Name: neonatal pial surface electroporation
Uploaded: 2014-05-07T14:00:00
Description: Watch this Scientific Journal Video about Neonatal Pial Surface Electroporation at JoVE.com

作者要感谢支持，从塞缪尔Oschin全面癌症研究所癌症研究论坛奖，以及资金的雪松，西奈半岛的再生医学研究所和卡介苗家庭。描述该项目由美国国家研究资源中心，格兰特UL1RR033176资助的CTSI核心券的形式得到了支持，并且现在正处于国家中心推进转化科学，格兰特UL1TR000124。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。

这个程序是按照雪松 - 西奈IACUC要求。调查人员应当确保机构IACUC符合前程序。所有工具和试剂应当在使用前灭菌。 1，工具的准备，解决方案和DNA混合物<ol><li>将100毫米火抛光的高硼硅玻璃毛细管成微量拉马。设置加热，以允许标准加权拉以形成约17.5毫米枪头。用锋利的手术剪刀的技巧在距离约8-9毫米的尖端开始，以创造大约100微米直径的开口。 </li><li>做一只股票1％w / v?...

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对软脑膜表面祖成功电穿孔的最关键方面是：1）靶向质粒组合到软脑膜表面; 2）避免血肿​​的发生在注射部位; 3）避免与电脑相关的死亡率。 适当地靶向软脑膜表面由头骨测量和小心刺入完成，以避免软膜表面的渗透。 1）位移后轻轻揉组织（ 即快绿是在皮肤和结缔组织头骨上面的本地化）的快速绿色染料与皮肤：针对不当上覆皮肤或基础脑室或脑实质将由证明或2...

神经干细胞和祖细胞是存在于整个哺乳动物CNS 1，2。它们的性质和在周围的脑和脊髓的脑室区域胚胎和成年生发区属性已被广泛地记载在过去的十年中1-3。在很大程度上，这是由于越来越精确的遗传工具，如两侧装接loxP等位基因或逆转录病毒谱系追踪4的神经系统特异性表达Cre重组的发展。然而，一个祖区域软脑膜表面祖区，最近才在任何细节5-7被描述并等待全面的检查。 脑的软膜表面被定义为在脑表面和周围脑膜8之间的界面。在发育过程中，神经上皮，以及后来的放射状胶质年底脚连接到这个表面9,10。有些杉木ST的神经元在人脑和神经细胞的多丝分裂在此区域11的观察。后来，在胚胎神经，皮质的interneurons被称为遍历软脑膜地区，除了在中间区和脑室下区12-14它们的迁徙路线。在此期间，干细胞可以从该区域中进行培养，它似乎是神经和gliogenesis 5的活性位点。在成人大脑，它已经报道的interneurons可以从软脑膜表面祖细胞缺氧的挑战7诞生。然而，该地区对他的胚胎和出生后发育过程中genensis的贡献由于专门调查这一区域6的难度依然晦涩的部分。在上丘和在大脑皮质，表面（或层我在皮层）的interneurons可以调节下层兴奋性神经元群体的电路的输出，从而有助于significantly这些结构的功能。特别是，第1层的interneurons在首要的位置，调节神经元的整定其广泛的连接性皮质列15,16的浅表和深部层大脑皮层的上层射击。以类似的方式，水平的interneurons接受兴奋性输入从皮 ​​质和视网膜纤维，项目在相对 ​​宽的区域，并推测介导的抑制神经元群体响应远程视觉刺激17,18的。另外，其形态是非常适合于在显影视觉系统19中的图案化波活动中发挥潜在作用。有趣的是，interneuron发育和成熟发生很大程度出生后。另外，该成熟过程中已经发现，通过神经细胞的活动进行调节，因此对电路功能20,21终身后果发育可塑性的基板。值得注意的是，正Ø促销员的描述，可以专门针对这些细胞植入基因。除以祖细胞可以靶向与逆转录病毒7，但病毒产量是耗时并需要技巧，得到所需要的细胞转导的高滴度。 电穿孔导致了文艺复兴时期的神经发育的研究，因为它允许在神经祖细胞4，22，23信号通路的快速，高效的基因审讯。电穿孔涉及到注射的质粒DNA，随后递送电脉冲的头部的外侧，以单向驱动DNA导入周围的脑室4，22，23的增殖祖细胞。电显示通过细胞周期进行质粒的转基因24的表达M期，要求细胞的转运。具体而言，已经发现，只细胞通过内质粒电穿孔的8小时M期会表达外源基因，尽管他们有效地提供心室壁24的所有细胞内〜160微米。据推测，这是由于需要为核膜破裂在允许的附加 ​​型质粒的核设施，如化学品导致的核通透性能诱导后有丝分裂细胞25质粒的表达。最初受聘于胚胎22，电穿孔，是深受26后，27适于用在出生后大脑。最近，我们已经适应了电穿孔的在软脑膜表面祖6遗传操作使用。此外，使用这种方法，我们已经表明，有明显的祖细胞的两个不同细胞系中此区域的元间和星形细胞6。该协议详细介绍了简单，快速，强大的方式来针对这些细胞进行审讯这些细胞的机制调节发展。

