Name: genetic manipulation of the mouse developing hypothalamus through in utero electroporation
Uploaded: 2013-07-24T12:00:00
Description: Watch this Scientific Journal Video about Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation at JoVE.com

这项工作是由德国研究基金会（德意志研究联合会）资助。

1。制备DNA和玻璃注射微量移液器<ol><li>质量好的玻璃的微量是必不可少的，降低了初始的高流产率，由于羊水流失。已经有据可查的程序拉玻璃微13,18,25。使用直径1.2mm的毛细管拉在常规萨特设定P = 500;热= 300 P-97的移动设备，上拉= 40，速度= 50;时间= 50。适合拉马用3毫米的“药谷”丝（萨特仪器FT330B）。 2毫米大小的细丝已经取得了对我们不太满意的结果。另一方面，坡口的微量提示似乎...

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关于麻醉：由于在子宫内的电穿孔进入下丘脑可以在技术上是艰巨而需要较长的昏迷时间，我们优选通过氧和异氟醚的混合物给药麻醉诱导和维持。根据我们的经验，动物可以保持适当的麻醉，在这样的时间长达一个小时，至少，母亲的恢复是非常快的，提高胚胎存活率。其他麻醉方法也可提供。最简单的程序由腹腔注射诱导和维持麻醉状态2.5 0.9毫升/千克体重的氯胺酮（25毫克/毫升），?...

遗传操纵胚胎小鼠大脑是首选的方法，了解发育调控。然而，突变小鼠线的产生是缓慢和昂贵。一个强大的方法来引入特定的基因哺乳动物的大脑发育中神经元的变化是在子宫内的电。本质上，该技术包括DNA转染到胚胎脑的神经上皮通过电脉冲，然后使胚胎存活了一段时间，收集大脑，并分析它的可能的新颖，翔实的表型。以这种方式，实验者可以几乎立即测试假设没有小鼠突变体的生产所需的等待时间长。 到胚胎发育开始的DNA转染在OVO电鸡胚1。基本概念证明型的鼠标进行文化<s高达2。这很快就被随后该技术在子宫内 3,4鼠标的第一个描述。 主要的问题是转染而不杀死它们，或者母亲的大脑在子宫内的胚胎发展。学习进行必要的手术（开腹手术，注射，电穿孔），需要一个漫长的训练期。一旦手术已经掌握的胚胎存活率比是可以接受的地步，接下来的关键问题是：大脑结构进行访问？这并不奇怪，首次发表的论文，得到的结果与在子宫内的电主要集中在皮质发育5-9。这是使用这种技术刊物的大部分仍然是正确的，因为该区域是在显影最访问外科手术的小鼠大脑皮质（ 图1）。已经描述了该程序为在子宫内的电穿孔到皮质在打印10 11-14视频。可以使用的技术的修改目标端 ​​脑腹侧部，在基底神经节15。 除了端脑，间脑（传统分为丘脑和下丘脑）是前脑更难以达到的区域。少数的论文报告，针对其背鳍和最容易的部分，丘脑16-19。 下丘脑是最腹侧的前脑的一部分，因此，一个本地化的最深刻的背侧表面（皮层）（ 图1）。 在子宫内基因操作的小鼠大脑的研究人员致力于该地区仍然是一个艰巨的挑战。据我们所知，只有极少数文章报道的在宫内转染到小鼠下丘脑20,21的结果。然而，canno下丘脑功能的重要性被夸大了，因为它规定的行为，如吃喝，交配，繁殖和养育子女的22。此外，改变下丘脑发展有助于以后的生活条件，如肥胖，高血压，糖尿病，性早熟的23起源。基因能够改变下丘脑在开发过程中会提供一个非常强大的工具来了解它。 基本手术开腹孕鼠，我们这里使用的是类似的协议，用于其他协议11,13,14,24。我们将在这里简要地描述他们的完整性。我们的程序的关键，在另一方面，是不同的麻醉，注射的地方，不同的电极的正极相对于胚胎的头部插入和位置。我们优选通过气体吸入超过简单的腹腔麻醉诱导和维持麻醉，因为前者允许有些较长的麻醉需要一个艰难的手术。吸入异氟醚麻醉恢复快，因为通常是母亲表现出正常的行为已经分钟后手术。最简单的点注入的DNA溶液的玻璃微侧脑室内，但它是完全不适合丘脑电穿孔。直接在第三脑​​室注射液确实是针对深间脑结构的关键。这是可能的转染与标准的，现成的电极从E12.0或E12.5下丘脑。我们已经发现了一些特别适合来此的目的NEPA基因（日本千叶）制造的电极。 随着我们的过程中，我们得到整个下丘脑神经上皮或部分区域转取决于电极方向转。在这里，我们展示了乳头体转染技术，可以说是最深和最凹进的所有丘脑核团。此外，我们详细的组织学分析转染细胞的细胞层次的解析。 在子宫内的电转鼠标在子宫内发育中的大脑与其他方法的比较，可以发现在讨论部分。

