Name: a pipeline using bilateral in utero electroporation to interrogate genetic influences on rodent behavior
Uploaded: 2020-05-21T14:45:00
Description: Watch this Scientific Journal Video about A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior at JoVE.com

我们感谢丽莎·克雷茨格对手稿的批判性反馈和编辑。我们感谢克鲁兹-马丁实验室的所有研究助理，他们在帮助进行注水和行为大脑细胞计数方面非常宝贵。我们感谢安德烈·克韦奇对三极电极设计的意见，以及托德·布鲁特和波士顿大学生物成像核心的使用。这项工作得到了NARSAD青年研究员赠款（AC-M，#27202）、布伦顿R.卢茨奖（ALC）、I.奥尔登·马奇奖（ALC）、NSF NRT UTB：神经光子学国家研究奖学金（ALC，#DGE1633516）和波士顿大学本科研究机会项目（WWY）的支持。资助者在研究设计、数据收集和分析、决定出版或编写手稿方面没有作用。

所有实验方案均根据美国国家卫生研究院（NIH）动物研究指南执行，并经波士顿大学机构动物护理和使用委员会（IACUC）批准。
1. DNA溶液准备
<ol><li>购买商业质粒或子克隆感兴趣的基因到质粒与所需的促进剂。在这里，使用了 CAG 推广器 （pCAG-EGFP） 下含有 EGFP 的质粒。 注：根据所需的表达水平确定所需的推广人。一般来说，CAG促进剂下的质粒可用于达到高水平的转基因?...

<ol>
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这里描述了一条管道，它结合了对大量前皮质神经元感兴趣的新基因的处理与小鼠的行为检测。此外，该管道允许对同一小鼠在产后发育早期和成年期间的行为进行纵向研究。这项技术绕过了依赖遗传动物模型的需要，这种模型在时间和费用方面可能代价高昂。这个协议的优点是，它可以用来研究神经发育和神经精神病，最近GWAS已经发现了新的遗传关联28，29。</sup...

全基因组关联研究（GWAS）推动了与大脑病理学1、2、3、4相关的新候选基因的发现。这些研究在理解精神分裂症（SCZ）等破坏性神经精神病方面特别有益，对新基因的研究成为新研究和治疗干预的起点。在产前和产后早期发育期间，SCZ的基因在前额皮层（PFC）中表现出偏颇的表达，这一区域与几种神经精神病的病理学有关。此外，小鼠模型的精神疾病表现出异常活动在PFC网络6，8，9。这些结果表明，SZC相关基因可能在这个区域的发展布线中发挥作用。需要使用动物模型进行进一步调查，以了解这些候选基因对PFC中建立联系的贡献，并确定这些基因是否在神经精神病的发病机制中具有因果关系。小鼠的基因操纵技术，允许在产前和产后发育期间研究特定神经元回路的基因表达变化，是了解将基因表达变化与PFC功能障碍联系起来的分子机制的有希望的方法。
遗传小鼠系提供了一种研究特定基因对大脑发育和功能的影响的方法。然而，依靠转基因小鼠可能是有限的，因为并不总是有商业上可用的线来检查特定基因对神经回路发展的影响。此外，开发自定义鼠标线可能非常昂贵和耗时。将转基因小鼠与病毒方法相结合的交叉基因操纵策略的使用彻底改变了对大脑10、11、12的理解。尽管取得了很大进展，病毒策略还是有一定的局限性，这取决于病毒载体类型，包括包装能力的限制，可以限制病毒表达13和细胞毒性与病毒表达14。此外，在大多数实验条件下，使用腺相关病毒（AAV）的强健基因表达大约需要2至4周15周，这使得常规的病毒策略在产后早期发育期间无法操纵基因。
在子宫电化（IUE）是一种替代方法，允许快速和廉价的基因转移16，17，当加上荧光标签和药理遗传或光遗传学方法，提供了一个强大的平台，解剖神经元电路的功能。此外，随着CRISPR-Cas9基因组编辑基因的发展，可以通过细胞型特异性敲击或敲除特定基因或通过18、19的调节来过度表达或精确改变。当基因对神经元电路的影响需要在狭窄的发育窗口20中进行测试时，使用IUE的基因操纵方法尤其有利。IUE 是一种多才多艺的技术，通过将基因插入特定促进器下的表达载体，可以轻松实现过度表达。通过使用不同强度的促进剂驱动表达，或使用能够暂时控制基因表达的诱发促进剂21，22，可以达到对基因表达的额外控制。此外，IUE允许在特定皮质层、细胞类型和大脑区域内定位细胞，这并不总是可行的使用其他方法5，17。基于三个电极的IUE配置的最新进展，产生了更高效的电场分布，扩展了这种方法的功能范围，使科学家能够瞄准新的细胞类型，提高可瞄准的23、24个电池的效率、准确性和数量。这项技术最近被用来确定补充组件4A（C4A）的因果作用，这是一种与SCZ相连的基因，在PFC功能和早期认知5中。
这里介绍的是一个实验管道，它结合了基因转移方法，以目标前额皮层（包括PFC）中的大量兴奋神经元为目标，以及行为范式，不仅能够研究细胞和电路水平的变化，而且允许在整个产后发育和成年后对行为进行监测。首先描述的是一种在正面皮质区域双边传染大量层（L）2/3金字塔神经元的方法。接下来，概述了检测幼鼠和成年小鼠社会行为的任务。细胞计数可以在完成行为任务后获得，以量化细胞转染的范围和位置。此外，转染的细胞数量可以与行为数据相关联，以确定更多的转染细胞是否会导致行为更大的扰动。

