Name: separation of mouse embryonic facial ectoderm and mesenchyme
Uploaded: 2013-04-12T13:00:00
Description: Watch this Scientific Journal Video about Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme at JoVE.com

作者要感谢艾琳在图1和实验室的其他成员的帮助和讨论，说明胚胎头彩。这项工作是由美国国立卫生研究院授予DE012728（TW）

1。准备Dispase酶II <ol><li>制备新鲜的盐水（HBS）的Hepes缓冲液（50mM HEPES / KOH pH为7.4，150mM的NaCl）。添加到16.8毫升超纯水H 2 O 2毫升0.5 M羟乙基哌嗪/ KOH pH值7.4和1.2毫升2.5 M氯化钠</li><li> 10毫克/毫升中性蛋白酶II。中性蛋白酶称取0.2 g II（罗氏，猫＃4942078001），和溶于20毫升哈佛商学院。准备在个别的Eppendorf管中并储存在-20℃的1.0毫升等分试样用于长期存储。 </li><li> 10毫克/毫升的中性蛋白酶I...

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该协议提供了一个简单的方法来分离面部小鼠胚胎外胚层和间充质的基础上Dispase酶II的初步处理步骤。在以前的研究中，我们进行了解剖的面部突起之前，中性蛋白酶II处理，但我们一直发现，间充质变成“粘”，更难以操纵。我们的新的协议，避免了这个问题。结合中性蛋白酶II处理与柔和的身体的力量，通过上下吹打分离的外胚层和间充质更容易。此外，该协议还可以很好地用于分离的胚胎外...

<table>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <td>Dispase II (neutral protease, grade II)</td>
 <td>Roche</td>
 <td>4942078001</td>
 <td>Make 10 mg/ml Dispase II stock solution in HBS. Aliquot and store at -20 &deg;C . </td>
 </tr>
 <td>RNAlater</td>
 <td>Ambion</td>
 <td>AM7021</td>
 <td>&nbsp;</td>
 </tr>
</table>

separation of mouse embryonic facial ectoderm and mesenchyme

口面裂是最常见的颅面畸形，从而影响1.5在1000新生儿全球1,2。颜面部clefting引起的脸部发育异常3。在人类和小鼠中，最初的增长和图案的脸依赖于几个小芽组织，面部突起4,5。面对来自6个主要的日珥：配对正面的鼻流程（FNP），上颌骨突起（MXP）和下颌骨突起（MDP）。这些日珥包括装在覆上皮细胞肿胀，间质细胞，。在多个物种的研究表明，信号之间的串扰面部外胚层和间充质是至关重要的塑造脸部6。然而，机械的细节关于这些信号继电器中所涉及的基因所缺乏的。的方式来获得一个全面的了解基因表达的转录因子结合，及相关的染色质标记的开发与平面外胚层和间充质细胞的分离和鉴定分离的组织车厢。 在这里，我们提出了一个方法，分离面外胚层和间充质细胞在胚胎天（E）10.5鼠标的面部的形成，融合的日珥之前，一个关键的发展阶段。我们的方法是适用的方法，我们以前用于解剖面部突起7。在这个早期的研究中，我们采用近交系C57BL / 6小鼠作为这株已成为标准的遗传学，基因组学和面部形态。在这里，虽然，由于可用组织更有限的数量，我们已利用远交的CD-1菌株，购买更便宜，更健壮的畜牧业，并趋向于产生更多的胚胎（12-18）比任何自交系每窝小鼠品系8。在胚胎隔离，中性蛋白酶中性蛋白酶用于治疗II全胚胎。然后，面部突起剖析编出来，面部外胚层分离的间充质细胞。这种方法保持完整的面部外胚层和间质。可用于蛋白质检测，染色质免疫沉淀法（ChIP）的检测，基因芯片的研究，和RNA-seq的技术，包括使用这种方法获得的样品。

中性蛋白酶II处理后的胚胎的外胚层往往是松散的。经过解剖（ 图2A）的面部突起应该是完整的。孤立的面部外胚层是明确的，免费的间充质组织（ 图2B）。的协议，以确定疗效和检测交叉污染检测前脑表达Zic3 9，外胚层具体的CDH1和间质特定的SOX10 10采用逆转录聚合酶链反应。我们的研究证实，这两个面部外胚层和间充质细胞表达预期的基因和无交叉污染...

Watch this Scientific Journal Video about Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme at JoVE.com

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

外胚层和间充质细胞的胚胎面部分离的协议。我们使用的是中性蛋白酶II对待整个胚胎首先，剖析整个面部突起，然后分离的面部外胚层和间充质细胞。

分离的小鼠胚胎面外胚层和间充质

 Orofacial clefts are the most frequent  craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal ...

Research

JoVE Journal

Biology

Electrophoretic Separation of Proteins

电泳分离蛋白质

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

科研

生物学

Isolation and Derivation of Mouse Embryonic Germinal Cells

小鼠胚胎生殖细胞的分离和推导

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

腺病毒介导的基因去除信号分子在原代培养的小鼠胚胎成纤维

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

从小鼠胚胎肾肾区细胞的分离和培养

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

偏光易位荧光蛋白爪蟾外胚层响应Wnt信号

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic ...

Electroporation of Craniofacial Mesenchyme

颅面间质细胞的电

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

适用于培养人类胚胎和诱导多能干细胞的小鼠胚胎成纤维细胞的制备

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

A System for ex vivo Culturing of Embryonic Pancreas

A System for ex vivo Culturing of Embryonic Pancreas

一个系统<em&gt;体外</em培养的胚胎胰腺

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

为外植体含量的颅间充质细胞行为评定

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...