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Acepromazine</td>
			<td>Sanofi GmbH</td>
			<td>&nbsp;</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>HDG9623</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Ketamin</td>
			<td>Pharma GmbH</td>
			<td>&nbsp;</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Fast Green</td>
			<td>Fluka</td>
			<td>44715</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rimadyl</td>
			<td>Pfizer</td>
			<td>&nbsp;</td>
			<td>non-steroidal anti-inflammatory</td>
		</tr>
		<tr>
			<td>Braunoderm</td>
			<td>Braun</td>
			<td>3887138</td>
			<td>povidone-iodine</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline PBS</td>
			<td>Gibco</td>
			<td>14190</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Temgesic (buprenorphine)</td>
			<td>Essex Pharma</td>
			<td>&nbsp;</td>
			<td>opioid analgesic</td>
		</tr>
		<tr>
			<td>Eye Ointment</td>
			<td>Pan-Ophtal</td>
			<td>7136926</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Anaesthetic Device Komesaroff Mark-5</td>
			<td>Medical Developments Australia</td>
			<td>ACN 004 903 682</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Capillary puller P-97</td>
			<td>Sutter Instrument Co.</td>
			<td>P-97</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Compresstome</td>
			<td>Precisionary Instr.</td>
			<td>VF-300</td>
			<td>Vibratome-type device</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Zeiss</td>
			<td>LSM700</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM3050S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY21EDIT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electrode 1</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY550-10</td>
			<td>Stainless Steel Needle Electrode, 10 mm-Tip, 0. 5 mm diam.</td>
		</tr>
		<tr>
			<td>Electrode 2</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY700P4L</td>
			<td>Cover Round Platinum Plate 4 mm diameter</td>
		</tr>
		<tr>
			<td>Fiberoptic cold light source</td>
			<td>Leica</td>
			<td>KL2500 LCD</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass capillaries</td>
			<td>Harvard Apparatus</td>
			<td>GC120T-15</td>
			<td>1. 2 mm O.D. x 0. 94 mm I.D.</td>
		</tr>
		<tr>
			<td>Glass bead sterilizer</td>
			<td>Fine Science Tools</td>
			<td>FST250</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Harvard Apparatus</td>
			<td>py872-5272</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Injection device</td>
			<td>World Precision Instruments</td>
			<td>Pneumatic Pico Pump PV820</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Suture Thread Coated Vicryl</td>
			<td>Ethicon</td>
			<td>V4914</td>
			<td>Peritoneal Suture</td>
		</tr>
		<tr>
			<td>Suture Thr. Supramid</td>
			<td>Serag Wiessner</td>
			<td>TO07171L</td>
			<td>Skin Suture</td>
		</tr>
	</tbody>
</table>

genetic manipulation of the mouse developing hypothalamus through in utero electroporation

基因改造发展哺乳动物大脑的特定区域，是一个非常强大的实验方法。但是，生成新的小鼠突变体往往是令人沮丧的缓慢。它已被证明， 在子宫内的合理的操作室后的生存期的小鼠脑开发的访问是可能的。不过，此过程的结果与已报道几乎完全发育中的大脑最肤浅和容易获得的部分， 即皮层。丘脑，更窄，更内侧的区域，已被证明更加困难的目标。转染到更深的原子核，特别是下丘脑，可能是最具挑战性的，因此很少的结果已被报道。在这里，我们展示了一个过程为目标的整个下丘脑神经上皮或它的一部分（下丘脑地区）通过电穿孔转染。我们的方法的关键是昏迷时间较长，注射在T希尔德心室，以及适当的种类和电极的定位。此外，我们将展示最凹陷的下丘脑核，乳头体定位和随后的病理分析结果。

大多数丘脑神经元之间E11.5到E15.2出生，根据在大鼠有点短的鼠标发展27,28翻译成26的出生约会分析。下丘脑神经发生的高峰期达到在E12.5 29-31。因此，在本研究转染选择的年龄（E12.5），相当大的比例的下丘脑神经元可以在任何给定的尾鳍rostro的水平标记。 分析E18.5厚vibratome的类型的段（ 图3A和3B）表明，下丘脑神经上皮的整个...

Watch this Scientific Journal Video about Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation at JoVE.com

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

尽管下丘脑的功能和医学重要性，<em&gt;在子宫内</em&gt;遗传操纵其发展已经很少有人尝试。我们显示的详细步骤<em&gt;在子宫内</em&gt;电到老鼠的下丘脑和展示具有代表性的结果整体和局部（区域）下丘脑转。

遗传操纵鼠标的发展下丘脑通过<em&gt;在子宫内</em&gt;电穿孔

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Research

JoVE Journal

Neuroscience

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

科研

神经科学

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 ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

高效的基因传递到多个中枢神经系统使用新界在子宫内电穿孔

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

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 ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

转基因小鼠内耳的发展<em&gt;在体内</em&gt;电穿孔

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

Cortex-，Hippocampus-，Thalamus-，下丘脑 - ，横向隔核 - 纹状体专用<em&gt;在子宫内</em&gt;电穿孔的C57BL / 6小鼠

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

在子宫内电途径研究神经元亚群的兴奋性和单细胞连接

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

延期共聚焦显像移植神经元在器官性切片培养胚胎小鼠脑使用<em&gt;在Utero</em&gt;电穿孔

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...