Name: single-cell electroporation across different organotypic slice culture of mouse hippocampal excitatory and class-specific inhibitory neurons
Uploaded: 2020-10-06T13:30:00
Description: Watch this Scientific Journal Video about Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons at JoVE.com

这项工作得到了国家卫生研究院（R01NS085215至K.F.，T32 GM107000和F30MH122146至A.C）的支持。作者感谢渡边奈女士娴熟的技术援助。

所有动物协议都经过马萨诸塞大学医学院机构动物护理和使用委员会（IACUC）的审查和批准。切片培养制备、质粒制备和电位也详细介绍了我们以前发布的方法，并可以参考其他信息10。
1. 切片文化准备
<ol><li>准备小鼠组织型海马片培养，如前所述11，使用产后6至7天大的男女小鼠。<ol>
<li>为由 （在 mM 中）： 238 蔗糖组成的有机切片培养物...

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+single-cell+manipulation+in+brain+tissue.">High-throughput single-cell manipulation in brain tissue.</a> PLoS One. 7 (4), 35603 (2012).</li><li>Keener, D. G., Cheung, A., Futai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+efficient+method+for+single-cell+electroporation+in+mouse+organotypic+hippocampal+slice+culture.">A highly efficient method for single-cell electroporation in mouse organotypic hippocampal slice culture.</a> Journal of Neuroscience Methods. 337, 108632 (2020).</li><li>Stoppini, L., Buchs, P. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+synaptic+scaling+induced+by+changes+in+postsynaptic+firing.">Rapid synaptic scaling induced by changes in postsynaptic firing.</a> Neuron. 57 (6), 819-826 (2008).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+on+microchips:+the+harmful+effects+of+pH+changes+and+scaling+down.">Electroporation on microchips: the harmful effects of pH changes and scaling down.</a> Science Reports. 5, 17817 (2015).</li><li>Karra, D., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+techniques+for+neuronal+cells.">Transfection techniques for neuronal cells.</a> Journal of Neuroscience. 30 (18), 6171-6177 (2010).</li><li>Taverna, E., Haffner, C., Pepperkok, R., Huttner, W. 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我们在这里描述一种电击方法，它以高效率和高精度同时转导兴奋神经元和抑制神经元。我们优化的电化方案有三个创新突破，以实现高效的基因转染。我们的第一个修改是增加移液器的大小相比，以前公布的协议3，5，6。这种变化使我们能够电化许多神经元，而无需移液器堵塞。此外，与以前的方法相比，低电阻移液器允许...

在分子生物学中，研究者最重要的考虑因素之一是如何将感兴趣的基因传递到细胞或细胞群中，以阐明其功能。不同的分娩方法可分为生物（如病毒载体）、化学（如磷酸钙或脂质）或物理（如电浸、微注射或生物毒性）1、2。生物方法效率高，可以是细胞类型特异性，但受特定遗传工具的发展的限制。化学方法在体外非常强大，但变染通常是随机的：此外，这些方法大多只保留给原细胞。在物理方法中，从技术角度来看，生物处理是最简单和最简单的，但再次以相对较低的效率产生随机转染结果。对于需要转移到特定细胞而不需要开发遗传工具的应用，我们期待单细胞电聚变3，4。
过去二十年来，电聚变只指场电电，而已开发出多个体外和体内单细胞电电化方案，以提高5、6、7的特异性和效率，证明电电化可用于将基因转移到单个细胞，因此，可以非常精确。然而，这些程序在技术上要求高、耗时、效率低下。事实上，最近的论文已经调查了机械化电镀钻机8，9的可行性，这可以帮助消除其中几个障碍，调查人员有兴趣安装这样的机器人。但对于那些寻找更简单的方法的人来说，电透电的问题，即细胞死亡、转染失败和移液器堵塞，仍然是一个问题。
我们最近开发出一种电透法，它使用大倾玻璃移液器、较温和的电脉冲参数和独特的压力循环步骤，在兴奋神经元中产生的转染效率比之前的方法高得多，并使我们能够首次在抑制性内窥镜中转染基因，包括小鼠组织性切片培养10海马CA1区域的索马托他汀表达抑制内周素。然而，这种电位法在不同抑制性内膜类型和神经元发育阶段的可靠性尚未得到解决。在这里，我们证明了这种电位技术能够将基因转化为兴奋神经元和不同类别的内窥神经。重要的是，无论在体外（DIV）切片培养年龄测试天数，转染效率都很高。这种既定的、用户友好的技术强烈推荐给任何有兴趣在体外小鼠脑组织环境中使用不同细胞类型的单细胞电聚变的调查员。