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			<td height="21" style="height:21px;width:215px;">13mm Silk Black Braided Suture</td>
			<td style="width:121px;">Havel&#39;s</td>
			<td style="width:161px;">SB77D</td>
			<td style="width:167px;">Suture skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adson Forceps</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">11006-12</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">C270 Webcam</td>
			<td style="width:121px;">Logitech</td>
			<td style="width:161px;">N/A</td>
			<td>Record behavior</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Electroporator</td>
			<td style="width:121px;">Custom-built</td>
			<td style="width:161px;">N/A</td>
			<td>See Figure 1 and 2 and Bullmann et al, 2015</td>
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		<tr height="21">
			<td height="21" style="height:21px;">EZ-500 Spin Column Plasmid DNA Maxi-preps Kit, 20preps</td>
			<td>Bio Basic Inc.</td>
			<td style="width:161px;">BS466</td>
			<td>Pladmid preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Fast Green FCF</td>
			<td style="width:121px;">Sigma</td>
			<td>F7252-5G</td>
			<td>Dye for DNA solution</td>
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			<td height="21" style="height:21px;width:215px;">Fine scissors- sharp</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">14060-09</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisherbrand Gauze Sponges</td>
			<td>Fisher Scientific</td>
			<td>1376152</td>
			<td>IUE</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Gaymar Heating/Cooling</td>
			<td style="width:121px;">Braintree</td>
			<td style="width:161px;">TP-700</td>
			<td style="width:167px;">Heating Pad</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glass pipette puller</td>
			<td>Sutter Instrument,</td>
			<td style="width:161px;">P-97</td>
			<td>IUE</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glass pipettes</td>
			<td>Sutter Instrument,</td>
			<td>BF150-117-10</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Hair Removal Lotion</td>
			<td style="width:121px;">Nair</td>
			<td style="width:161px;">N/A</td>
			<td>Hair removal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hartman Hemostats</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">13002-10</td>
			<td>IUE</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Open field maze- homemade acrylic arena</td>
			<td style="width:121px;">Custom-built</td>
			<td style="width:161px;">N/A</td>
			<td>50 &#215; 50 &#215; 30 cm length-width-height</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">pCAG-GFP</td>
			<td style="width:121px;">Addgene</td>
			<td>11150</td>
			<td>Mammalian expression vector for expression of GFP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Picospritzer III</td>
			<td>Parker Hannifin</td>
			<td style="width:161px;">N/A</td>
			<td>pressure injector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Retractor - 2 Pronged Blunt</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">17023-13</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Ring forceps</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">11103-09</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Sterilizer, dry bead</td>
			<td style="width:121px;">Sigma</td>
			<td>Z378569</td>
			<td>sterelize surgical tools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SUTURE, 3/0 PGA, FS-2, VIOLET FOR VET USE ONLY</td>
			<td>Havel&#39;s</td>
			<td>HJ398</td>
			<td>Suture muscle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Water bath</td>
			<td style="width:121px;">Cole-Parmer</td>
			<td>EW-12105-84</td>
			<td style="width:167px;">warming sterile saline</td>
		</tr>
	</tbody>
</table>

a pipeline using bilateral in utero electroporation to interrogate genetic influences on rodent behavior

随着全基因组关联研究揭示了许多神经系统疾病的异质遗传基础，研究特定基因对大脑发育和功能的贡献的必要性增加。依靠小鼠模型来研究特定基因操纵的作用并不总是可行的，因为转基因小鼠系成本很高，而且许多与疾病相关的新基因还没有商业上可用的基因系。此外，创建鼠标线可能需要多年的开发和验证。在子宫电镀中，一种相对快速和简单的方法，以细胞类型特定的方式在体内操纵基因表达，只需要开发DNA质粒来实现特定的基因操作。子宫内电子化的双边可用于瞄准大量前额皮层金字塔神经元。将这种基因转移方法与行为方法相结合，可以研究基因操纵对前额皮层网络功能以及幼鼠和成年小鼠社会行为的影响。

成功开发并实施了定制电镀器和三爪电极。 对于 IUEs，根据先前描述的设计27（图 1A和图2）构建了廉价的定制电子增压器。一个三爪电极是23，24使用塑料钳子与2个负电极连接到爪尖和正极连接到牙刷手柄的末端（图1B）。对电极和三个爪电极进行了测?...

Watch this Scientific Journal Video about A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior at JoVE.com

A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior

最近发现的疾病相关基因在神经精神病发病机制中的作用仍然模糊不清。子宫电化技术的改性双边技术允许在大量神经元中进行基因转移，并检查基因表达变化对社会行为的因果关系。

使用双边子宫内电渗透的管道，以询问遗传对啮齿动物行为的影响

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution ...

Research

JoVE Journal

Biology

In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos

E16大鼠胚胎在子宫内侧脑室注射和电穿孔

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors ...

科研

生物学

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

E15小鼠胚胎在子宫内侧脑室注射和电穿孔

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

Method for Culture of Early Chick Embryos ex vivo New Culture

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

双整装原位杂交技术在早期鸡胚

Method for Whole Mount Antibody Staining in Chick

整装在鸡的抗体染色方法

Assay for Neural Induction in the Chick Embryo

在鸡胚神经诱导的含量为

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

在子宫内和前体内的基因表达在小鼠视网膜神经节细胞的电穿孔

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

监测内质网钙稳态使用<em&gt; Gaussia</em&gt;荧光素酶SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...