Name: in ovo expression of microrna in ventral chick midbrain
Uploaded: 2013-09-16T07:00:00
Description: Watch this Scientific Journal Video about In ovo Expression of MicroRNA in Ventral Chick Midbrain at JoVE.com

我们承认K.米基克，谁促成了这部电影和M.尼古列斯库为的miR图片的初始阶段。 C.胡贝尔是由Universtitätsklinikum蒂宾根大学的IZKF，A.奥威炳廷阿南德由Universtitätsklinikum图宾根的财富计划的奖学金支持。

1。用于在OVO电要求<ol><li> 制备的载体构建：的miR义和反义引物设计后Ambion公司的指示，退火并连接到的ApaI / EcoRI位消化的pSilencer U6.1载体。成功转型后所产生的克隆人被种植和使用Quiagen公司的midi制备试剂盒的pSilencer质粒分离碱裂解法。 </li><li> 电极：电极可以购买或容易建立13。我们使用铂丝的自制电极（Φ= 0.2，0.25或0.5毫米的直径），或者在某些情况?...

<ol>
	<li>Kutter, C., Svoboda, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNA,+siRNA,+piRNA:+Knowns+of+the+unknown.">miRNA, siRNA, piRNA: Knowns of the unknown.</a> RNA Biol. 5, 181-188 (2008).</li><li>Szell, M., Bata-Csorgo, Z., Kemeny, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enigmatic+world+of+mRNA-like+ncRNAs:+their+role+in+human+evolution+and+in+human+diseases.">The enigmatic world of mRNA-like ncRNAs: their role in human evolution and in human diseases.</a> Semin. 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该视频演示了转染质粒转化的小鸡脑的特定区域的神经上皮细胞的有效方法。低电压的矩形电脉冲可以将DNA引入小鸡神经管细胞大毛 6,16。但是，DNA的定位精度由宽电场，其上升通过比较大的电极（Φ= 0.5mm）的经常受阻。我们试图通过下面的百濑等 8的指引，使用电极直径小，以解决这个问题。对于腹侧中脑转染，我们将阴极区带蛋白或膜下如果胚胎的年龄允许它正?...

小非编码RNA作为额外的球员基因表达的识别推出了新的复杂的基因组编程/基因调控。不同品种的非编码RNA在神经细胞，包括非编码小分子RNA 1-4功能的重要性。小分子RNA（MIR或miRNA）为例说明在开发大脑5个不同的和不断变化的表达谱。有针对性的鸡胚蛋内电穿孔提供了开发过程中的基因表达和沉默的时间和空间控制一个独特的机会。 本视频演示了不同的步骤，使用在OVO电6-10在鸡脑的特定区域进行miR的异位表达。为了确保在这些细胞中的小非编码RNA的效果持久，miR的DNA序列克隆到单或双顺反子载体。 在OVO电中，miR含v厄克托基是通过使一个小窗口，在蛋壳后露出的胚胎注入到中脑的神经管。转染的脑小加（阳极）和减号（阴极）的特定区域的铂电极被放置在特定的位置上。对于腹侧中脑的转染，所述阳极被置于左侧中脑腹侧和上面的中脑的右半边的阴极施加电流前下方。在蛋壳的开口被封闭，磁带和胚胎孵育只要所需的任何分析。该方法最初是由村松等 6所描述和特定区域的转染改善了由百濑等 8。 <img src="/files/ftp_upload/50024/50024schematic1.jpg" fo:content-width-="6in" fo:src="/files/ftp_upload/50024/50024schematic1highres.jpg"/> 原理概述。 <ol><li>在鸡蛋胚胎是通过切割一个小窗口，进入次曝光Ë蛋壳。 </li><li>溶解的载体（s）是注入到使用微毛细管中脑。 </li><li>两个电极 - 平行放置，或根据与上面的胚胎 - 产生一个脉冲电场。 </li><li>电场在时间上产生毛孔中的细胞膜，从而促进进入细胞由带负电荷的DNA（或RNA）被吸引到阳极11,12。 </li></ol>