<table>
	<tbody>
		<tr>
			<td>Plasmid preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid Purification Kit</td>
			<td>Qiagen</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Organotypic slice culture preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6 Well Plates</td>
			<td>GREINER BIO-ONE</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5/45 Forceps</td>
			<td>FST</td>
			<td>#5/45</td>
			<td>Angled dissection forceps for organotypic slice culture preparation</td>
		</tr>
		<tr>
			<td>Flask Filter Unit</td>
			<td>Millipore</td>
			<td>SCHVU02RE</td>
			<td>Filtration and storage of culture media</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Binder</td>
			<td>BD C150-UL</td>
			<td></td>
		</tr>
		<tr>
			<td>McIlwain Tissue Chopper</td>
			<td>TED PELLA, INC.</td>
			<td>10180</td>
			<td>Tissue chopper for organotypic slice culture preparation</td>
		</tr>
		<tr>
			<td>Millicell Cell Culture Insert, 30 mm</td>
			<td>Millipore</td>
			<td>PIHP03050</td>
			<td>Organotypic slice culture inserts</td>
		</tr>
		<tr>
			<td>Osmometer</td>
			<td>Precision Systems</td>
			<td>OSMETTE II</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE coated spatulas</td>
			<td>Cole-Parmer</td>
			<td>SK-06369-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>FST</td>
			<td>14958-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Vacuum Filtration System</td>
			<td>Millipore</td>
			<td>SCGPT01RE</td>
			<td>Filtration and storage of aCSF</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary Glasses</td>
			<td>Warner Instruments</td>
			<td>640796</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipetter Puller</td>
			<td>Sutter Instrument</td>
			<td>P-1000</td>
			<td>Puller</td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Binder</td>
			<td>BD (E2)</td>
			<td></td>
		</tr>
		<tr>
			<td>Puller Filament</td>
			<td>Sutter Instrument</td>
			<td>FB330B</td>
			<td>Puller</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single-cell electroporation and fluorescence imaging #1</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3.5 mm Falcon Petri Dishes</td>
			<td>BD Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>Airtable</td>
			<td>TMC</td>
			<td>63-7512E</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Q Imaging</td>
			<td>Retiga-2000DC</td>
			<td>Camera</td>
		</tr>
		<tr>
			<td>Electroporation System</td>
			<td>Molecular Devices</td>
			<td>Axoporator 800A</td>
			<td>Electroporator</td>
		</tr>
		<tr>
			<td>Fluorescence Illumination System</td>
			<td>Prior</td>
			<td>Lumen 200</td>
			<td></td>
		</tr>
		<tr>
			<td>Manipulator</td>
			<td>Sutter Instrument</td>
			<td>MPC-385</td>
			<td>Manipulator</td>
		</tr>
		<tr>
			<td>Metamorph software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Image acquisition</td>
		</tr>
		<tr>
			<td>Peristaltic Pump</td>
			<td>Rainin</td>
			<td>Dynamax, RP-2</td>
			<td>Perfusion pump</td>
		</tr>
		<tr>
			<td>Shifting Table</td>
			<td>Luigs &#38; Neuman</td>
			<td>240 XY</td>
			<td></td>
		</tr>
		<tr>
			<td>Speaker</td>
			<td>Unknown</td>
			<td></td>
			<td>Speakers connected to the electroporator</td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>SZ30</td>
			<td></td>
		</tr>
		<tr>
			<td>Table Top Incubator</td>
			<td>Thermo Scientific</td>
			<td>MIDI 40</td>
			<td></td>
		</tr>
		<tr>
			<td>Upright Microscope</td>
			<td>Olympus</td>
			<td>BX61WI</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence imaging #2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>All-in-One Fluorescence Microscope</td>
			<td>Keyence</td>
			<td>BZ-X710</td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell electroporation across different organotypic slice culture of mouse hippocampal excitatory and class-specific inhibitory neurons

电电已成为将特定基因转移到细胞中以了解其功能的关键方法。在这里，我们描述了一种单细胞电聚技术，它最大限度地提高了小鼠组织型海马切片培养中兴奋和类特异性抑制神经元的体外基因转染效率（+80%）。我们使用大玻璃电极、含四毒素的人工脑脊液和轻度电脉冲，将感兴趣的基因传递到培养的海马CA1金字塔神经元和抑制内窥镜中。此外，电聚可以在培养海马切片中进行长达21天的体外，而不会降低转染效率，从而可以研究不同的切片培养发育阶段。随着对检查不同细胞类型基因的分子功能的兴趣日益浓厚，我们的方法演示了一种可靠而直接的方法，可以在小鼠脑组织中进行体外基因转换，该方法可以使用现有的电生理学设备和技术进行。

我们的单细胞电位能够精确地将基因输送到视觉识别的兴奋和抑制神经元中。我们在三个不同的时间点对三种不同的神经元细胞类型进行电击。帕瓦尔布明 （Pv） 或车辆谷氨酸类型 3 （VGT3） 表达神经元通过交叉 Pvcre （JAX #008069） 或 VGT3cre （JAX #018147） 线与 TdTomato （红色荧光蛋白的变体） 记者线 （Jax #007905）， 分别命名为 Pv/TdTomato 和 VGT3/TdTomato 线。从C57BL/6J、Pv/TdTomato 和 VGT3/Td...

Watch this Scientific Journal Video about Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons at JoVE.com

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

这里介绍的是一个单细胞电位化协议，可以在一系列体外海马切片培养年龄内提供兴奋和抑制神经元的基因。我们的方法提供精确和高效的基因表达在单个细胞，可用于检查细胞自主和细胞间功能。

小鼠河马兴奋和类特异性抑制神经元的不同组织型切片培养的单细胞电位

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a ...

Research

JoVE Journal

Neuroscience

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

海马脑片的器官型检查神经元损伤的文化模式

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

科研

神经科学

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

器官型切片文化绿色荧光蛋白表达的实时成像周围神经生长的小鼠胚胎

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Nucleofection和胚胎小鼠海马和皮层神经元的原代培养

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Organotypic Hippocampal Slice Cultures

器官型海马切片培养

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

从产前小鼠海马神经元的分离和培养

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Primary Culture of Mouse Dopaminergic Neurons

小鼠多巴胺能神经元的原代培养

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

器官切片文化研究少突胶质细胞动力学和髓鞘

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

在器官型海马脑片搭配全细胞记录

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

神经血管重塑的分析Entorhino，海马脑切片培养

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

器官海马切片培养模型研究肿瘤细胞的神经保护和侵袭性

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...