涉反射成像传感器（IRIS）

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

科研

生物工程

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

无试剂检测核酸，蛋白质和小分子的电化学DNA生物传感器的制备

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蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

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简单的微流控芯片的<em在体内</em&gt;成像<em&gt; C.线虫</em&gt;<em&gt;果蝇</em&gt;和斑马鱼

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

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The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

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一个完整的皮肤缺损模型，以评估生物材料的血管化<em&gt;体内</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

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无标记技术的单细胞分泌物的时空成像

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

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中空纤维生物反应器的<em&gt;在体内</em&gt;般的哺乳动物组织培养

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

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3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

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间质干细胞培养和交付自体条件：用于骨科应用程序的智能方法

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on ...