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			<td height="21" style="height:21px;">Fire Polished Borosilicate Tubing</td>
			<td>World Precision Instruments, Inc.</td>
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			<td>Sutter Instruments Company</td>
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			<td><a name="RANGE!B6">Harvard Apparatus, BTX Instrument Div</a></td>
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neonatal pial surface electroporation

在过去的几年中软脑膜表面已被确定为重要的生发利基胚胎，围产期和成年神经和gliogenesis期间，包括伤后。然而，对于基因询问这些祖人口和跟踪他们的谱系方法已经由于缺乏特异性或耗时的生产病毒的限制。因此，在该区域的进展一直只有少数这个位置的调查相对较慢。在出生后的大脑电已经使用了十多年，研究在胚胎神经干细胞的特性，以及最近。这里我们描述了一种高效，快速和简单的技术的基础上适合电穿孔的方法软膜表面祖细胞的遗传操作。软膜表面电允许这些祖细胞的浅显遗传标记和操纵，从而代表为研究这些细胞的一个省时省力又经济的方法。

软膜表面电穿孔结果中质粒DNA的细胞中，主要是祖细胞，在室温或接近软脑膜表面6的表达。更具体地，所述电极的定向是在听写质粒运动和随后的表达式的方向是至关重要的。因此，在双电极配置，该质粒被定向在负和正电极之间的大致的直矢量。因此，如果负极放置在注射部位和正极是腹侧向负极，质粒将被压入软膜表面。假定定向的电极以产生电流矢量垂直于表面将是非常理想的。?...

Watch this Scientific Journal Video about Neonatal Pial Surface Electroporation at JoVE.com

Neonatal Pial Surface Electroporation

软脑膜表面是受到越来越多的关注，在中枢神经系统的唯一祖区。在此，我们详细为这个祖区使用改进的电穿孔法快速基因操作的方法。此过程可用于细胞和分子细胞谱系及参与细胞分化信号通路的调查，并阐明子细胞的命运和属性。

新生儿软脑膜表面电穿孔

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and ...

Research

JoVE Journal

Neuroscience

In vivo Electroporation of Developing Mouse Retina

In vivo Electroporation of Developing Mouse Retina

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

科研

神经科学

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

在子宫内电穿孔：鼠标控制时空基因Transefection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Neonatal Subventricular Zone Electroporation

新生儿脑室下区电穿孔

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

Subtype-selective Electroporation of Cortical Interneurons

皮质的interneurons的亚型选择性电穿孔

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

脊髓神经元从新生小鼠中分离和培养

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

新生小鼠脑巴细胞基因调控的立体定向手术

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Cochlear Surface Preparation in the Adult Mouse

成年小鼠中的科利耳表面制备

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice

新生小鼠缺氧缺血期间的连续视频脑电图

Hypoxia ischemia is the most common cause of neonatal seizures. Animal models are crucial for understanding the mechanisms and physiology underlying ...

Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin

通过脑室内注射血红蛋白模拟新生儿脑室内出血

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and ...

Mouse Neonatal Brain Micro-CT Scanning and Image Processing

Micro-CT Imaging and Morphometric Analysis of Mouse Neonatal Brains

Author Spotlight: High-Resolution Imaging of Mouse Neonate Brains &#8211; A Micro-CT Protocol with Lugol&#39;s Solution Contrast Agent

Neuroimages are a valuable tool for studying brain morphology in experiments using animal models. Magnetic resonance imaging (MRI) has become the standard ...