<table border="1">
<tbody>
 <tr>
 <td>Name </td>
 <td>Company</td>
 <td></td>
 <td>Model</td>
 </tr>
 <tr>
 <td>Borosillicate glass capillaries </td>
 <td>Hartenstein</td>
 <td></td>
 <td>Model: 0.9 mm</td>
 </tr>
 <tr>
 <td>Microcapillary puller</td>
 <td>WPI, Berlin</td>
 <td></td>
 <td>Model: Pul1-E</td>
 </tr>
 <tr>
 <td>Electroporator</td>
 <td>Intracel</td>
 <td></td>
 <td>Model: TSSIC</td>
 </tr>
 <tr>
 <td>Stereomicroscope - fluorescence </td>
 <td>LEICA</td>
 <td></td>
 <td>Model: MZFLIII</td>
 </tr>
 <tr>
 <td>Stereomicroscope</td>
 <td>Zeiss</td>
 <td></td>
 <td>Model: Stemi</td>
 </tr>
 <tr>
 <td>Camera and software</td>
 <td>Zeiss</td>
 <td></td>
 <td>Model: Axiocam MRc/ Axiovision Re. 4.8</td>
 </tr>
 </tbody>
</table>

in ovo expression of microrna in ventral chick midbrain

非编码RNA是另外玩家在调节基因表达。针对在特定领域OVO电提供了空间和时间的控制异位microRNA表达谱的独特工具。不过，腹脑结构，如中脑腹侧是相当难以达成任何操作。在这里，我们展示了使用薄的铂电极电穿孔的miRNA进入中脑腹侧一种有效的方式。这种方法提供了一种可靠的方法转染中脑和体内研究的有用工具的特定区域。

转染了miR中脑区域的范围是可见的，通过GFP的从沿与所述miR表达载体（ 图2）喷射的报告基因载体的表达。使用铂电极的直径为0.5mm且平行放置脑引线的AP轴通常为大面积的转染沿DV-和AP-轴，包括后脑，中脑和间脑有时（ 图2A和B）。的电极的结果在宽的电场的大小施加到大脑区域，因此广泛染区。 它难以精确地定位，比如例如在腹侧中脑区域。降低0.5...

Watch this Scientific Journal Video about In ovo Expression of MicroRNA in Ventral Chick Midbrain at JoVE.com

In ovo Expression of MicroRNA in Ventral Chick Midbrain

异位表达的是一种技术来阐明大脑发育的小分子RNA的作用。但是，使用在OVO电针对特定领域是具有挑战性的。这里，我们表明一个有效的方式来选择性电穿孔腹侧和背侧中脑区域。

在微RNA在腹鸡中脑大毛表达

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

Research

JoVE Journal

Neuroscience

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

胚胎腹侧中脑器官型片文化：一个系统来研究多巴胺能神经元发展在体外</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

科研

神经科学

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

在OVO在鸡胚中脑多巴胺神经元发育中的基因功能研究电穿孔

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

在黑质和腹侧被盖区多巴胺调控的综合性能分析

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

对基因差异表达的RNA序列中电穿孔鸡胚脊髓分析

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

中脑多巴胺神经元的数目在成年小鼠环境的调变

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

生活多巴胺能神经元可靠的鉴定中脑培养使用RNA测序和TH-启动子驱动的EGFP表达

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

An In Ovo Model for Testing Insulin-mimetic Compounds

An In Ovo Model for Testing Insulin-mimetic Compounds

用于测试胰岛素模拟化合物的蛋模型

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and ...

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

在河马区文特尔区谷氨酸终端的 Vivo 解剖和功能跟踪组合

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a ...

Loss-of-Function Approach in the Embryonic Chick Retina by Using Tol2 Transposon-Mediated Transgenic Expression of Artificial microRNAs

使用Tol2转座子介导的人工microRNA的转基因表达在胚胎雏鸡视网膜中实现功能丧失的方法

The chick retina has long been an important model system in developmental neurobiology, with advantages including its large size, rapid development, and ...